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  • 1990-1994  (1,483)
  • 1991  (1,483)
  • Cell & Developmental Biology  (1,482)
  • Nuclear reactions
  • 201
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 3-4 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
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  • 202
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 5-6 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
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  • 203
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 7-8 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
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  • 204
    ISSN: 0730-2312
    Keywords: oncogenes ; osteoblasts ; osteocalcin ; alkaline phosphatase ; collagen ; transcription ; gene expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: There is a generalized reciprocal relationship between cell growth and expression of genes that occurs following completion of proliferation, which supports the progressive development of cell and tissue phenotypes. Molecular mechanisms which couple the shutdown of proliferation with initiation of tissue-specific gene transcription have been addressed experimentally in cultures of primary diploid osteoblasts that undergo a growth and differentiation developmental sequence. Evidence is presented for a model which postulates that genes transcribed post-proliferatively are suppressed during cell growth by binding of the Fos/Jun protein complex to AP-1 Promoter sites associated with vitamin D responsive elements of several genes encoding osteoblast phenotype markers (Type I collagen, alkaline phosphatase, osteocalcin).
    Additional Material: 2 Ill.
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  • 205
    ISSN: 0730-2312
    Keywords: stromal cells ; cytokines ; synergy ; high proliferative potential stem cell ; Dexter culture ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The intramedullary control of marrow cell production has been a difficult area to approach experimentally. The introduction by Dr. Dexter and colleagues of long-term stromal dependent culture systems for murine marrow and the adaptation of these systems to human marrow growth have allowed for in-vitro studies of stromal dependent hemopoiesis. Despite some controversy in this area, most studies appear to show that adherent murine or human stromal cells are capable of producing a relatively large number of hemopoietic growth factors including G-CSF, GM-CSF, CSF-1, IL-6 and, at least by PCR analysis, IL-3. Other work indicates that the most primitive hemopoietic cells which appear to be multifactor responsive adhere directly to these stromal cells presumably through mediation of various adherence proteins.An early acting, multilineage factor termed hemolymphopoietic growth factor-1 (HLGF-1) has been isolated from a murine stromal cell line and may be identical to the recently described ligand for the c-kit receptor. This may represent an important early survival/maintenance factor for stem cells in this system.Studies on primitive stem cells, especially the high proliferative potential colony forming cell (HPP-CFC), indicate that they are responsive to varying combinations of growth factors and that with increasing numbers of growth factors, as studied in serum-free systems, decreasing concentrations of the factors may be biologically active.These observations altogether suggest that intramedullary hemopoiesis may be regulated by the positioning of early multifactor responsive stem cells via adherent proteins in juxtaposition to synergistically acting combinations of grwoth factors attached to stromal cell surfaces or the extracellular matrix. In addition, selective production of different growth factors from different subsets of cells may create growth factor gradients and explain the spacial distribution of different cell types within the marrow cavity.
    Additional Material: 2 Ill.
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  • 206
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 303-310 
    ISSN: 0730-2312
    Keywords: hepatoma-associated antigens ; nonhistone proteins ; nuclear matrix ; polyclonal antibodies ; Morris hepatoma 7777 ; Kirkman-Robbins hepatomas ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Polyclonal antibodies generated against a group of high molecular weight nonhistone proteins from Morris hepatoma 7777 were used in immunological studies of hepatoma-associated nonhistone proteins in rat and hamster. We revealed the presence of cross-reactive antigens in rat Morris hepatomas 7777 and 8994, and in hamster Kirkman-Robbins hepatoma, but not in normal rat or hamster livers. These specific nonhistone proteins were found to be preferentially localized in the nuclear matrix of rat Morris hepatoma 7777 as well as hamster Kirkman-Robbins hepatoma.
    Additional Material: 4 Ill.
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  • 207
    ISSN: 0730-2312
    Keywords: preadipocyte ; growth hormone ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In the present study, monoclonal antibodies were produced using porcine adipocyte extracts as the immunogen. Two of the monoclonal antibodies, designated CB6 and IB4, exhibited reactivity toward only cells containing lipid in stromal-vascular cell cultures. The antigens recognized by the CB6 and IB4 monoclonal antibodies were 50 kD and 55 kD proteins, respectively. In vivo, IB4 immunoreactivity was detected only in lipid-containing cells, whereas immunofluorescence using CB6 was also detectable around muscle fiber bundles underlying the subcutaneous mesenchyme. In fetal subcutaneous mesenchyme, CB6 and IB4 immunoreactivities toward lipid-containing cells increased with developmental age, but each was not detectable in cells containing the smallest lipid droplets. In stromal-vascular cultures containing adipocytes, 48 hour treatment with the anti-lipogenic agent, growth hormone, only slightly altered CB6 immunoreactivity, whereas IB4 immunoreactivity was reduced by more than sixfold. The exact identity of the CB6 and IB4 antigens was not determined, but each may be useful as markers for studying regulation of adipocyte metabolism.
    Additional Material: 4 Ill.
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  • 208
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 279-283 
    ISSN: 0730-2312
    Keywords: immortalization ; cell lines ; astrocytes ; retroviruses ; grafts ; temperature sensitive ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Immortalized cell lines can serve as model systems for studies of neuronal development and restoration of function in models of neurological disease. Cell lines which result from spontaneous or experimentally-induced tumors have been used for these purposes. More recently, the techniques of genetic engineering have resulted in the production of cell lines with specific desired characteristics. This has been accomplished by insertion of a desired gene into a pre-existing immortal cell or by immortalizing primary cells. The production of immortal cell lines using temperature-sensitive immortalizing genes offers an additional method of controlling gene expression, and thereby controlling cell proliferation and differentation. In the nervous system, these techniques have produced immortal cell lines with neuronal and glial properties.
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  • 209
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 311-316 
    ISSN: 0730-2312
    Keywords: 2,6,10,14-tetramethylpentadecane ; histone ; DNA conformation ; pristane ; lymphocyte ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effects of pristane on the conformation of chromatin in cells isolated from the lymphoid tissues of pristane-treated Copenhagen rats were examined by flow cytometry, thermal denaturation, sensitivity to enzymatic digestion, and histone protein analyses. Decreases were observed in the fluorescent intensities of propidium iodide (PI) stained nuclei isolated from lymphoid cells of pristane-treated rats when compared with normal rat lymphoid nuclei. Studies to address the possible basis for the pristane-induced changes in the DNA staining characteristics of lymphocytes demonstrated that (1) there were no decreases in the amount of DNA present in the nuclei, (2) nuclei isolated from pristane treated rats were less sensitive to thermal denaturation, as well as DNase I enzymatic digestion, and (3) there were apparent increases in the expression of the H1 histone proteins. Collectively, these results suggest that pristane elicits a conformational change in the chromatin which may be mediated by altered expression of nuclear-associated histone proteins.
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  • 210
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991) 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 211
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 319-326 
    ISSN: 0730-2312
    Keywords: tissue repair ; macrophage ; fibroblast ; extracellular matrix ; growth factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.
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  • 212
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 317-318 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 213
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 292-302 
    ISSN: 0730-2312
    Keywords: apatite ; bone matrix ; foreign body giant cells ; implants ; extracellular matrix ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Specific cellular interactions with components of the extracellular matrix can influence cellular differentiation and development of many tissues. The extracellular matrix of bone is composed of organic constituents and a solid phase of calcium and inorganic phosphate (apatite). When implanted subcutaneously in rats, particles of bone matrix (BPs) recruit progenitors that differentiate into multinucleated cells with osteoclastic features. Because BPs deficient in osteocalcin, a bone matrix protein, were less efficient at promoting osteoclast formation than were normal BPs, we directly examined the influence of osteocalcin on osteoclast differentiation. We evaluated tissue responses to particles of synthetic crystalline apatite alone (Ap), having many of the features of native apatite of mature bone, or to apatite prepared with osteocalcin (Ap/OC), bovine serum albumin (Ap/BSA) or rat bone collagen (Ap/Col). Twelve days after subcutaneous implantation in normal rats, Ap, Ap/BSA, and Ap/Col particles generated a mild foreign body reaction with multinucleated cells in direct contact with the particles; these cells were negative for tartrate-resistant acid phosphatase (TRAP) activity and lacked ruffled borders. In contrast, Ap particles containing approximately 0.1% osteocalcin were partially resorbed and they generated more multinucleated cells that were TRAP-positive, were immunoreactive with an antibody against tartrate-resistant purple acid phosphatase, and displayed ultrastructural features of active osteoclasts including ruffled borders and clear zones. These data support the hypothesis that osteocalcin may function as a matrix signal in the recruitment and differentiation of bone-resorbing cells.
    Additional Material: 7 Ill.
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  • 214
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 335-339 
    ISSN: 0730-2312
    Keywords: cytotoxic lymphocyte regulation ; IL-2 activated lymphocytes ; cytokine networks ; tumor necrosis factor ; interleukin-1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: High dose [〉 1 nM or 30 IU] interleukin-2 (IL-2) can induce MHC -unrestricted killing from various lymphoid populations. Although it is well established that CD16+ NK cells are the major source of blood-derived LAK precursors, other lymphoid cells, including several CD3+ T subsets can be a source of precursor activity. We hypothesize that most, if not all, lymphocytes with cytolytic potential may eventually express MHC-unrestricted killing, when provided with adequate IL-2 to intiate required secondary cytokine production. This perspective article presents our cumulative data supporting the role of secondary cytokines in the IL-2 initiated activation of MHC-unrestricted killing, first by our observations of synergy with the exogenously added TNFs or IL-1s in combination with low dose IL-2, and then by the evidence of endogenous cytokine production and response in lymphocytes stimulated with high dose IL-2.Understanding the amplification mechanism(s) of the various effector arms of the immune system is critical to the eventual regulation of graft rejection, autoimmune phenomena, and potentially to the treatment of cancer. Our studies have focused on the cytotoxic lymphocyte effector system, and have addressed the molecular pathways by which IL-2 induced cytokines influence the quantity and quality of the cytotoxic lymphocyte response.This article will review the pivotal role that IL-2 plays in the development of CTL (MHC-restricted antigen-specific cytotoxic lymphocytes), followed by the description of how studies in the CTL system led to the observation that IL-2 alone can activate a heterogeneous collection of MHC-unrestricted killer lymphocytes, originally known as “Lymphokine Activated Killers” or LAK. We will then describe experiments performed in out laboratory over the past several years demonstrating the positive regulation of LAK activity by exogenous addition of TNF-α, TNF-β, IL-1α or IL-1β. Finally, we will summarize our data and propose, in the context of the current literature, the endogenous autocrine/paracrine amplification network of secondary cytokines operative in the generation of LAK.
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  • 215
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 353-358 
    ISSN: 0730-2312
    Keywords: bone marrow ; peripheral blood ; gene transfer ; IL-2 ; neo gene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human T-lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T-cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T-lymphocytes were propagated in culture for up to 14 weeks with interleukin-2 (IL-2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T-cells are capable of long-term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.
    Additional Material: 3 Ill.
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  • 216
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 327-334 
    ISSN: 0730-2312
    Keywords: multiple-cytokine responsive enhancer (MRE) ; glucocorticoid repression ; promoter occlusion ; keratinocytes ; breast carcinoma cell lines ; cell proliferation ; cell motility ; cell-cell association ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Epithelial cells both produce and are affected by interleukin-6 (IL-6). Experiments with an adenocarcinoma-derived cell line (HeLa) reveal that activation of the transfected human IL-6 promoter occurs largely through two partially overlapping second messenger (cAMP, phorbol ester)- and cytokine (IL-1, TNF, serum)-responsive enhancer elements (MRE I, -173 to -151 and MRE II, -158 to -145). MRE I contains the typical GACGTCA cAMP and phorbol ester-responsive (CRE/TRE) motif, whereas MRE II defines a new CRE/TRE motif that contains an imperfect dyad repeat. The mechanism of dexamethasone-mediated repression of IL-6 gene expression in epithelial cells involves occlusion of the entire MRE enhancer region and of the core-promoter elements (TATA-box and RNA start site) by ligand-activated glucocorticoid receptor. Enhanced levels of IL-6 expression are observed in many solid tumors and in the hyperprolifer active (and glucocorticoid-suppressible)lesions of psoriasis. In cell culture, IL-6 enhances, inhibits, or has no effect on the proliferation of epithelial cells depending upon the cell-type examined IL-6 enhances proliferation of keratinocytes but inhibits that of breast carcinoma cell lines ZR-75-1 and T-47D. In these breast carcinoma cells, IL-6 elicits a major change in cell phenotype which is characterized by a fibroblastoid morphology, enhanced motility, increased cell-cell separation, and decreased adherens type junctions (desmosomes and focal adhesions). The new data identify IL-6 as a regulator of epithelial cell growth and of cell-cell association.
    Additional Material: 3 Ill.
