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  • 1
    Electronic Resource
    Electronic Resource
    Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes (1992)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 111-122 
    Details
    ISSN: 0886-1544
    Keywords: cardiac muscle ; actin dynamics ; α-actinin ; vinculin ; microinjection ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, α-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells'native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, α-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throug hout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC3[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970210204
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  • 2
    Electronic Resource
    Electronic Resource
    Talin dynamics in living microinjected nonmuscle cells (1989)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 14 (1989), S. 271-287 
    Details
    ISSN: 0886-1544
    Keywords: actin-membrane interaction ; adhesion plaque ; vinculin ; integrin ; fibroblasts ; epithelial cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To investigate the role of talin in the anchoring of actin-containing stress fibers to the cell membrane of nonmuscle cells, a fluorescent analog of the adhesion plaque protein talin was developed, characterized, and microinjected into living cells. Purified chicken gizzard talin was covalently labeled with the fluorescent dye lissamine rhodamine B sulfonyl chloride. The fluorescently labeled protein was then chromatographed on Sephadex G-25 and DEAE-cellulose in order to remove free dye and denatured protein. The fluorescent talin was able to bind purified vinculin and was localized in adhesion plaques, membrane ruffles, microspikes, and polygonal networks in acetone-permeabilized nonmuscle cells. In cells that were double-stained with fluorescent talin and an affinity-purified anti-talin an-tibody, a one-to-one correspondence of adhesion plaque staining was seen. Living epithelial cells (PtK2) were microinjected during interphase with fluorescent talin. Computer-enhanced video microscopy was used to document adhesion plaque dynamics such as (1) changes in plaque shape, (2) alterations in plaque positions, and (3) the appearance, growth, and dissolution of plaques. In cells that were followed during mitosis, the adhesion plaques disappeared during cell rounding and then subsequently reappeared upon spreading of the two daughter cells. Treatment of microinjected cells with DMSO in order to disassemble stress fibers resulted in an altered localization of the fluorescent talin. Upon recovery of the cell from the drug, the talin was visualized in its characteristic submembraneous position. These results are the first to document the role and distribution of talin in dynamic processes occurring in living microinjected nonmuscle cells.
    Additional Material: 13 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970140213
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  • 3
    Electronic Resource
    Electronic Resource
    Intact alpha-actinin molecules are needed for both the assembly of actin into the tails and the locomotion of Listeria monocytogenes inside infected cells (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 97-107 
    Details
    ISSN: 0886-1544
    Keywords: Listeria ; actin ; alpha-actinin ; vinculin ; talin ; filopodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: After the infectious bacterium, Listeria monocytogenes, is phagocytosed by a host cell, it leaves the lysosome and recruits the host cell's cytoskeletal proteins to assemble a stationary tail composed primarily of actin filaments cross-linked with alpha-actinin. The continual recruitment of contractile proteins to the interface between the bacterium and the tail accompanies the propulsion of the bacterium ahead of the elongating tail. When a bacterium contacts the host cell membrane, it pushes out the membrane into an undulating tubular structure or filopodium that envelops the bacterium at the tip with the tail of cytoskeletal proteins behind it. Previous work has demonstrated that alpha-actinin can be cleaved into two proteolytic fragments whose microinjection into cells interferes with stress fiber integrity. Microinjection of the 53 kD alpha-actinin fragment into cells infected with Listeria monocytogenes, induces the loss of tails from bacteria and causes the bacteria to become stationary. Infected cells that possess filopodia when injected with the 53 kD fragment lose their filopodia. These results indicate that intact alpha-actinin molecules play an important role in the intracellular motility of Listeria, presumably by stabilizing the actin fibers in the stationary tails that are required for the bacteria to move forward. Fluorescently labeled vinculin associated with the tails when it was injected into infected cells. Talin antibody staining indicated that this protein, also, is present in the tails. These observations suggest that the tails share properties of attachment plaques normally present in the host cells. This model would explain the ability of the bacterium (1) to move within the cytoplasm and (2) to push out the surface of the cell to form a filopodium. The attachment plaque proteins, alpha-actinin, talin, and vinculin, may bind and stabilize the actin filaments as they polymerize behind the bacteria and additionally could also enable the tails to bind to the cell membrane in the filopodia. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280202
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  • 4
    Electronic Resource
    Electronic Resource
    Contractile protein dynamics of myofibrils in paired adult rat cardiomyocytes (1993)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 26 (1993), S. 301-312 
    Details
    ISSN: 0886-1544