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  • 217
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 359-365 
    ISSN: 0730-2312
    Keywords: extracorporeal liver assist device ; hepatocyte transplantation ; acute liver failure ; hollow fiber ; P450 ; hepatocyte culture ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Technological advances in the separation and culture of mammalian hepatocytes have facilitated the use of these cells as the foundation for either hepatocyte transplantation or hepatocyte-seeded hollow fiber liver assist devices (LAD). To fully appreciate the practical applications of these tissue engineering solutions, it is necessary to understand the types of human liver failure as well as the corresponding animal models. The most immediate application of this type of technology is the treatment of hepatic encephalopathy (HE), an acute and highly fatal complication of fulminant hepatic failure. Although the pathogenesis of HE is unknown, failure of the detoxification function of the liver is accepted as playing an important role in this disorder. Consequently, the assaying and preservation of P450 activity in the grafted cells or in the LAD must be among the main targets of this research. This review explores the problems in hepatocyte transplantation and culture that deserve special consideration and emphasizes the conditions contributing to the vitro maintenance of phenotypic expression of these cells.
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  • 218
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 340-345 
    ISSN: 0730-2312
    Keywords: controlled release ; sustained release ; drug delivery ; proteins ; polymer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The development of biocompatible, controlled release systems for macromolecules has provided the opportunity for researchers and clinicians to target and deliver, on site, biologically active factors. This advance has also facilitated the purification and characterization of a number of important biomolecules. These systems include controlled release delivery systems which release proteins through porous polymer matrices, degradable polymeric delivery systems, and modulated polymer release systems. These areas of research will be reviewed with regards to their design, release kinetics, and biocompatibilities. The utilization of these systems to release such biologically important polypeptides as growth factors (e.g., fibroblast growth factor, epidermal growth factor, transforming growth factor-B) as well as a number of important inhibitory factors (e.g., nitrosoureas, angiogenesis inhibitors) in both in vivo and in vitro studies will be discussed.
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  • 219
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 346-352 
    ISSN: 0730-2312
    Keywords: growth factors ; wound healing ; receptors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Wound healing is a localized process which involves inflammation, wound cell migration and mitosis, neovascularization, and regeneration of the extracellular matrix. Recent data suggest the actions of wound cells may be regulated by local production of peptide growth factors which influence wound cells through autocrine and paracrine mechanisms. Two peptide growth factors which may play important roles in normal wound healing in tissues such as skin, cornea, and gastrointestinal tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α). EGF/TGF-α receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract. In addition, EGF or TGF-α are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. Healing of a variety of wounds in animals and patients was enhanced by treatment with EGF or TGF-α. Epidermal regeneration of partial thickness burns on pigs or dermatome wounds on patients was accelerated with topical application of EGF or TGF-α, and EGF treatment accelerated healing of gastroduodenal ulcers. EGF also increased tensile strength of skin incisions in rats and corneal incisions in rabbits, cats, and primates. Additional research is needed to better define the roles of EGF, TGF-α, and their receptor in normal wound healing, to determine if alterations have occurred in the EGF/TGF-α system in chronic wounds, and to optimize vehicles for effective delivery of peptide growth factors to wounds.
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  • 220
    ISSN: 0730-2312
    Keywords: HIV-1 infection of promyelocytes ; unintegrated HIV-1 DNA ; azidothymidine ; CD4 ; clonal analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In this report, we describe a human immunodeficiency virus type-1 (HIV-1)-infected promyelocytic cell line, OM, derived from HL-60 cells. Although the OM cell line was biologically cloned twice, the pattern of HIV-1 expression during culture appeared analogous to a classical acute spreading infection and was inhibited by both azidothymidine and recombinant soluble CD4 treatment. The number of OM cells actually expressing HIV-1 at the beginning of culture was 0%, reached a peak of nearly 100% at 6 weeks, and then fell to 〈 10% HIV-1+ cells by 10 weeks. Clonal analysis of the surviving cells verified that stable HIV-1+ OM cells resulted from the spreading infection. Southern analysis confirmed the transmission of HIV-1 through these OM cultures and the occurrence of stable clones which resulted. The initial percentage of OM cells actually harboring the HIV-1 genome was 〈0.1%, indicating nonfaithful transmission of an unintegrated HIV-1 genome during clonal expansion. These results demonstrate that extrachromosomal HIV-1 DNA can contribute to the spread of HIV-1 infection and give rise to cells which have stably integrated HIV-1 provirus.
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  • 221
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 374-380 
    ISSN: 0730-2312
    Keywords: lipogenesis ; lipolysis ; protein phosphorylation ; second messenger ; glycogen synthesis ; adipocytes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The pleiotropic nature of insulin action suggests diverse mechanisms of signal transduction for the hormone. The specific protein phosphatase inhibitor, okadaic acid, is utilized to differentiate metabolic pathways that may be regulated by phosphorylation or dephosphorylation of key enzymes. In H-35 hepatoma cells, okadaic acid inhibits insulin-stimulated glycogen synthesis with an IC50 of 400 nM. In contrast, activation of lipogenesis by insulin is inhibited with an IC50 of 50 nM okadaic acid. The toxin also inhibits stimulation of lipogenesis in these cells by the insulin-sensitive inositol glycan enzyme modulator. In isolated rat adipocytes, insulin-stimulated lipogenesis is also inhibited by okadaic acid with an IC50 of approximately 1,700 nM. The antilipolytic effect of insulin in these cells is more sensitive to okadaic acid, exhibiting an IC50 of 150 nM. Maximal activation of lipogenesis by insulin is dramatically reduced by okadaic acid with no effect on the concentration required for half-maximal activation, whereas the sensitivity of insulin-induced antilipolysis is attenuated by okadaic acid, with no apparent reduction in the maximal effect of the hormone. Taken together, these data suggest that specific phosphatases may be differentially involved in some of the metabolic pathways regulated by insulin.
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  • 222
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 391-400 
    ISSN: 0730-2312
    Keywords: phosphorylation ; linear kinetics ; T antigen peptide ; whole cell lysates ; mitotic cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H-A-D-A-Q-H-A-T-P-P-K-K-K-R-K-V-E-D-P-K-D-F-OH (T antigen) and H-K-R-A-L-P-N-N-T-S-S-S-P-Q-P-K-K-K-P-L-D-G-E-Y-NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 μM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexs of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2-binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinases and their regulation throughout the cell division cycle.
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  • 223
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 46 (1991) 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 224
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 401-411 
    ISSN: 0730-2312
    Keywords: neutral endopeptidase ; uterus contractions ; oxytocin ; kidney enkephalinase ; uterus enzyme ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the latter stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.
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  • 225
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    Journal of Cellular Biochemistry 46 (1991), S. 1-2 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 226
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    Journal of Cellular Biochemistry 46 (1991), S. 3-8 
    ISSN: 0730-2312
    Keywords: glioma ; cytogenetics ; molecular genetics ; tumor progression ; glioblastoma staging ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The tendency of human cancers to progress towards a more malignant state over time is a well described biological phenomenon. Recent investigations elucidating the genetic nature of malignancy and the possible mechanisms responsible for this evolution have suggested that a sequential pathway may exist whereby a cell population accumulates a nested set of genetic aberrations which endow it with the ability to overwhelm other populations and dominate the tumor. Human astrocytomas are a dramatic case in point, where specific genetic events of amplification and deletion are seemingly related to the stages of malignancy. The identification of these aberrations represents the first stage in the dissection of the temporal process of this cancer. Its augmentation with functional analyses will likely allow a fuller genetic description of in vivo transformation.
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  • 227
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    Journal of Cellular Biochemistry 46 (1991), S. 9-20 
    ISSN: 0730-2312
    Keywords: polyinosinic acid: polycytidylic acid ; antiviral ; antiproliferation ; gene induction ; immunomodulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The study of double-stranded RNA (dsRNA) encompasses a variety of fields. Basic research in this area has contributed to a greater mechanistic understanding of gene induction, tumor cell growth arrest, the establishment of antiviral states, and immunomodulation. Because of the possible clinical value of these molecules, physicians are now exploring the use of synthetic dsRNA to treat patients with cancer, HIV-1 disease, and immune dysfunction. Continued studies of the mechanisms of action of dsRNA are likely to suggest an even wider scope of clinical applications.
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  • 228
    ISSN: 0730-2312
    Keywords: ADCC ; cytokine ; macrophages ; tumors ; human colon cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mononuclear phagocytes participate in host immunological defense against tumors. We have investigated the role of selected recombinant cytokines on human macrophage-mediated tumor cytotoxicity in vitro utilizing a human colon cancer cell line target, SW1116, and murine monoclonal antibody 17-1A. Blood monocytes were kept in continuous culture to allow differentiation into macrophages. Maximum antibody dependent cellular cytotoxicity (ADCC) as measured in a 3H-thymidine release assay occurred after culturing the monocytes for 5-7 days. Human recombinant macrophage colony stimulating factor (CSF) (1,000 U/ml) did not increase ADCC above control levels whereas recombinant human granulocyte-macrophage colony stimulating factor, interleukin 4, and interleukin 3 were all capable of increasing ADCC. Antibodies to the CD11/CD18 integrin receptors did not significantly inhibit ADCC. When the ADCC incubation occurred in the presence of antibodies to the human Fc receptors, ADCC was inhibited significantly only by anti-FcRIII (3G8). A role for tumor necrosis factor alpha or other soluble mediators of cytotoxicity was not demonstrable in this system. These studies suggest avenues for manipulation and augmentation of macrophagemediated antitumor ADCC.
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  • 229
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    Journal of Cellular Biochemistry 46 (1991), S. 21-26 
    ISSN: 0730-2312
    Keywords: hemopoiesis ; ES cells ; myoblasts ; osteoblasts ; hepatocytes ; neurones ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The notion that a single hormone may exert a broad range of effects has become well established. As such, leukemia inhibitory factor (LIF) is a prime example. LIF was initially described, purified, and genetically cloned on the basis of its ability to induce the differentiation and suppress the clonogenicity of the monocytic leukemia cell line, M1. Subsequently, it has become apparent that in vitro LIF inhibits the differentiation of pluripotential ES cells, stimulates the synthesis of hepatic acute-phase proteins, induces a switch in neurotransmitter phenotype from adrenergic to cholinergic, suppresses adipocyte lipoprotein lipase activity, and results in an increase in bone resorption. Moreover, elevation of LIF levels in vivo has a number of patho-physiological consequences, many of which parallel those effects observed in vitro. The challenge that lies ahead is to determine whether other sites of LIF action exist and to define more clearly the physiological role LIF plays in vivo.A major mechanism of cell-cell communication is by the production and secretion of polypeptide hormones by one cell type, which act either systemically or locally, via interaction with specific receptors on the surface of responsive cells. Recently, it has become apparent that hormones initially described and named, on the basis of a specific action, in many cases exert a spectrum of effects on a broad range of cell types. Moreover, the effects exerted are often mimicked closely by other hormones. Hormones that act in a pleiotropic manner are, for example, transforming growth factor-β (TGF-β), the various fibroblast growth factors (FGFs), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF). This review will focus on the various biological effects ascribed to LIF.
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  • 230
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    Journal of Cellular Biochemistry 46 (1991), S. 37-47 
    ISSN: 0730-2312
    Keywords: nucleic acid conformation ; DNA ; DNA conformation ; polyamines ; spermine ; spermidine ; DNA bending ; A DNA ; B DNA ; Z DNA ; anthracyclines ; DNA/ligand interactions ; polyamine function ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Modeling, x-ray diffraction, and solution studies have contributed to the understanding of interactions between polyamines and nucleic acids. Polyamines stabilize a variety of unusual DNA structures and conformations in vitro, including both the left-handed Z and the right-handed A DNA. In addition, polyamines condense DNA and may be important in bending specific sequences. Investigations into the mechanisms of these effects provide support for both specific and nonspecific interactions between polyamines and DNA. Although exact relationships between the binding of polyamines and conformational changes in nucleic acids are still being clarified, polyamines remain important candidates for regulators of DNA conformation in vivo.
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  • 231
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    Journal of Cellular Biochemistry 46 (1991), S. 54-59 
    ISSN: 0730-2312
    Keywords: Na+/nucleoside cotransport ; AE-1 nucleoside transport mutant ; S49 mouse lymphoma cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: S49 murine lymphoma cells were examined for expression of various nucleoside transport systems using a non-metabolized nucleoside, formycin B, as substrate. Nitrobenzylthioinosine (NBTI)-sensitive, facilitated transport was the primary nucleoside transport system of the cells. The cells also expressed very low levels of NBTI-resistant, facilitated nucleoside transport as well as of Na+-dependent, concentrative formycin B transport. Concentrative transport was specific for uridine and purine nucleosides, just as the concentrative nucleoside transporters of other mouse and rat cells. A nucleoside transport mutant of S49 cells, AE-1, lacked both the NBTI-sensitive, facilitated and Na+-dependent, concentrative formycin B transport activity, but Na+-dependent, concentrative transport of α-aminoisobutyrate was not affected.