    Keywords: intercated discs ; microinjection ; actin ; alpha-actinin ; vinculin ; myosin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The purpose of this study was to determine how quickly contractile proteins are incorporated into the myofibrils of freshly isolated cardiomyocytes and to determine whether there are regions of the cells that are more dynamic than others in their ability to incorporate the proteins. Paired cardiomyocytes joined at intercalated discs and single cells were isolated from adult rats, and microinjected 3 hours later with fluorescently labeied actin, alpha-actinin, myosin light chains, and vinculin. The cells were fixed and permeabilized at various period, 5 seconds and longer, after microinjection. Actin became incorporated throughout the I-Bands in as short a time as 5 seconds. The free edges of the cells, which were formerly intercalated discs, exhibited concentrations of actin greater than that incorporated in the I-Bands. This extra concentration of actin was not detected, however, at intact intercalated discs connecting paired cells. Alpha-actinin was incorporated immediately into Z-Bands and intercalated discs. Vinculin, also, was localized at the Z-Bands and at intercalated discs, but in contrast to alpha-actinin, there was a higher concentration of vinculin in the region of the intact intercalated discs. Both alpha-actinin and vinculin were concentrated at the free ends of the cells that were formerly parts of intercalated discs. Myosin light chains were observed to incorporate into the A-Bands in periods as short as 5 seconds. These results suggest that the myofibrils of adult cardiomyocytes may be capable of rapid isoform transitions along the length of the myofibrils. The rapid accumulation of fluorescent actin, alpha-actinin, and vinculin in membrane sites that were previously parts of intercalated discs, may reflect the response to locomotory activity that is initiated in these areas as cells spread in culture. A similar response after an injury in the intact heart could allow repair to occur. © 1993 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970260405
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  • 5
    Electronic Resource
    Electronic Resource
    “Molded” rods of macroporous polymer for preparative separations of biological products (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 476-480 
    Details
    ISSN: 0006-3592
    Keywords: continuous medium ; molded column ; macroporous polymer ; liquid chromatography ; proteins ; preparative HPLC ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A continuous rod of porous poly(glycidy1 methacrylate-co-ethylene dimethacrylate) has been prepared by a free radical polymerization within the confines of a 16-mm-i.d. glass column. The epoxide groups of the rod have been modified in situ by their reaction with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydroxypropyl functionalities that are required for the ion-exchange chromatographic mode. The bimodal pore size distribution curve typical for other molded separation media also prevail for the preparative-size rod. The column has been used successfully for the chromatographic separation of a mixture of standard proteins and yeast enzymes. The column exhibits a dynamic capacity that exceeds 420 mg of bovine serum albumin at a flow velocity of 60 cm/h. © 1995 John Wiley & Sons, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260480509
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  • 6
    Electronic Resource
    Electronic Resource
    Immobilization of trypsin onto “molded” macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) rods and use of the conjugates as bioreactors and for affinity chromatography (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 355-363 
    Details
    ISSN: 0006-3592
    Keywords: trypsin ; immobilization ; molded support ; poly(glycidyl methacrylate-co-ethylene dimethacrylate) ; porous materials ; affinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Trypsin immobilization onto continuous “molded” rods of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) and some applications of the conjugate have been studied. The rods polymerized within a tubular mold (chromatographic column), were treated in situ with ethylenediamine, activated with glutaraldehyde and finally modified with trypsin. The performance of the trypsin-modified rods was evaluated and compared to that of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads, modified with the same enzyme. Overall the enzyme-modified rods performed substantially better than the corresponding beads. In particular, the performance of the molded supports as enzymatic reactors or as chromatographic media benefits greatly from the enhanced mass transfer that is characteristic of the molded rod at high flow rates. © 1996 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    New formats of polymeric stationary phases for HPLC separations: Molded macroporous disks and rods (1996)
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 9 (1996), S. 326-334 
    Details