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  • 232
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    Journal of Cellular Biochemistry 46 (1991), S. 27-36 
    ISSN: 0730-2312
    Keywords: atherosclerosis ; EDHF ; endothelial dysfunction ; endothelin ; flow ; hypoxia ; leukocytes ; mechanoreception ; muscarinic receptor subtypes ; nitric oxide ; platelets ; pressure ; S-nitroso-L-cysteine ; thrombosis ; vasospasm ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Key discoveries in the past decade revealed that the endothelium can modulate the tone of underlying vascular smooth muscle by the synthesis/release of potent vasorelaxant (endothelium-derived relaxing factors; EDRF) and vasoconstrictor substances (endothelium-derived contracting factors; EDCF). It has become evident that the synthesis and release of these substances contribute to the multitude of physiological functions the vascular endothelium performs. Accumulating evidence suggests that at least one of the EDRFs is identical with nitric oxide (NO) or a labile nitroso compound, which is produced from L-arginine by an NADPH- and Ca2+-dependent enzyme, arginine oxidase. The existence of more than one chemically distinct EDRF has been proposed, including an endothelium-derived hyperpolarizing factor (EDHF). The target of EDRF (NO) is soluble guanylate cyclase (increase in cyclic GMP) while EDHF appears to activate a K+-channel in vascular smooth muscle. Recent data suggest that muscarinic receptor subtypes selectively mediate the release of EDRF(NO) (M2) and EDHF (M1). EDRF(NO) affects not only the underlying vascular smooth muscle, but also platelets, inhibiting their aggregation and adhesion to the endothelium. The antiaggregatory effect of EDRF is synergistic with prostacyclin, so their combined release may represent a physiological mechanism aimed at preventing thrombus formation. An additional proposed biological function of EDRF(NO) is cytoprotection by virtue of scavenging superoxide radicals. The endothelium can also mediate vasoconstriction by the release of a variety of endothelium-derived contracting factors (EDCF). Other than the unique peptide endothelin, the nature of EDCFs has not yet been firmly established. Autoregulation of cerebral and renal blood flow and hypoxic pulmonary vasoconstriction may represent the physiological role of endothelium-dependent vasoconstriction. Growing evidence indicates that the endothelium can serve as a unique mechanoreceptor, sensing and transducing physical stimuli (e.g., shear forces, pressure) into changes in vascular tone by the release of EDRFs or EDCFs. In physiological states, a delicate balance exists between endothelium-derived vasodilators and vasoconstrictors. Alterations in this balance can result in local (vasospasm) and generalized (hypertension) increase in vascular tone and also in facilitated thrombus formation. Endothelial dysfunction may also contribute to the pathophysiology of angiopathies associated with hypercholesterolemia and atherosclerosis.
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  • 233
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    Journal of Cellular Biochemistry 46 (1991), S. 60-68 
    ISSN: 0730-2312
    Keywords: bactericidal activity ; macrophages ; neutrophils ; β-1,3-D-polyglucose ; microbeads ; IL1 ; TNF ; PGE2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Beta-1,3-D-polyglucose derivatives protect mice against otherwise lethal bacterial infections. This protective effect has been considered to be mediated through mononuclear phagocytes. By using radioactive labelling, we localized the β-1,3-D-polyglucose derivatized microbeads (GDM) during the period following injection. The GDM was recovered mainly in the milky spots of the omentum. In animals treated with GDM, the total white cell number was significantly increased in peritoneal fluid of mice before and after challenge with E. coli. Bacterial counts in peritoneal fluid of GDM treated animals declined to zero after 24 h. In untreated animals there was a slight increase in bacterial counts until the animals died after about 12 h. Mouse peritoneal macrophages stimulated with GDM released significant amounts of IL-1 and PGE2. There was no significant release of TNF. Levels of IL-1 and PGE2 in peritoneal fluid increased significantly during the first 48 h after treatment with GDM. There was no increase of levels of TNF. After challenge with E. coli, the levels of IL-1, TNF, and PGE2 were significantly lower compared with control animals. In untreated animals the levels of IL-1 and TNF remained elevated until the animals died after about 12 h. These studies demonstrate that the raised levels of arachidonic acid metabolites after pretreatment with GDM or AG seems to inhibit the otherwise lethal elevation of IL-1 and TNF in body fluids which is seen in untreated animals.
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  • 234
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    Journal of Cellular Biochemistry 46 (1991), S. 48-53 
    ISSN: 0730-2312
    Keywords: HSPG synthesis ; isolation of basal extracellular HSPG ; primary cultures ; serum-free medium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Primary cultures of rat hepatocytes maintained as monolayer in a serum-free medium synthesise and secrete sulphated proteoglycans. Nearly 5% of the total 35(S)-sulphated material was obtained in a soluble form from beneath the cell layer. A shift in gel filtration pattern on β-elimination with alkali suggested that it is a sulphated proteoglycan. On ion exchange chromatography over Dowex AG 1×2, the major fraction was eluted with 1.25 M NaCl. Further, nearly 80% of the 35(S)-labeled material was susceptible to nitrous acid degradation and more than 90% of the material was resistant to chondroitinase ABC digestion suggesting that it is predominantly a heparan sulphate proteoglycan (HSPG). Since HSPG is a major component of basement membrane, its binding with collagen was studied by a solid phase binding assay. About 75% of the 35(S) HSPG bound to wells coated with type IV collagen whereas only about 20% bound to type I collagen at physiological pH. Binding to collagen IV was reduced by about 50% when free GAG chains were used indicating that the protein core is also involved in interaction with the collagen. These results indicate the possible role of this basal extracellular heparan sulphate proteoglycan in the basal lamina formation.
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  • 235
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    Journal of Cellular Biochemistry 46 (1991), S. 69-77 
    ISSN: 0730-2312
    Keywords: cytokines ; cell proliferation ; polyamines ; cytotoxicity ; growth factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The activity of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), has been shown to be rapidly modulated by a variety of growth regulatory molecules. In this report the effect of the growth modulatory peptide, tumor necrosis factor, on ODC activity was examined on two cell lines which express equivalent TNF binding properties, but differ in their growth response when exposed to this factor. TNF treatment of WI-38 fibroblasts stimulated both their growth and induced ODC activity 5-10-fold when measured 6-24 h after TNF incubation. TNF induced cytotoxicity in ME-180 cervical carcinoma cells and, interestingly, stimulated both ODC activity (3-6-fold) and putrescine accumulation when measured prior to the onset of cytotoxicity. Induction of ODC was TNF concentration-dependent and paralleled the concentration-dependency for cytotoxicity. Based upon studies with cycloheximide, de novo protein biosynthesis was required for TNF-mediated ODC induction in ME-180 cells.The effects of other growth inhibitory peptides and growth factors were analyzed for their combined effect on ODC activity in TNF-treated or untreated ME-180 cells. Interferon gamma treatment had no significant effect on basal ODC activity but inhibited TNF-mediated ODC induction by ∼50%. EGF treatment resulted in a potent stimulation of ODC activity which was not effected by TNF pre-treatment or coadministration on ME-180 cells. These results suggest that TNF has properties which are similar to those of a growth factor and distinct from those of other growth inhibitory peptides. The early growth factor-like actions of TNF occur on both normal fibroblasts and some tumor cells and evidence suggests that these effects are antagonistic to the antiproliferative effects of TNF.
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  • 236
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    Journal of Cellular Biochemistry 46 (1991), S. 94-94 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 237
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    Journal of Cellular Biochemistry 46 (1991), S. 78-85 
    ISSN: 0730-2312
    Keywords: sarcoplasmic reticulum ; calcium regulation ; calcium/phospholipid-binding protein ; calcium-release/uptake ; immunolocalization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Annexin VI is a member of a Ca2+-dependent, phospholipid-binding protein family. Although functions for this annexin have been proposed from in vitro studies, most remain controversial. Díaz-Muñoz et al. (J Biol Chem 265:15894, 1990) demonstrated that annexin VI modified, in a Ca2+-dependent manner, the gating behavior of the sarcoplasmic reticulum Ca2+-release channel, reconstituted into artificial bilayers, by increasing both the open probability and the mean open time. This effect was specific to the trans chamber, which represents the luminal side of the sarcoplasmic reticulum. In agreement with those findings, we show herein that annexin VI produced no effect on Ca2+-uptake or -release by intact heavy sarcoplasmic reticulum vesicles (analogous to the cis chamber). We also used monospecific antibodies to evaluate the subcellular localization of annexin VI by immunofluorescent microscopy. Studies in rat skeletal muscle suggest that annexin VI is present surrounding individual myofibrils. Double immunolocalization studies with cultured muscle cells (chick myotubes) using anti-annexin VI and anti-SR Ca2+-ATPase antibodies demonstrated superimposable staining patterns. In non-muscle tissue (normal rat kidney (NRK) cells), a punctate, perinuclear anti-annexin VI staining pattern was observed. Collectively, these data suggest that annexin VI may play a regulatory role in the Ca2+-release/uptake cycle in the sarcoplasmic reticulum as well as in non-muscle organelles, a key process in stimulus-response systems.
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  • 238
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    Journal of Cellular Biochemistry 46 (1991) 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 239
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    Journal of Cellular Biochemistry 46 (1991), S. 86-93 
    ISSN: 0730-2312
    Keywords: Ca2+-dependent ; phospholipid-binding ; proteolysis ; purification ; repeats ; immunoreactivity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Annexin VI has eight highly conserved repeated domains; all other annexins have four. Díaz-Muñoz et al. (J Biol Chem 265:15894, 1990) reported that annexin VI alters the gating properties of the ryanodine-sensitive Ca2+-release channel isolated from sarcoplasmic reticulum. To investigate the domain structure of rat annexin VI (67 kDa calcimedin) required for this channel regulation, various proteolytic digestions were performed. In each case, protease-resistant core polypeptides were produced. Annexin VI was digested with V8 protease and two core polypeptides were purified by Ca2+-dependent phospholipid binding followed by HPLC. The purified fragments were shown to be derived from the N- and C-terminal halves of annexin VI, and demonstrated differential immunoreactivity with monoclonal antibodies to rat annexin VI. While both core polypeptides retained their ability to bind phospholipids in a Ca2+-dependent manner, they did not regulate the sarcoplasmic reticulum Ca2+-release channel as did intact annexin VI.
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  • 240
    ISSN: 0730-2312
    Keywords: terminal differentiation ; senescence ; C-Ha-ras oncogene ; growth regulation ; inositol triphosphate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Extracellular levels of calcium at 1.05 mM or higher induce terminal differentiation and senescence in the mortal (MCF-10M) line of human breast epithelial cells, but does not retard the growth or induce differentiation in the immortal (MCF-10A) and oncogene transformed (MCF-10AneoT) lines. Intracellular levels of calcium and inositol triphosphate were determined in MCF-10M, MCF-10A, and MCF-10AneoT, under conditions of low and high extracellular calcium. We hereby report that increases in extracellular calcium is translated into significant increases in intracellular levels of calcium and inositol triphosphate in MCF-10M, but not in MCF-10A and MCF-10AneoT. This difference in the apparent calcium buffering capacity between the mortal and the immortalized human breast epithelial cells could account for the latter's unperturbed growth potential in high extracellular calcium environment.
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  • 241
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    Journal of Cellular Biochemistry 46 (1991), S. 242-249 
    ISSN: 0730-2312
    Keywords: hEGF ; site-directed mutagenesis ; receptor affinity ; receptor kinase ; mitogenesis ; NMR ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Seven site-specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K½ (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity. Substitutions are tolerated to different degrees, depending upon hydrophobicity and size of the side chain. Substitution with ionic residues led to the most drastic reduction in activity. One-dimensional nuclear magnetic resonance spectroscopy, at physiological pH, of several of the mutants did not detect any major structural perturbations which would account for the loss of activity. The results suggest that the side chain of leucine 47, because of its charge neutrality, size, and hydrophobicity, is highly important, although not absolutely essential for the interaction of EGF with its receptor. A striking finding was the lower (compared with wild type) Vmax values of the mutants in the tyrosine kinase reaction, but these low Vmax mutants, in cell culture experiments, were able to stimulate at high concentrations a growth response equivalent to wild type EGF.
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  • 242
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    Journal of Cellular Biochemistry 46 (1991) 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 243
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    Journal of Cellular Biochemistry 46 (1991), S. 284-290 
    ISSN: 0730-2312
    Keywords: erythrocytes ; magnesium ; echinocyte ; calcium ; plasma membrane ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Intact human erythrocytes, initially depleted of Mg2+ by EDTA incubation in the presence of A23187, exhibit Mg2+-dependent phosphate production of around 1.5 mmol per liter cells · h, half-maximally activated at around 0.4 mM added free Mg2+. This appears to correspond to Mg2+-stimulated adenosine triphosphatase (Mg2+-ATPase) activity found in isolated membranes, which is known to have a similar activity and affinity for Mg2+. Vanadate (up to 100 μM) inhibited Mg2+-dependent phosphate production and ATP breakdown in intact cells. Over a similar concentration range vanadate (3-100 μM) transformed intact cells from normal discocytes to echinocytes within 4-8 h at 37°C, and more rapidly in Mg2+-depleted cells. The rate of Ca2+-induced echinocytosis was also enhanced in Mg2+-depleted cells. These results support previous studies in erythrocyte ghosts suggesting that vanadate-induced shape change is associated with inhibition of Mg2+-ATPase activity localized in the plasma membrane of the red blood cell.