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A molding process has been used for the preparation of separation media in different shapes such as rods and flat membrane-like disks. The polymerization is carried out using a mixture of monomers, porogenic solvent and free-radical initiator under conditions that afford macroporous materials with through-pores or channels large enough to provide the high flow characteristics required for applications in chromatography. In contrast to classical suspension polymerization, the solubility of monomers in water does not restrict their use. The versatility of the preparation technique is demonstrated in polymerizations involving both hydrophobic and hydrophilic monomers such as styrene, chloromethylstyrene, glycidyl methacrylate, alkyl methacrylates and acrylamide. Techniques have been developed that allow fine control of the porous properties of the polymers. These, in turn, determine the hydrodynamic properties of the separation devices that contain the molded media.Since all the mobile phase must flow through the separation medium, the mass transport within the molded media is accelerated considerably by convection. Therefore, the separations can be performed at much higher flow rates than in packed columns. This is particularly important for separations of large molecules such as proteins for which diffusion is a serious problem that significantly slows down the separation processes.The molded separation media have been used for the separation of biological compounds using gentle chromatographic modes such as hydrophobic interaction, ion-exchange and affinity chromatography during which the biological activity of the separated compounds is completely retained.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    PEGA supports for combinatorial peptide synthesis and solid-phase enzymatic library assays (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Peptide Science 4 (1998), S. 195-210 
    Details
    ISSN: 1075-2617
    Keywords: PEGA ; solid-phase enzyme assay ; PEG ; MMP-9 ; fluorescence quenched peptides libraries ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Permeable resins cross-linked with long PEG chains were synthesized for use in solid-phase enzyme library assays. High molecular weight bis-amino-polyethylene glycol (PEG) 4000, 6000, 8000 were synthesized by a three-step reaction starting from PEG-bis-OH. Macromonomers were synthesized by partial or di-acryloylation of bis-amino-PEG derivatives. Bis/mono-acrylamido-PEG were copolymerized along with acrylamide by inverse suspension copolymerization to yield a less cross-linked resin (Type I, compounds 6-9). Furthermore, acryloyl-sarcosin ethyl ester was co-polymerized along with bis-acrylamido PEG to obtain more crosslinked capacity resin (Type II, compounds 13-19). N,N-Dimethylacrylamide was used as a co-monomer in some cases. The polymer was usually obtained in a well-defined beaded form and was easy to handle under both wet and dry conditions. The supports showed good mechanical properties and were characterized by studying the swelling properties, size distribution of beads, and by estimating the amino group capacity. Depending on the PEG chain length, the monomer composition and the degree of cross-linking the PEGA supports showed a high degree of swelling in a broad range of solvents, including water, dichloromethane, DMF, acetonitril, THF and toluene; no swelling was observed in diethyl ether. The PEGA resins (Type I) with an amino acid group capacity between 0.07 and 1.0 mmol/g could be obtained by variation of the monomer composition in the polymerization mixture. Fluorescent quenched peptide libraries were synthesized on the new polymer using a multiple column library synthesizer and incubated with the matrix metalloproteinase MMP-9 after it had been activated by 4-aminophenyl mercuric acetate resulting in 67/83 kDa active enzyme. The bright beads were separated manually under a fluorescence microscope and sequenced to obtain peptide substrates for MMP-9. After treatment with ethylene diamine, high-loaded resins (Type II) have been employed in continuous flow peptide synthesis to yield peptides in excellent yield and purity. © 1998 European Peptide Society and John Wiley & Sons, Ltd.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Anomalous conductivity zones in electrophoresis. III. Experimental tests of the theory (1983)
    Weinheim : Wiley-Blackwell
    Electrophoresis 4 (1983), S. 46-52 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: An assumption used in developing the basic theory, that ionic retardation factors are substantially independent of solute concentration, is tested and found valid. The results of electrophoresis through 15% polyacrylamide gels are found to be in agreement with the theory. Zones of altered concentration appear in the presence of spermine tetrahydrochloride, sodium phosphate buffer (pH 7.2) or Tris-HCl (pH 7.0). The use of ionic retardation factors and transport numbers deduced from conductivity measurements leads to correct prediction of the sign of concentration change in each case. The direction and velocity of migration of a low-concentration boundary can also be predicted, together with associated changes in pH. Further confirmation comes from a detailed analysis of published work on electrophoresis through sucrose gradients. The theoretical treatment is suitable for application to other systems (such as isoelectric focusing and isotachophoresis) where a gel is used as a stabilizing medium, and where effects of the kind discussed may produce unexpected distubances.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150040107
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  • 10
    Electronic Resource
    Electronic Resource
    A program in BASIC for the determination of molecular weights and linear graphic representation of an electrophoretic protein pattern: Application to serotypes A and B of Candida albicans studied by polyacrylamide gel electrophoresis and immunoblotting (1986)
    Weinheim : Wiley-Blackwell
    Electrophoresis 7 (1986), S. 316-323 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A program in BASIC Microsoft for a microcomputer is described for the analysis of electrophoretic patterns obtained by polyacrylamide gel electrophoresis. The program enables a rapid calculation of relative molecular masses and designs three types of graphs: the protein pattern, the linear representation of molecular masses and the linear regression curve calculated from several standard proteins. The application of this program for the study of serotypes A and B of the yeast Candida albicans is shown.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150070706
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