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  • 244
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    Journal of Cellular Biochemistry 46 (1991), S. 277-283 
    ISSN: 0730-2312
    Keywords: transformation ; malignancy ; metastasis ; gene regulation ; cancer ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Changes in the quantitative expression of certain genes or in the amounts of their products can quickly stimulate progression to the metastatic phenotype. This has been done experimentally by transferring dominantly acting oncogenes such as c-H-rasEJ into susceptible cells or more recently by interfering with metastasis suppressor genes. In vivo such rapid qualitative changes in dominantly acting oncogenes or suppressor genes occur only rarely, and progression to highly metastatic phenotypes is thought to occur through a process involving the slow stepwise progression of a subpopulation of neoplastic cells to more malignant states. Such slow changes can be reversible and need not involve known dominantly acting oncogenes or metastatic suppressor genes, consistent with clinical and experimental observations on naturally occuring, highly advanced metastatic tumors. An important element in the natural progression of tumors to more malignant states may be their ability to circumvent host environmental controls that regulate growth and cellular diversity. They also evolve into heterogeneous cellular phenotypes, a process that appears to mainly involve quantitative changes in gene expression but can be rapidly stimulated in cell culture by the introduction of a dominantly acting oncogene or inhibited by the introduction of a suppressor gene. The oncogenes and suppressor genes that affect malignancy may control important steps in the quantitative regulation of sets of genes that are ultimately responsible for the cellular alterations seen in adhesion receptors, cell motility responses, cell-cell communication components, degradative enzymes and their inhibitors, growth factor receptors, components that aid in escape from host surveillance mechanisms and others that are important in malignancy. Highly malignant cells that have slowly evolved in vivo may contain only a few qualitative gene changes but have undergone extensive cycles of diversification and accumulation of quantitative changes in the expression of genes that encode products that are related to malignancy and metastasis. Thus highly malignant cells can arise quickly due to specific qualitative changes in critical controlling genes or more slowly by less critical qualitative genetic changes together with cycles of cellular diversification and accumulation of quantitative changes in gene expression.
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    Journal of Cellular Biochemistry 46 (1991), S. 291-301 
    ISSN: 0730-2312
    Keywords: protein phosphorylation ; phosphotyrosine ; phosphotyrosyl protein phosphatase ; orthovanadate ; cell proliferation ; canine prostate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The ability of dividing canine prostatic epithelial cells in primary monolayers to phosphorylate protein tyrosyl residues was evaluated by metabolic studies performed through incorporation of [32P]-phosphate into alkali-resistant phosphoproteins and by the assay of their tyrosine protein kinase activity. The presence of sodium orthovanadate during cell incubation with [32P]-phosphate greatly enhanced the relative labelling intensity of a 44 kDa alkali-resistant phosphoprotein and the total cellular content of phosphotyrosine in proteins; in this respect, growth factors such as epidermal growth factor, insulin, and insulin-like growth factor I, and the steroids dihydrotestosterone and estradiol were inactive. When the cells were solubilized, sodium orthovanadate stimulated their tyrosine protein kinase activity and inhibited their phosphotyrosine phosphatase activity. To characterize the tyrosine protein kinase of these cultured cells, conditions for optimal activity were established using the substrate poly [Glu80Na, Tyr20]. The subcellular localization of the enzyme was determined upon cell fractionation: 88% of the kinase activity was associated with the particulate fraction and 30% of this activity was partially solubilized with 0.5% Triton X-100; this solubilization was improved to 83% in the presence of 0.25 M KCl. The enzyme directly solubilized from prostatic cells with Triton X-100 (38% of activity) mainly catalyzed the alkali-resistant phosphorylation of pp63, pp59, and pp44, which contained phosphotyrosine. These proteins were also phosphorylated by the major peak of kinase activity which was eluted at an apparent molecular weight of 300-350 kDa upon gel filtration. On a cell basis, the kinase activity was four- to eleven-fold higher in the dividing epithelial cells in culture compared to quiescent secretory and non-secretory epithelial cells, freshly isolated from dog prostates. It is proposed that this tyrosine protein kinase is implicated in the regulation of the proliferation of prostatic epithelial cells.
    Additional Material: 6 Ill.
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  • 246
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 156-161 
    ISSN: 0730-2312
    Keywords: selectins ; inflammation ; hemostasis ; leukocytes ; cell-cell interactions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: GMP-140 is a membrane glycoprotein located in secretory granules of platelets and endothelium. When these cells are activated by agonists such as thrombin, GMP-140 is rapidly translocated to the plasma membrane. GMP-140, along with ELAM-1 and the peripheral lymph node homing receptor, defines the selectin family of structurally related molecules that regulate interactions of leukocytes with the blood vessel wall. Each of these molecules contains an N-terminal lectin-like domain, followed by an EGF-like region, a series of consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. The genomic structures of the selectins suggest that they arose by duplication and modification of exons encoding specific structural domains. GMP-140 is a receptor for neutrophils and monocytes when it is expressed on activated platelets and endothelium. This property facilitates rapid adhesion of leukocytes to endothelium at regions of tissue injury as well as platelet-leukocyte interactions at sites of inflammation and hemorrhage. Like other leukocyte adhesion molecules, GMP-140 may also participate in pathologic inflammation, thrombosis, and tumor metastasis. Confirmation of such pathologic roles may lead to design of new drugs that block adhesive receptor function in human disease.
    Additional Material: 4 Ill.
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  • 247
    ISSN: 0730-2312
    Keywords: endothelial cells ; neovascularization ; heparan sulfate ; heparanase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Despite the ubiquitous presence of basic fibroblast growth factor (bFGF) in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues, suggesting that the extracellular matrix (ECM) may serve as a reservoir for bFGF. Moreover, functional studies indicated that bFGF is an ECM component required for supporting endothelial cell proliferation and neuronal differentiation. We have found that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by heparanase expressed by normal and malignant cells (i.e. platelets, neutrophils, lymphoma cells). It is proposed that restriction of bFGF bioavailability by binding to ECM and local regulation of its release provide a novel mechanism for neovascularization in normal and pathological situations. The subendothelial ECM contains also tissue type-and urokinase type-plasminogen activators which participate in cell invasion and tissue remodeling. These results and studies on the properties of other ECM-immobilized enzymes (i.e. thrombin, plasmin, lipoprotein lipase) and growth factors (GM-CSF, IL-3, osteogenin), suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized and presistent mode of action, as compared to the same molecules in a fluid phase.
    Additional Material: 5 Ill.
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  • 248
    ISSN: 0730-2312
    Keywords: neuroendocrine ; cytokines ; adhesion ; transin ; stromelysin ; collagenase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Endothelial cells play a fundamental role in the pathogenesis of chronic inflammatory arthritis in humans such as rheumatoid arthritis (RA), as well as experimental animal models such as streptococcal cell wall (SCW) arthritis in Lewis (LEW/N) rats. This review summarizes data in support of this concept. The earliest apparent abnormalities in synovial tissues of patients with RA and Lewis rats with SCW arthritis appear to reflect microvascular endothelial cell activation or injury. At the molecular level, the abnormalities include enhanced expression by endothelial cells of activation markers such as class II major histocompatibility complex antigens, phosphotyrosine, leukocyte adhesion molecules, oncoproteins such as c-Fos and c-Myc, and metalloproteinases such as collagenase and transin/stromelysin. The development of severe, chronic, destructive arthritis is dependent upon thymic-derived lymphocytes and is accompanied by tumorlike proliferation of cells in the synovial connective tissue stroma (blood vessels and fibroblastlike cells), which results in resorptive destruction of bone and cartilage. Multiple criteria support the analogy to a neoplastic process. Paracrine and autocrine factors such as interleukin-1 (IL-1), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and heparin-binding fibroblast growth factors (HBGF, FGF) appear to play important roles in the generation of these lesions. Finally, in addition to the autocrine and paracrine regulatory factors, neuroendocrine factors, particularly the hypothalamic-pituitary-adrenal axis, appear to be involved in the counterregulation of the inflammatory process. The counterregulatory effects are mediated, in part, by inhibition of endothelial cell activation by corticosteroids.
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  • 249
    ISSN: 0730-2312
    Keywords: estrogen dependent ; tumor cell heterogeneity ; postconfluent growth ; antiestrogen ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice.These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.
    Additional Material: 8 Ill.
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  • 250
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 188-195 
    ISSN: 0730-2312
    Keywords: leukemia ; differentiation ; growth factors ; HL-60 ; cell growth ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Downregulation of the c-myc gene in HL-60 cells is associated with growth inhibition and induction of differentiation. Previous studies have reported that the growth inhibitors TGFβ and TNFα downregulate c-myc mRNA levels, suggesting the possibility that these agents may exert some of their phenotypic effects via c-myc downregulation. Our study demonstrates that although both growth inhibitors produce a similar decrease in c-myc protein synthesis, TNFα produces a greater growth inhibition and differentiation induction in HL-60 cells. Combined addition of anti-myc oligomer with either growth inhibitor produces no additive effect. In fact, 4 μM anti-myc oligomer produces the same growth and differentiation effects as does 10 ng/ml TGFβ1. We conclude that downregulation of c-myc expression represents a common mechanism of growth inhibition by TGFβ and TNFα, but that TNFα possesses an additional effect that is independent of c-myc expression.
    Additional Material: 5 Ill.
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  • 251
    ISSN: 0730-2312
    Keywords: epidermis ; skin ; skin graft ; cell culture ; in vitro ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The technology for culture of epidermis is one of the most advanced to date for generation of a tissue in vitro. Cultured epidermis is already used for a number of applications ranging from use as a permanent skin replacement to use as an organotypic culture model for toxicity testing and basic research. While simple epidermal sheets have been grafted successfully, more advanced models for skin replacement consisting of both dermal and epidermal components are in development and being tested in a number of laboratories. One of the most advanced in vitro models is the living skin equivalent, an organotypic model consisting of a collagen lattice contracted and nourished by dermal fibroblasts overlaid with a fully formed epidermis.
    Additional Material: 3 Ill.
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  • 252
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    Journal of Cellular Biochemistry 45 (1991), S. 258-260 
    ISSN: 0730-2312
    Keywords: chondrogenesis ; chondrocyte ; cell culture ; joint repair ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Articular cartilage is notoriously defective in its capacity for self-repair, making joints particularly sensitive to degenerative processes. However, methods are now available for the preparation of large numbers of differentiated chondrocytes from a small biopsy sample from any patient. The cells are amplified by proliferation as fibroblast-like cells that will re-express the cartilage phenotype when placed in suspension or gel culture. The chondrocytes can be collected from gel cultures after agarase treatment and reconstituted into cartilage tissue in pellet cultures. In addition, these chondrocytes can be suspended in an appropriate delivery vehicle and implanted into defect sites with a high reparative success rate in an animal model. Appropriate procedures can now be tested in appropriate patient populations.
    Additional Material: 2 Ill.
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  • 253
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    Journal of Cellular Biochemistry 45 (1991), S. 261-267 
    ISSN: 0730-2312
    Keywords: tissue transplantation ; catecholamines ; dopamine ; L-DOPA ; genetic engineering ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In animal models, grafts derived from several different tissues, principally fetal substantia nigra and adrenal medulla from young adults, have been found to be effective in alleviating some of the manifestations of lesions of the substantia nigra. It has been suggested that these grafts function by diffusely secreting dopamine, by exerting trophic effects on the host brain, or by producing a new innervation of the host corpus striatum. Evidence for each of these modes of action is briefly reviewed. Several brain tissue transplantation techniques have been described. Each of these techniques has significant limitations in animal models. The significance of these limitations for human application is described, and possibilities for improving the efficacy of brain tissue transplantation in animal models and for human application are discussed.
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  • 254
    ISSN: 0730-2312
    Keywords: retrovirus ; fibroblast ; brain grafting ; tyrosine hydroxylase ; Parkinson's disease ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Neural transplantation, a mode of cellular replacement, has been used as a therapeutic trial for Parkinson's disease. Studies indicate that tonic release of the metabolites from the graft that can be utilized by the host brain, is likely to be the major mechanism responsible for the therapeutic effect. The use of fetal tissue is complicated by ethical controversy and immunological incompatibility. Autografting adult tissue has not been successful mainly due to poor survival. Genetically engineered cells are promising alternative sources of donor cells. We have investigated the potential of primary skin fibroblasts as donor cells for intracerebral grafting. Primary skin fibroblasts survive in the brain and remain in situ. A number of genes (nerve growth factor, tyrosine hydroxylase, glutamic acid decarboxylase, and choline acetyltransferase) have been successfully introduced and expressed in the primary fibroblasts. The L-dopasecreting primary fibroblasts exhibited a behavioral effect in a rat model of Parkinson's disease up to 8 weeks after being grafted into denervated striatum. Factors that can maximize gene transfer, transgene expression, and fibroblast survival in the brain make up the future direction of investigation.
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  • 255
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    Journal of Cellular Biochemistry 45 (1991), S. 268-272 
    ISSN: 0730-2312
    Keywords: hematopoiesis ; stem cell ; perfusion ; hematopoietic growth factor ; genetic engineering ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM-CSF and IL-3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.
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  • 256
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    Journal of Cellular Biochemistry 46 (1991), S. 143-151 
    ISSN: 0730-2312
    Keywords: interleukin-1 ; growth factor ; cytokine ; proliferation ; host defence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Lymphocyte activating factors (LAFs), e.g., interleukin-1 (IL-1) and IL-1-like factors, have previously been demonstrated outside the immune system in the skin, thymus epithelium, and the human and rat testis. We have studied the presence of LAFs in normal tissues of the adult rat, utilizing a highly IL-1 sensitive murine thymocyte proliferation assay. We have demonstrated high amounts of LAF activity in the tongue, esophagus, proventricular part of the stomach, and the liver. Some activity was also demonstrated in the duodenum, placenta, spleen, Peyer's patches, glandular stomach, and jejunum, but no bioactivity was present in other gastrointestinal, endocrine, lymphoid, or haematopoietic tissues. We were also unable to detect any LAF activity in the reproductive organs (except for the testis), urinary tract, skeletal and muscular tissues, brain, eyes, salivary glands, or lung. In the esophagus the activity was mainly localized to the mucosa. The LAF activity in the skin was partly inhibited by treatment with a mixture of antibodies against human IL-1α and IL-1β Dose response curves and gel filtration on a Sephacryl S-200 column suggested the presence of a high molecular weight (90,000-100,000 Da) LAF inhibitory factor in the liver. In all positive tissues, the demonstrated LAFs had a molecular weight of 15,000-25,000 Da, as determined by Sephacryl S-200 gel filtration. Of the positive tissues, the skin, tongue, esophagus, and the proventricular part of the stomach all contain stratified squamous epithelium. It is tempting to suggest that the detected LAFs have a similar function in these barrier tissues, e.g., to serve as host defence factors, or, alternatively or additionally, as tissue growth factors.
    Additional Material: 8 Ill.
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  • 257
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    Journal of Cellular Biochemistry 46 (1991), S. 152-165 
    ISSN: 0730-2312
    Keywords: drug-induced gingival fibrosis ; extracellular matrix ; collagen ; collagenase ; tissue inhibitor of metalloproteinases ; cell subpopulations ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: To investigate the mechanism of cyclosporine (CS)-induced fibrotic gingival enlargement, the effect of CS on the collagenolytic activities of 14 different human gingival fibroblast strains derived from healthy individuals with non-inflammed gingiva was examined in vitro. There was marked heterogeneity among individuals in basal levels of collagenase activity, and there was also variation among the subpopulations derived from one strain. Fibroblasts from different individuals also varied markedly in their collagenolytic response to CS (0.1 to 0.75 μg/ml). In most strains, CS decreased collagenase activity, but in some, the drug caused no change or significantly increased activities. In most of the subpopulations CS significantly decreased collagenolytic activity.Two of the fibroblast strains and the subpopulations described above were examined for the production of immunoreactive collagenase and tissue inhibitor of metalloproteinase (TIMP). The two strains made similar amounts of collagenase, but differed markedly in TIMP levels; CS affected their collagenase production differently but had similar effects on TIMP. Among the subpopulations there was variation in the production of collagenase, although none made detectable levels of TIMP; they also varied in the production of both proteins in response to CS. In two of the subpopulations and in both strains at some concentrations, the effect of CS on the relative levels of collagenase and TIMP could account for the decreased collagenase activity; i.e., the level of collagenase was unchanged or decreased, and TIMP production was unchanged or increased.This study demonstrates the variation among individuals as well as intrastrain heterogeneity of human gingival fibroblasts with regard to collagenase activity and the production of collagenase and TIMP. The heterogeneity of the collagenolytic response of different gingival fibroblast strains and their subpopulations to CS treatment may partly explain the susceptibility of only some individuals to CS-induced gingival enlargement.
    Additional Material: 6 Ill.
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  • 258
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    Journal of Cellular Biochemistry 46 (1991), S. 190-190 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 259
    ISSN: 0730-2312
    Keywords: chromatin structure ; gene expression ; streptavidin-biotin affinity chromatography ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,37apos;-dithiopropionyl-3-aminoallyl]-2' -deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragements which had bound to the affinity complex. Western blot assessment for ubiquitinated histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.
    Additional Material: 5 Ill.
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  • 260
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    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 46 (1991) 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 261
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    Journal of Cellular Biochemistry 46 (1991), S. 234-241 
    ISSN: 0730-2312
    Keywords: ADP-ribosylation ; CS histone variants ; cell cycle ; sea urchin zygotes ; 3 aminobenzamide ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The patterns of poly(ADP-ribosylation) in vivo of CS (cleavage stage) histone variants were compared in sea urchin zygotes at the entrance and the exit of S1 and S2 in the initial developmental cell cycles. This post-translational modification was detected by Western immunoblots with rabbit sera anti-poly(ADP-ribose) that was principally reactive against ADP-ribose polymers and slightly against ADP-ribose oligomers. The effect of 3 aminobenzamide (3-ABA), an inhibitor of the poly(ADP-ribose) synthetase, on S phase progression was determined in vivo by measuring the incorporation of 3H thymidine into DNA. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) in a cell cycle dependent manner. A significantly positive reaction of several CS variants with sera anti-poly(ADP-ribose) was found at the entrance into S phase, which decreases after its completion. The incubation of zygotes in 3-ABA inhibited the poly(ADP-ribosylation) of CS variants and prevented both the progression of the first S phase and the first cleavage division. These observations suggest that the poly(ADP-ribosylation) of atypical CS histone variants is relevant for initiation of sea urchin development and is required for embryonic DNA replication.
    Additional Material: 3 Ill.
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  • 262
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    Journal of Cellular Biochemistry 46 (1991), S. 260-265 
    ISSN: 0730-2312
    Keywords: rough membranes ; smooth membranes ; structural transitions ; transformation ; spin probes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: This paper deals with microviscosity parameters and thermoinduced structural transitions in the lipids of smooth and heavy rough endoplasmic reticulum membranes isolated from Krebs II ascites cells incubated with the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate. The phorbol ester was found to bring about a threefold increase in the microviscosity of the lipids in heavy rough membranes. Spin probe I (2,2,6,6-tetrahydro-4-capryloyl-oxypiperidine-1-oxyl), localized in the surface layer of the membrane lipids, gave results which indicate an increased number of thermoinduced structural transitions in the smooth membranes in the treated cells due to the transitions occurring at relatively low temperature and a decreased number of such transitions in the heavy rough fraction especially at high temperature. For 5,6-benzo-2,2,4,4-tetramethyl-1,2,3,4-tetrahydro-γ-carboline-oxyl, probe II, mainly distributed in the annular lipids, a decrease in the number of low temperature transitions in the smooth fraction was observed, while an increase occurred in the heavy rough one. The results obtained are discussed in terms of the effect of phorbol esters as promoters of tumor progression.
    Additional Material: 3 Ill.
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  • 263
    ISSN: 0730-2312
    Keywords: protein kinase C ; flow cytometry ; image cytometry ; fluorescence anisotropy ; fluorescence recovery after photobleaching ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N′-di(2-hydroxyethyl)amino)-7-nitrobenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25°C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.
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  • 264
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    Journal of Cellular Biochemistry 46 (1991), S. 321-330 
    ISSN: 0730-2312
    Keywords: secretion ; leucine binding protein ; leader peptidase ; signal sequence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The leucine-specific binding protein, encoded by the livK gene, is located in the periplasm of E. coli. The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm. C-terminal deletions or fusions of the livK gene to trpA (encoding the α subunit of tryptophan synthetase) were secreted with little loss of efficiency [1]. A series of deletions was constructed at the unique Sphl site within livK, near the 5' end of the region coding for the mature protein. Between 16 and 113 amino acids were deleted in the amino-terminal one-third of the protein. A few of these deletions were located within a few amino acids of the signal sequence processing site. Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal. Secondary structure predictions suggested that the α-helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components. These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion. During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS-PAGE, was much greater than could be accounted for by processing of the signal sequence. This anomalous mobility on gels, however, could be eliminated by performing SDS-PAGE in the presence of 6 M urea.
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  • 265
    ISSN: 0730-2312
    Keywords: T cells ; aging ; IL-2 ; IL-4 ; IFNγ ; CD45RB ; 3G11 ; 6C10 ; CD44 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Splenocytes from young adult or old C57BL/6NNia mice were stimulated in vitro with the anti-CD3∊ mAb, 145-2C11, in either soluble (2C11s) or plate-bound (2C11i) form. In the young group, each mode of cell activation resulted in peak DNA synthesis at ∼48 h of culture; at this time point, the old group exhibited response levels to 2C11s or 2C11i that were ∼40% of those in the young group. However, in the presence of 2C11i, splenocytes from old donors showed a delayed peak response which approached the peak levels attained in the young group. To analyze the responsiveness of the CD4+ T cell subpopulation, this cell type was isolated from spleens of young or old mice and was stimulated in vitro with 2C11s or 2C11i, in the presence or absence of added accessory cells (T cell-depleted, irradiated splenocytes). The induction of DNA synthesis by 2C11s was accessory cell dependent, and the response in the old group were markedly reduced in comparison to those in the young group. In contrast, stimulation of DNA synthesis with 2C11i was relatively accessory cell independent, resulted in higher response levels in both age groups, and lessened the disparity between age groups. The analysis of IL-2 and IL-4 secretion by stimulated CD4+ cells revealed that, in response to 2C11s and accessory cells, only IL-2 accumulation was detectable and the levels in the young group were ∼10-fold higher than the IL-2 levels in the old group. However, stimulation of CD4+ cells with 2C11i and accessory cells yielded improved IL-2 production and a detectable IL-4 response in the old group, whereas the young group exhibited a response profile similar to that induced by 2C11s. Further analysis of the IL-2, IL-4, and IFNγ mRNA levels in 2C11i-stimulated CD4+ cells revealed that old donor cells accumulated similar levels of IL-2 transcripts, but higher levels of IL-4 and IFNγ transcripts, than young donor CD4+ cells. Finally, we analyzed splenic CD4+ cells for membrane expression of four molecules - 3G11, 6C10, CD45RB, and CD44 - thought to demarcate CD4+ cell subsets with restricted patterns of cytokine production. The CD4+ cell fraction of individual mice contained higher percentages of cell phenotypes associated with increased IL-4:IL-2 production ratios (i.e., 3G11lo, CD45RBlo) and with increased IFNγ synthesis (i.e., CD44hi). Taken together, these data show marked alterations in the CD4+ cell subset composition in old mice, detected at the levels of subset marker expression and profiles of cytokine production. Moreover, conclusions regarding CD4+ cell competency in old donors can differ depending on the choices of stimuli and readouts for cell function in the experimental design. Therefore, age-related differences in T cell reactivity in vitro may be partially explained by the shifts in the representation of individual CD4+ subsets, each with potentially unique activation requirements and functional attributes.
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  • 266
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    Journal of Cellular Biochemistry 46 (1991), S. 366-366 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 267
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    Journal of Cellular Biochemistry 46 (1991), S. 345-350 
    ISSN: 0730-2312
    Keywords: SMCs ; mRNA ; mitochondrial ADP/ATP carrier ; calcyclin ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vascular smooth muscle cells (SMCs) play a key role in the development of major arteries. Furthermore, abnormal growth of vascular smooth muscle cells has been implicated in the progression of major diseases of the cardiovascular system. Here, we report detection in primary cultures of bovine vascular smooth muscle cells of mRNA for two growth-state-related genes, 2F1 and 2A9, which code for a mitochondrial ADP/ATP carrier and calcyclin, respectively, and on the characterization of their cell cycle expression. Cultures of exponentially growing smooth muscle cells were made quiescent by serum deprivation. Upon readdition of serum, cells entered the cell cycle synchronously; DNA synthesis began 12 h post-serum addition. Levels of 2F1 and 2A9 RNA were low in quiescent cells and increased between 2 and 4 h post-serum addition. No changes in the rates of transcription of the 2F1 or 2A9 genes were detected by nuclear run-off assays during the time course. Thus the regulation of changes in expression of 2F1 and 2A9 in early G1 is mediated post-transcriptionally.
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  • 268
    ISSN: 0730-2312
    Keywords: bone resorption ; osteoclast ; superoxide dismutase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Large multinucleated osteoclasts are the major cells responsible for bone breakdown and have been reported to produce high levels of superoxides which may contribute to the process of bone resorption (Key et al.: J Bone and Mineral Res 4 [suppl. 1]:S206, 1989). Osteoclasts also possess high levels of superoxide dismutase, a protective enzyme capable of converting toxic superoxides to less toxic H2O2 (Fridovich: J Biol Chem 264:7761-7764, 1989). The amino acid sequence of manganese and/or iron superoxide dismutase has a conserved region which exhibits substantial homology with a fragment obtained from a high molecular weight osteoclast surface marker glycoprotein which is reactive with monoclonal antibody 121F. In this report, evidence is presented substantiating immunological, biochemical, and functional similarities between the osteoclast membrane antigen recognized by the 121F monoclonal antibody and superoxide dismutase. Western blot and immunoprecipitation studies show that a monospecific polyclonal antibody generated against immunoaffinity purified antigen is cross-reactive with superoxide dismutase. Both the antigen and a high molecular weight superoxide dismutase activity have been detected in osteoclast plasma membrane preparations. The levels of superoxide dismutase activity and the membrane antigen have been found to correlate in antigen depletion studies and in western blots probing osteoclasts and closely related marrow-derived giant cells. Moreover, regions of osteoclast superoxide dismutase activity identified by electrophoretic zymogram analysis have been shown by gel electrophoresis and western blots to contain the high molecular weight antigen, or complexes of the antigen with the 121F monoclonal antibody when these were premixed prior to nondenaturing electrophoresis. It is proposed that the osteoclast plasma membrane possesses a high molecular weight superoxide dismutase activity. Furthermore, it appears that this activity is associated with the osteoclast antigen recognized by the 121F monoclonal antibody.
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  • 269
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    Journal of Cellular Biochemistry 47 (1991) 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 270
    ISSN: 0730-2312
    Keywords: chromatin ; chromatin remodeling ; fertilization ; embriogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western imunoblot analysis with policlonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.
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  • 271
    ISSN: 0730-2312
    Keywords: MGP ; chondrogenesis ; osteogenesis ; gene expression ; vitamin D ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Matrix Gla protein (MGP), a vitamin K dependent protein, has recently been identified in many tissues. However, it is accumulated only in bone and cartilage suggesting that the expression of MGP may be related to the development and/or maintance of the phenotypic properties of these tissues. We systematically evaluated MGP mRNA expression as a function of bone and cartilage development and also as regulated by vitamin D during growth and cellular differentiation. Three experimental models of cartilage and bone development were employed:colon; an in vivo model for endochondral bone formation, as well as in primary cells of normal diploid rat chondrocyte and osteoblast cultures. MGP was expressed at the highest level during cartilage formation and calcification in vivo during endochondral bone formation. In chondrocyte cultures, MGP mRNA was present throughout the culture period but increased only after 3 weeks concomitantly with type I collagen mRNA. In osteoblast cultures, MGP mRNA was expressed during the proliferative period and exhibited increased expression during the period of matrix development. In contrast to osteocalcin (bone Gla protein), this increase was not dependent on mineralization but was related to the extent of differentiation associated with and potentially induced by extracellular matrix formation. During the proliferative period, type I collagen mRNA peaked and thereafter declined, while type I collagen protein steadily accumulated in the extracellular matrix. Constant MGP levels were maintained in the mineralization period of osteoblast differentiation in vitro which is consistent with the constant levels found during the osteogenic period of the in vivo system. MGP mRNA levels in both osteoblasts and chondrocytes in culture were significantly elevated by 1,25-(OH)2D3 (10-8 M, 48 h) throughout the time course of cellular growth and differentiation. Interestingly, when MGP mRNA transcripts from vitamin D treated and untreated chondrocytes and osteoblasts were analyzed by high resolution Northern blot analysis, we observed two distinct species of MGP mRNA in the vitamin D treated chondrocyte cultures while all other cultures examined exhibited only a single MGP mRNA transcript. Primer extension analysis indicated a single transcription start site in both osteoblasts and chondrocytes with or without vitamin D treatment, suggesting that the lower molecular weight MGP message in vitamin D treated chondrocytes may be related to a modification in post-transcriptional processing. In conclusion, these results show that the selective accumulation of MGP in bone and cartilage tissues in vitro may be related to the development and/or maintance of a collagenous matrix as reflected by increases in MGP mRNA during these periods. Moreover, our data suggest that cartilage and bone MGP mRNA may in part be selectively regulated by 1,25-(OH)2D3 at the post-transcriptional level.
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  • 272
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    The @Anatomical Record 229 (1991), S. 195-202 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The morphology of spermatozoa and the initial stages of spermegg fusion at fertilization were investigated ultrastructurally in the rose bitterling, Rhodeus ocellatus ocellatus. Each spermatozoon is composed of a spherical head without an acrosome, two centrioles, a large mitochondrion, and a flagellum. Freeze-fracture of spermatozoa illustrates that specialized arrays of intramembranous particles (IMPs) are present on the protoplasmic facing (PF) surface of the head plasma membrane at the portion slightly in front of the centrioles. The specialized arrays, whose functions are uncertain, are parallelogram-like in shape. The distribution of the particles is random and less compact in other areas of the head plasma membrane. The number of particles on the PF surface is larger than that on the extracellular facing (EF) surface. The complementary structures of the specialized arrays are also found on a similar portion of the EF surface. An ultrastructural study clearly shows fusion of gamete plasma membranes at the initial stages of sperm entry into the egg. Membrane fusion is first observed in eggs fixed 10 seconds after insemination in fresh water. The fusion site is the microvillus membrane of a sperm entry site on the egg and the head membrane of the spermatozoon. The plasma membrane fusion of gametes is discussed relative to the distribution of the IMPs and the fusion site.
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  • 273
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    The @Anatomical Record 229 (1991), S. 203-208 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The ischiourethralis (IU), a striated perineal muscle presumed to be involved in sexual reflexes, was studied in the rat. The paired muscle arises from the penile crus and the penile bulb and unites in a raphe over the deep dorsal vein of the penis. Retrograde tracing studies show that the muscle is innervated by neurons in the dorsolateral nucleus of the lumbar spinal cord, a pudendal nerve motor nucleus which also innervates the ischiocavernosus muscle. Excision of the IU muscle did not interfere with the ability of males to display normal copulatory behavior, nor did it affect significantly the number and intensity of reflexive erections. It nevertheless remains possible that the IU may contribute to intense glans erection by compressing the deep dorsal vein.
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  • 274
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    The @Anatomical Record 229 (1991), S. 315-320 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The scanning and transmission electron microscopes were used to examine the processes of spermiation and sperm maturation in the marmoset. We observe that the heads of late spermatids are embedded in the apical aspect of the large sleeve-like columnar portion of Sertoli cells. As spermiogenesis progresses, spermatids become associated with numerous small apical Sertoli cell extensions. These finger-like processes undergo a sequence of changes during spermiation. Spermatozoa from the caput, corpus, and cauda epididymides were examined. In caput epididymis of marmoset, the apical segment of the spermatozoa extends well beyond the rostral edge of the nucleus and folds back on itself. In sagittal sections, the acrosome exhibits a distinct hook shape. In the corpus, the distinctive hookshaped apical segment of the acrosome is observed in some spermatozoa, but the apical extension is significantly smaller or projects out only slightly beyond the nucleus. In cauda epididymis, the extension is absent. A similar acrosomal hook has been reported in the pigtailed monkey, which is an Old World species. We suggest that changes in acrosome structure during sperm maturation may be fairly widespread among primates.
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  • 275
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    The @Anatomical Record 229 (1991), S. 334-338 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Using polyclonal antibodies against a 13 KD human testicular inhibin, immunocytochemical localization studies were carried out in marmoset monkey testes. The pattern as well as the intensity of immunocytochemical staining for inhibin vary substantially during development. In early development (day 1 to 2 months) Leydig cells are the predominant cell types showing intense staining which reaches its nadir at 3 months. Subsequently both Sertoli cells as well as Leydig cells show equal intensity of inhibin staining. Testicular inhibin is likely to play a vital role in cell to cell communication.
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  • 276
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cathepsin D (EC3.4.23.5) is an aspartyl endopeptidase involved in lysosomal proteolysis. Its functional role is uncertain. This study was undertaken to determine the cellular and subcellular distribution of cathepsin D in the normal rat ventral prostate and its possible role in the castration-induced atrophy of the gland. Cathepsin D was localized immunohistochemically to perinuclear lysosomes in secretory cells, in capillary endothelial cells, and, occasionally, in stromal cells of the untreated animal. Castration resulted in an increased number of cathepsin D-positive cells in the stroma within 24 hr. By 48 hr after castration autophagolysosomes formed in secretory cells and apoptotic bodies appeared in the epithelium. Although apoptotic bodies generally contained immunoreactive cathepsin D, a subpopulation of larger apoptotic bodies, which commonly rested on the basement membrane and contained multiple inclusions, were more variable in cathepsin D expression. The induction of cathepsin D in dendritic cells basally oriented in the epithelium was noted at 4 days of castration. These cells had a phagocytic phenotype, were distributed periodically along the basement membrane, and were not found in ductal epithelia. Treatment with actinomycin D or hydrocortisone to reduce the rate of regression of the ventral prostate blocked the appearance of these cathepsin D-positive, basally oriented epithelial cells. Our data indicate that this cathepsin D-positive, phagocytic cell differentiates from a cell resident in the prostatic epithelium. We suggest that it differentiates from basal cells in the secretory tubuloalveolar portion of the gland and that it is involved in the destruction of regressed secretory cells.
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  • 277
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The spatial distribution of α- and β-myosin heavy chain isoforms (MHCs) was investigated immunohistochemically in the embryonic human heart between the 4th and the 8th week of development. The development of the overall MHC isoform expression pattern can be outlined as follows: (1) In all stages examined, β-MHC is the predominant isoform in the ventricles and outflow tract (OFT), while α-MHC is the main isoform in the atria. In addition, α-MHC is also expressed in the ventricles at stage 14 and in the OFT from stage 14 to stage 19. This expression pattern is very reminiscent of that found in chicken and rat. (2) In the early embryonic stages the entire atrioventricular canal (AVC) wall expresses α-MHC whereas only the lower part expresses β-MHC. The separation of atria and ventricles by the fibrous annulus takes place at the ventricular margin of the AVC wall. Hence, the β-MHC expressing part of the AVC wall, including the right atrioventricular ring bundle, is eventually incorporated in the atria. (3) In the late embryonic stages (approx. 8 weeks of development) areas of α-MHC reappear in the ventricular myocardium, in particular in the subendocardial region at the top of the interventricular septum. These coexpressing cells are topographically related to the developing ventricular conduction system. (4) In the sinoatrial junction of all hearts examined α- and β-MHC coexpressing cells are observed. In the older stages these cells are characteristically localized at the periphery of the SA node.
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  • 278
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The epithelial cell types present in respiratory (= distal alveolarized) and terminal (= distal nonalveolarized) bronchioles in adult human lung were characterized with scanning and transmission electron microscopy (SEM, TEM) and light microscopic cytochemistry, using specific antibodies against surfactant protein SP-A and mucins, and Alcian blue/periodic acid-Schiff (AB/PAS) staining.In the respiratory bronchiole, two epithelial cell populations share the same basal lamina: one pseudostratified columnar with ciliated, secretory, and basal cells and the other predominantly simple cuboid with some interspersed flat (type I) cells. The columnar secretory cells show the ultrastructure of mucous cells. Light microscopically, they react with mucin antibodies and contain primarily periodatereactive acid mucins. The mucous cells are the distal secretory cells described by Clara (1937). The cuboid cells are identified as type II (precursor) cells based on ultrastructural criteria for embryonic type II cells (Ten Have-Opbroek et al., 1988a, 1990a), including a cuboid cell shape, a large and roundish nucleus, rough and smooth endoplasmic reticulum (ER), osmiophilic multivesicular bodies, and dense bodies. These dense bodies in turn frequently exhibit - like those in embryonic type II cells - internal vesicles or lamellae, variability in size and shape, a specific relationship to ER and a widespread cytoplasmic distribution. Finally, the cuboid cells show a cytoplasmic staining pattern for SP-A. The terminal bronchiole is lined by the columnar cell population.In the respiratory bronchiole, the columnar (bronchial) and cuboid (alveolar) cell populations occupy distinctly different zones (pulmonary artery zone versus remaining wall). The alveolar part of the respiratory bronchiole (called alveolar tubule) defines the proximal border of a true respiratory unit.
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  • 279
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    The @Anatomical Record 229 (1991), S. 369-383 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The de-epithelialization of cells of the upper layer during the phenomena of polyingression and primitive streak ingression was studied by analyzing, from the time of laying to the end of gastrulation, the ultrastructure of the basal lamina underlying the upper layer. The electron density of the basal lamina and associated extracellular materials was enhanced by addition of tannic acid to the fixative. Special attention was also paid to the spatial and temporal distribution of blebs at the basal surface of the upper layer, and to the contribution of the de-epithelialized cells to the formation of the deep layer. The results indicate that a nascent basal lamina is already present at the time of laying, especially beneath regions of the area pellucida where polyingression is not apparent. From the onset of incubation, the basal lamina rapidly develops, and it is interrupted by a large number of blebs. However, during the first 6-8 h of incubation, i.e., stages 1-2 of Vakaet (Arch. Biol. (Liège) 81:387-426, 1970), a downward movement of deepithelialized cells that insert into the deep layer and form the endophyll persists cranially. This phenomenon of polyingression, which starts during the intrauterine period, probably extends from caudal to cranial and comes to an end by stage 3. During these first three stages, the number of blebs progressively decreases, especially in the cranial part of the area pellucida, and a thicker, continuous basal lamina associated with numerous interstitial bodies is laid down. The caudal part of the upper layer is still actively blebbing at that time. Due to the convergence of this area toward the axis of the blastoderm, which leads to ingression at and elongation of the primitive streak up to and including stage 6, the number of blebs at the basal surface of the upper layer progressively decreases. From stage 7 on, blebs are virtually absent; shortening of the primitive streak and formation of the head process begin. At the level of the head process, primitive streak ingression has ceased and a novel basal lamina is progressively deposited beneath the upper layer. By stage 9, a thick, smooth basal lamina physically separates the upper layer from the head mesenchyme. Summarizing, at the time of gastrulation, the presence of blebs that perforate the basal lamina is correlated with the de-epithelialization of cells. Before incubation, however, de-epithelialization of upper-layer cells occurs before the assembly of the basal lamina. Up to stage 3, the de-epithelializing cells, randomly distributed in an area in front of the limit of primitive streak ingression, insert into the deep layer and form endophyll. Behind this limit, the de-epithelialization occurs at the level of the primitive streak only and leads to the formation of mesoblast and, from stage 4 on, of definitive endoblast.
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  • 280
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    The @Anatomical Record 229 (1991), S. 407-414 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.
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  • 281
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The location of the motoneurons supplying the rabbit pharyngeal constrictor muscles (the superior and the middle constrictors, and the thyropharyngeus and the cricopharyngeus; the last two collectively compose the inferior constrictor) was investigated with intramuscular injection of HRP or the fluorescent tracer nuclear yellow into the individual muscles. Moreover, the peripheral course of their axons was investigated by injection of HRP into all of the pharyngeal constrictors in conjunction with intracranial severing of either the vagus or the glossopharyngeal nerves. The pharyngeal constrictor motoneurons were ipsilaterally located within a subdivision of the nucleus ambiguus which is formed by a compact arrangement of the smallest neurons of the nucleus and situated in the rostral half of the nucleus. We named that subdivision the compact cell group (CoG). Axons of the pharyngeal constrictor motoneurons traversed the vagal rootlets. The rostrocaudal extent of the pharyngeal constrictor motoneurons covered almost the entire length of CoG at a level from about 500 to 2,900 μm rostral to the obex, with their number being most numerous in the middle one-third level of the CoG. Although the motoneurons of the superior constrictor, those of the middle constrictor, and those of the thyropharyngeus and the cricopharyngeus overlapped considerably in location, they tended to be arranged rostrocaudally in that order. At the middle one-third level of the CoG, where the CoG is subdivided into dorsomedial and ventrolateral subgroups of neurons, the superior and the middle constrictor motoneurons were confined to the medial portion of the dorsomedial subgroup, while the inferior constrictor motoneurons were distributed throughout its entirety.
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  • 282
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    The @Anatomical Record 229 (1991), S. 434-438 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 283
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    The @Anatomical Record 229 (1991), S. 415-426 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A procedure has been developed for the three-dimensional immunoelectron microscopic localization of cytoskeletal filaments by a deep-etching replica method in combination with immunogold labeling and/or myosin subfragment 1 (S1) decoration techniques. Neonatal hamster heart cells grown on glass coverslips were extracted with Triton X-100 or physically permeabilized by breaking open the cell membranes. S1 decoration was performed on some specimens immediately after the permeabilization. After prefixation in formaldehyde, samples were immunostained with poly- or monoclonal antibodies to desmin or vimentin, and indirectly tagged with colloidal gold probes by the biotin-streptavidin method. After postfixation with glutaraldehyde, tannic acid and osmium tetroxide, the cells were freeze-etched and rotary-replicated with platinum and carbon in a freeze-fracture apparatus. Replicas were viewed with a transmission electron microscope using a tilting specimen stage to obtain stereo images. The procedure made it possible to identify the specific filaments within the complex cytoskeletal networks in cultured hamster heart muscle and nonmuscle cells at high resolution and in three dimensions. The method has advantages in its three-dimensionality and feasibility to evaluate the data by comparing them with those obtained by alternative light microscopic methods. Details of the protocol and a description of the results of using three different antibodies are given.
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  • 284
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    The @Anatomical Record 229 (1991) 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 285
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    The @Anatomical Record 229 (1991), S. 427-433 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The present study describes a method of culturing chick embryos together with their surrounding area vasculosa on two different culture media in succession. Embryos in the 2nd day of incubation (stages 13, 14, 15 according to Hamburger and Hamilton, 1951) were explanted from the yolk with the aid of a ring of filter paper and transferred dorsal side up to a silicone culture dish containing the first culture medium (89.5 % L-15, 10 % fetal calf serum, 0.5 % Antibiotics). The paper ring was clamped onto the wall of the culture dish by a steel ring so that the embryo was fixed for the culture period. After 4 ± 1, 8 ± 1, 12 ± 1 hrs, the embryos were taken from the culture dishes and transferred to others containing a yolk-albumen mixture as culture medium; 81.2 % of embryos survived the first phase of culture. On the second medium 50.3 % of explanted embryos were still alive at stage 20 (HH), and 7.9% of them reached the 5th day of development (St 25 HH).The average length of survival in vitro was found to be influenced by both the length of the first culture phase and the stage at which embryos were explanted.This culture method may be useful for teratological tests, since in the first phase of culture, concentrations of test substances and the time of exposure can be exactly adjusted, and in the second phase, the embryo is allowed to develop quite normally, under conditions similar to those in ovo.
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  • 286
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    The @Anatomical Record 229 (1991), S. 439-446 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In the present study, we have localized desmin and α-smooth-muscle actin in the ovaries of the zebrafish, the axolotl, and the red-eared turtle, using the unlabelled antibody peroxidase-antiperoxidase technique. In the axolotl ovary both smooth-muscle markers were demonstrated in cord-like structures, extended along ovarian blood vessels, and in some inner ovarian epithelium cells. In the ovaries of the teleost, smooth-muscle-like cells are detected in a suspensory apparatus formed by venous cords, the tunica albuginea, and the coat around the ovarian artery. Also, in the turtle ovary, smooth-muscle-like cells were found in a suspensory apparatus formed by chordae, the tunica albuginea, and the theca externa of the ovarian follicles. At the present time, the prevailing hypothesis is that, in addition to a role in the mechanical support, the smooth-muscle-like cells in the ovaries of these vertebrates seem to be important with respect to ovarian contractile processes.
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  • 287
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    The @Anatomical Record 229 (1991), S. 447-452 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cartilage canals are present in the epiphyseal cartilage of most mammals and birds. They are considered necessary for the maintenance of chon-drocytes and for the formation of epiphyseal ossification centers. The epiphyseal cartilage of marsupials was recently shown not to contain cartilage canals, and placental rats appear not to have cartilage canals, although some confusion exists in the literature. The present study examines the cartilaginous epiphyses and physes from the knee and hip of the rat and the two Australian monotremes (platypus, Ornithorhynchus anatinus and echidna, Tachyglossus aculeatus). In all three species, cartilage canals were absent. Vessels to epiphyseal ossification centers were present, however. In the center of the cartilaginous femoral head of the echidna, but not in the platypus or rat, there was a large cavity, which contained connective tissue and was lined by an endochondrium of chondroproginator cells. These appeared to be contributing to growth of the cartilaginous epiphysis. No similar structure has previously been described in the cartilaginous epiphysis of other species. There was no ligament of the femoral head in the hip joints of the monotremes, and it is suggested the absence of a ligament may be significant in the development of the cavity. It was noted in all specimens that despite being avascular the epiphyseal and physeal cartilage appeared viable and functionally normal. The small size of the cartilaginous epiphyses of the rat may account for their avascularity; but the epiphyses of the monotremes were much larger, especially the echidna, yet still avascular. These features provide strong evidence for fundamental differences between the avascular cartilage of monotremes and the vascular cartilage of most mammals.
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  • 288
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The morphology of the metaphyseal microvasculature at the epiphysis was examined at both the light and electron microscopic level in rickets and rachitic reversal. The animals studied were normal, rachitic, and rachitic reversed at 8, 24, and 96 hours post-vitamin D administration. The overall architecture of the metaphyseal vessels was significantly altered throughout the intervals examined. In the rachitic animal, arterioles, venules, and capillaries were found adjacent to the growth plate, either directly apposed to the hypertrophic chondrocytes or separated from them by bone-forming cells. These vessels are in many ways similar to the larger arterioles and venules that normally supply the metaphyseal capillary sprouts, but in the normal growing animal are usually located 350-500 μm from the epiphyseal cartilage. The rachitic capillaries appear relatively well differentiated with a partial basement membrane and a perivascular cell lining. In early rachitic reversal, small vascular projections are induced to grow from the large diameter venules that border upon the hypertrophic chondrocytes. These vascular sprouts that invade the epiphyseal cartilage are quite undifferentiated, with no basement membrane or pericyte lining at the sprout apex and occasional abluminal endothelial cell projections. Within 96 hours, the metaphyseal microvasculature has returned to an apparently normal state with only capillaries at the cartilage-vascular interface and larger vessels (arterioles and venules) located several hundred microns deeper into the metaphysis. The sequential processes of differentiation and cessation of capillary growth followed by dedifferentiation and reinitiation of microvascular growth make the rachitic system a unique one in which to study angiogenesis.
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  • 289
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    The @Anatomical Record 230 (1991), S. 468-472 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Using specific polyclonal antibodies generated against a 13 KD human testicular inhibin, immunocytochemical localization of inhibin was carried out in different regions of human epididymis. The concentrations of inhibin were greater in caput and corpus regions as compared to the caudal region. The epididymal inhibin was found to be bioactive, since it suppressed specifically the FSH levels of rat pituitaries in vitro. Spermiophage/macrophage cells exhibited strong staining for inhibin which were suggestive of a possible role of inhibin in modulation of immune function. In view of the known activities of inhibin in cellular growth, differentiation, and steroidogenesis, epididymal inhibin could have a role in acquisition of sperm fertilizing capabilities.
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  • 290
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    The @Anatomical Record 230 (1991), S. 473-480 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Interstitial cells of Leydig characteristically occur in clusters around blood vessels. Often these clusters remain intact when interstitial tissues are mechanically separated from other components of the testis. The presence of strong intercellular attachments is most likely one of the factors responsible for Ground Squirrel Testis Vancouver, British Columbia, Canada V6T 1Z3, maintaining the integrity of Leydig cell clusters. In many tissues, actin associated adhesion junctions commonly provide intercellular attachment. To determine if actin associated adhesion junctions are present between Leydig cells, we have used 1) immunofluorescence to probe for two components that characterize these junctions in other tissues and 2) electron microscopy to examine areas of intercellular contact for evidence of microfilament related adhesion junctions.Isolated clusters of unsectioned cells, which had been fixed and detergent extracted, were probed with the F-actin specific stains rhodamine phalloidin and NBD-phallacidin and with an affinity purified primary antibody raised against human platelet vinculin. In regions of intercellular contact, fluorescence staining with the actin probes was intense and appeared as a solid linear band. Similar regions also stained with the vinculin probe. In double label experiments, actin and vinculin probes were co-distributed at sites of intercellular contact.Zones of intercellular contact, apparently similar to those detected with fluorescence microscopy, were observed a t the ultrastructural level. At these sites, subsurface filaments, interpreted by us as actin, formed a dense carpet adjacent to the lasma membrane on each side of the junction. These filaments appeared to be organized into networks rather than discrete bundles.Our observations that 1) probes for actin and vinculin codistribute at certain sites of intercellular contact and 2) a layer of microfilaments is associated with the plasma membrane in electron micrographs of these contact regions support the conclusion that Leydig cells, at least in the ground squirrel, may possess actin associated adhesion junctions similar to those described between cells of numerous other types.
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  • 291
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Calbindin-D 28 kDa (CaBP 28 kDa), a vitamin D-dependent calcium-binding protein, has been associated with calcium handling by cells. We have investigated the expression of this protein in the rat incisor enamel organ, an epithelium interposed between a mineralizing matrix and connective tissue rich in blood vessels, by radioimmunoassay (RIA), Western blotting, and quantitative protein A-gold immunocytochemistry with antibodies to rat kidney CaBP 28 kDa. RIA of cytosolic extracts showed that enamel organs contained relatively high concentrations of CaBP 28 kDa (compared to kidney; see review by Christakos S., C. Gabrielides, and W.B. Rhoten 1989 Endocr. Rev., 10:3-25). Immunoblotting of proteins extracted from enamel organ strips revealed an intensely-stained band near 28 kDa throughout amelogenesis following ameloblast differentiation. Immunocytochemically, CaBP 28 kDa was localized exclusively within ameloblasts. The density of labelling increased from the presecretory stage to the secretory stage and fluctuated across the maturation stage in relation to ameloblast modulation. Ruffle-ended ameloblasts consistently showed the most intense immunoreaction. Gold particles were present throughout the cytoplasm and nuclei of ameloblasts but regions rich in rough endoplasmic reticulum or cell webs showed a higher immunolabelling. Some gold particles were also associated with the external face of the rough endoplasmic reticulum. Multivesicular bodies in maturation stage ameloblasts were occasionally immunoreactive. These data suggest that the intracellular concentration of CaBP 28 kDa is regulated throughout amelogenesis reflecting a stage-specific control of calcium homeostasis in ameloblasts.
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  • 292
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    The @Anatomical Record 230 (1991), S. 164-168 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Microridges produce a characteristic fingerprint-like pattern on the surface of fish oral mucosa. The cytoskeleton in these microridges was examined by immunofluorescence microscopy and transmission electron microscopy after detergent extraction and decoration with myosin subfragment 1. The effect of cytochalasin B on microridges was probed with scanning electron microscopy. Immunofluorescence microscopy revealed that actin filaments were present throughout the periphery of the epithelial cells and were especially localized beneath the free surface of the epithelium. In thin sections treated with Triton X-100, the majority of filaments in the microridges and their bases were found to be actin filaments and a plexus of keratin filaments that underlay the network of actin filaments. A part of the plexus of keratin filaments entered the microridges. After extraction with Triton X-100 and decoration with myosin subfragment 1, decorated actin filaments were found in the microridge cores, connected to the keratin filaments. The keratin filaments aggregated in the pattern of microridges and a few of them protruded into the microridges. Treatment with cytochalasin B caused microridges to disappear or to become thinner and lower or to change short or microvillus-like microridges. When most microridges disappeared, the surface of the superficial cells was prominently swollen, but the cell boundaries were fastened, and the microridges in the periphery were preserved. On the basis of these observations, the possible roles of actin and keratin filaments in the maintenance and the formation of microridges are discussed.
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  • 293
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    The @Anatomical Record 230 (1991), S. 175-182 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The fibronexus is a close transmembrane association between fibronectin filaments and actin microfilaments. It has been found at the surfaces of fibroblasts in tissue culture, as well as within contracting granulation tissue. This specialized connection has been proposed to play an important role in the adhesive properties of fibroblasts. The purpose of this study is to determine whether the fibronexus is present in other contracting tissues besides granulation tissue, specifically in Dupuytren's diseased tissue. Dupuytren's disease is a pathologic condition in which the palmar aponeurosis becomes shortened leading to irreversible flexion of the digits. Shortening of the aponeurosis is believed to be an active cellular process. Extracellular filaments and actin microfilaments form close transmembrane associations at the surfaces of actin-rich fibroblasts in Dupuytren's disease. Extracellular filaments extend from the cell surface into the surrounding tissue connecting fibroblasts with collagen fibrils and adjacent cells. In this study we have used immunoelectron microscopy to demonstrate that the extracellular filaments that participate in these close transmembrane associations contain fibronectin. High voltage electron microscopy has been used to examine the three-dimensional relationships between the cytoskeleton and fibronectin filaments in Dupuytren's diseased tissue. We propose that the fibronexus is a dominant adhesive structure at the surface of fibroblasts in Dupuytren's diseased tissue. The fibronexus, by mediating cell-to-cell and cell-to-matrix attachments, may serve to transmit contractile forces generated by actin microfilaments in these cells throughout the diseased tissue.
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  • 294
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    The @Anatomical Record 230 (1991), S. 169-174 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present a rapid technique for determining cancellous bone mineral changes in small experimental animals. We used the distal centimeter of the right femur from ovariectomized (OX) (N = 30) and shamovariectomized (ShOX) (N = 28) rats, aged 90 days at surgery and killed at times from 125-540 days postsurgery. We used dual photon absorptiometry to scan the segment three times: intact, after parasagittal splitting, and after removing all cancellous bone. We equated the difference between the second and third scans to cancellous bone mineral content (Cn.BMC). To validate this, we compared it with histomorphometrically determined bone volume (BV/TV) of the proximal tibial metaphysis of the same rat. Parasagittally splitting the segment removed no detectable mineral. OX rats had 40% less Cn.BMC than ShOX rats. However, OX rats had 80% lower BV/TV than ShOX rats. The subtraction technique not only makes a rapid, reasonable assessment of cancellous bone loss in OX rats but permits a smaller sample size than histomorphometry. The histomorphometric technique finds a greater difference between OX and ShOX rats because it examines a region where cancellous bone loss is more marked than does the scanning technique. The current technique measures bone of not only the central secondary spongiosa but also the juxtacortical region and the primary spongiosa, where OX-related differences are less prominent. The principles of this subtraction technique proved workable. However, for the future, we recommend a two-scan technique using a dual energy X-ray scanner. It is likely to take only 20-30 min per specimen to assess cancellous bone mineral.
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  • 295
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    The @Anatomical Record 230 (1991), S. 183-198 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Muscle spindles in the tenuissimus muscle of mature golden Syrian hamsters were examined by conventional and high-resolution scanning electron microscopy (HRSEM). For conventional SEM, entire muscles were first fixed in 2.5% buffered glutaraldehyde. Spindles were then isolated with a dissecting microscope under darkfield illumination and postfixed in 1.0% OsO4. Some spindles were treated with 8 N HCl at 60°C to clearly expose intrafusal fiber surfaces once the outer capsular sheath was mechanically disrupted. Preparation for HRSEM included aldehyde/osmium fixation and freeze-cleavage in liquid N2. The cytosol and certain cellular elements were also selectively extracted by immersion in 0.1% OsO4 for varying time intervals. In these preparations, the capsular sleeve showed a multilayered pattern of vesicle-laden cells with variant surface topography in different regions, including filopodia and small bristle-like surface-projections. An interlacing three-dimensional network of collagen fibrils intervened between the capsular lamellae. Within the spindles, sensory and fusimotor nerve endings closely adhered to the outer surfaces of intrafusal fibers. Sensory nerve terminals were enveloped by a prominent external lamina, and those that were cleaved open contained a plethora of elongated mitochondria that ran parallel with the longitudinal axis, along with vesicles, axoplasmic filaments, and lysosomes. Multiple adhesion sites between the sensory nerve membrane and the underlying sarcolemma of the intrafusal fiber were also observed in select regions. Fusimotor nerve endings were covered externally by processes of Schwann cells and their axoplasm was filled with a multitude of cellular organelles and synaptic vesicles. Dilated cisternae of the sarcoplasmic reticulum and numerous mitochondria were, in addition, observed below the postjunctional sarcolemma at the neuromuscular interface. The methodology used in this study provides a novel view of the exquisite three-dimensional architecture of this complex neuromuscular receptor.
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  • 296
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    The @Anatomical Record 230 (1991), S. 209-217 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The distribution of actin in spermatogenic cells and epididymal spermatozoa of the opossum, Monodelphis domestica, was examined by immuno-fluorescence microscopy to identify its potential function in the major structural events of sperm development. In spermatogenic cells actin was located at the site of initial interaction between the nucleus and acrosome and remained present through subsequent acrosome morphogenesis. Actin was also associated both with the posterior pole of the nucleus, at the site of flagellar attachment, and with the manchette. Thus actin may play a role in establishing the specific associations of spermatid organelles and in the streamlining of the cells' architecture. In epididymal spermatozoa two sites of actin localization are present. The first site is surrounding the connecting piece where it may participate in the characteristic 90° rotation of the head. The second site was a ring of actin surrounding the lateral boundary of the acrosome where it may play a role in the sperm pairing process which also occurs in the epididymis.
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  • 297
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The effect of tunicamycin (TM) on testicular cord organization in the fetal mouse was examined in vitro at light and electron microscopic levels, with special reference to the glycoprotein functions during Sertoli cell differentiation.In testicular explants treated with TM, testicular cord organization was inhibited. TM treatment affected basal lamina formation by Sertoli cells, resulting in a discontinuous basal lamina or none at all in certain areas. The disorganized Sertoli cells were amorphous in shape, exhibited poor epithelial polarity, and were irregularly arranged in the testicular parenchyma. Extracellular matrix and collagen fibers were often observed in the intercellular spaces between the disorganized Sertoli cells. Lectin histochemical observation revealed that the number of wheat germ agglutinin binding sites on the plasma membrane and basal lamina of disorganized Sertoli cells was significantly decreased by TM treatment. However, junctions were normally observed in the plasma membrane between disorganized Sertoli cells. Leydig cells showed a normal differentiation in the testicular parenchyma in the presence of TM.These observations suggest that basal lamina formation of Sertoli cells and/or the expression of their cell surface glycoconjugates may be crucial for the establishment of Sertoli cell polarity and/or the Sertoli-Sertoli cell interactions required for proper testicular cord formation. Sertoli cell organization into testicular cords and Leydig cell differentiation may be controlled by different regulatory mechanisms.
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  • 298
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    The @Anatomical Record 230 (1991), S. 218-224 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The cytoplasmic distribution of poly(A)+ mRNA and its relationship to annulate lamellae were examined in developing Necturus maculosus oocytes by in situ hybridization with [3H]poly(U). The specificity of [3H]poly(U) binding was tested by incubating control ovarian sections with either KOH or RNase A before in situ hybridization. In both experiments, the silver grain densities were markedly reduced. Poly(A)+ RNA is uniformly distributed in the cytoplasm until the mid-growth phase and then later in vitellogenesis becomes localized in the subcortical ooplasm. The silver grain density in the cytoplasm varied during oogenesis and was greatest in previtellogenic oocytes. Annulate lamellae commonly are observed with the light microscope in oocytes prior to vitellogenesis. In such oocytes, the labeled mRNA probe is observed over cytoplasmic regions of annulate lamellae. The results suggests that a differential localization of messenger RNA occurs during oogenesis in Necturus maculosus. Furthermore, poly(A)+ RNA is present in cytoplasmic regions of annulate lamellae.
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  • 299
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    The @Anatomical Record 230 (1991), S. 225-234 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Development of the fetal mouse esophageal epithelium was followed using light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and radioautography. At 15 days of gestation in the cervical (C), mediastinal (M), and abdominal (A) segments of the esophagus, the epithelium was two or three cells thick, and only cells located in the basal (germinal) layer incorporated tritiated thymidine. Ciliated cells were sparse in all three segments. At 17 days of gestation, longitudinal mesenchymal ridges became more differentiated in the distal segment. Labeling indices were lower than at preceding stages in each segment. Ciliated cells had increased in number and appeared to be evenly distributed along the whole esophagus. In periodic acid-Schiff (PAS)-stained sections, an increasing proximodistal distribution of glycogen stores was observed, with greatest concentrations found in segment A. At 18 days of gestation, labeling indices were comparable in segments C and M (11.7% ± 2.9% and 12.8% ± 1.9%, respectively) but remained higher in segment A (17.9% ± 2.0%). Ciliated cells were still present. At this stage, transverse circular furrows and ridges started to appear. They increased in number at 4 days after birth and were very closely distributed in the adult. In longitudinal sections, these ridges corresponded to projections of stratum granulosum and of the overlying stratum corneum. After birth, ciliated cells desquamated rapidly but some patches were still present at 4 days. At 8 days, the esophageal epithelium was not yet keratinized.
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  • 300
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    The @Anatomical Record 231 (1991), S. 193-200 
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We studied in the rat epididymis the presence of membrane-bounded vesicles in the stereociliar areas of the epithelial cells. The intimate contact between principal cell stereocilia and luminal spermatozoa was also explored.The epididymidis of adult male albino rats were fixed with Mollenhauer's fixative via the thoracic aorta; they were removed and the caput and the cauda were separated and fixed for 4 additional hours at 4°C. After fixation, the samples were processed with routine techniques for transmission and scanning electron microscopy.The study showed membrane-bounded vesicles in the lumen of the caput and cauda epididymidis. They are present between stereocilia, in the most peripheral regions of the epididymal lumen, and in a stereocilia-free zone in the apical plasma membrane of the principal cells. The smaller vesicles are located near the apical surface of the latter, and the larger ones are located near the tips of the stereocilia. Their contents are electron lucent in some images and electron dense in others. In several thin sections some of the vesicles are observed to have a stalk. This suggests that the possible mode of production may be an exocytotic process. Some membrane-bounded vesicles were found to be in contact with the head or the tail of maturating spermatozoa.Moreover, an intimate contact was found to exist in the epididymidis between the plasma membranes of the spermatozoa and the stereocilia.These observations seem to suggest two possible mechanisms for sperm-epididymal cell relations: 1) release of a secretion product via the membrane-bounded vesicles and 2) direct contact between stereocilia and spermatozoa.
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