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  • 1
    ISSN: 1432-1106
    Keywords: Key words Acetylcholine receptor ; Nicotinic ; Denervation supersensitivity ; Neuromuscular junction ; α-Bungarotoxin ; Immunocytochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Through the use of biotinylated-bungarotoxin and monoclonal antibodies, the nicotinic acetylcholine receptor (nAChR) was localized in the subneural apparatus of mammalian motor end plates of the flexor digitorum brevis muscle of the adult rat at the light and electron microscopic levels. Under normal conditions, nAChR was located in the primary post-synaptic membrane of the neuromuscular junction, and the depths of the junctional folds constituting the secondary post-synaptic membrane did not contain any nAChR. Up to 75 days after repeated transection of the related motor nerve (sciatic), there was no major alteration in the light-microscopic localization of junctional nAChR in the subneural apparatus, except for a moderate shrinkage and increased immunocytochemical reactivity of the subneural apparatus. At the electron microscopic level, however, immunocytochemical reactivity gradually occupied the entire extent of the secondary post-synaptic membrane, including the depths of the junctional folds, which exhibited extensive branching. In non-innervated portions of the muscle fibers, nAChR receptor appeared in a linear localization on the surfaces of denervated muscle fibers. This linear reaction was not continuous with the nAChR reaction of the motor end plates. It is concluded that denervation supersensitivity might not be due to spreading of junctional nAChR from the end-plate area, but rather to expression of nAChR in non-innervated portions of the muscle fiber and to the infraterminal (subsynaptic) spreading of nAChR into the depths of junctional folds.
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  • 2
    ISSN: 1437-773X
    Keywords: Key words Gastrointestinal stromal tumor ; Gastrointestinal autonomic nerve tumor ; Electron microscopy ; Immunohistochemistry ; Gastrointestinal tract
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Sixteen gastrointestinal stromal tumors (GISTs) were studied by immunohistochemical analysis and an ultrastructural procedure. The tumor locations were as follows: esophagus (2), stomach (7), small intestine (3), and large intestine (4). Four of the lesions were classified as malignant, 2 as borderline, and 10 as benign. On the basis of the immunohistochemical analysis, the tumors were classified as follows: 1 as myogenic type, 2 as Schwann cell type, 8 as Cajal cell type (including 2 gastrointestinal autonomic nerve tumors, GANTs), and 5 as mixed-cell type. In each subtype the phenotype was compared to the ultrastructural findings. Myogenic and Schwann cell type revealed ultrastructurally smooth muscle differentiation and schwannian tumor. All 8 tumors of the Cajal cell type revealed interdigitating cytoplasmic processes with occasional clusters of filopodia. Two tumors were subdivided as GANT. Five tumors of mixed-cell type were composed of a mixture of cells with variable myogenic features or variable neural differentiation. We confirmed in this study that immunohistochemical analysis reflected electron microscopic findings.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Medical electron microscopy 32 (1999), S. 209-212 
    ISSN: 1437-773X
    Keywords: Key words Keratin ; Bile canaliculi ; Ecto-ATPase ; Transgenic mice ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Keratin 8 (K8) and keratin 18 are the intermediate filament proteins that are expressed in hepatocytes. A K8-deficient FVB/N mouse is a unique animal model for assessing the contribution of keratin intermediate filaments (IFs) to the structural and functional integrity of hepatocytes. Hepatocytes from homozygous (−/−) K8-deficient mice manifest a reduced bile acid secretion and an increased fragility to mechanical stress and hepatotoxic drugs. Hepatocytes from heterozygous (+/−) mice are more susceptible to drug-induced injury. Immunofluorescent microscopy revealed that hepatocytes from (+/−) mice maintained K8 IFs and F-actin that are similar to those in wild-type (+/+) mouse hepatocytes. In (−/−) mouse hepatocytes, K8 protein was negative and F-actin presented a coarse and irregular pattern. Ecto-ATPase, detected by enzyme histochemistry and observed by electron microscopy, was reduced in the bile canaliculi of both (+/−) and (−/−) mouse livers, in comparison with that of (+/+) mouse livers. These results reveal for the first time different microscopical findings regarding the livers of these three genotypes. They also suggest that the reduction of ecto-ATPase plays a role in the increased fragility of (+/−) and (−/−) mouse livers.
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  • 4
    ISSN: 1437-773X
    Keywords: Key words Small cell neuroendocrine carcinoma ; Parotid gland ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A case of small cell neuroendocrine carcinoma of the parotid gland is presented with immunohistochemical and electron microscopic studies. Small cell neuroendocrine carcinoma is extremely rare and is often difficult to distinguish from malignant lymphoma, adenoid cystic carcinoma, and undifferentiated carcinoma. Under light microscopy, the tumor cells consisted of solid sheets and nests of small tumor cells. Immunohistochemically, they were positive for KL-1 and EMA, and focally positive for NSE and synaptophysin. Observation using an electron microscope showed membrane-bound neuroendocrine granules in some tumor cells. Histological evaluation indicated that the present case was small cell carcinoma of the parotid gland, showing a neuroendocrine variety.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Medical electron microscopy 32 (1999), S. 167-174 
    ISSN: 1437-773X
    Keywords: Key words Apoptosis ; Docetaxel ; Human leukemia cell ; DNA fragmentation ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We induced apoptosis in cells of the human leukemia cell line HL-60 using an antitumor agent, docetaxel (Taxotere), and investigated apoptosis in various aspects using in situ end-labeling (ISEL) of DNA, DNA fragmentation assay, flow cytometry, and electron microscopy. Because it inhibits depolymerization of tubulin, docetaxel is thought to arrest the cell cycle at the mitotic stage and to exert an antitumor effect. In this study, accumulation of docetaxel-treated cells at the G2/M phase was detected using flow cytometry. On ISEL of DNA, DNA fragmentation was observed at the mitotic stage. On electron microscopy, the nuclei of apoptotic cells lost their nuclear membranes, as do cells at mitosis, demonstrating that the cells were arrested mainly at the M phase in the cell cycle.
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  • 6
    ISSN: 1432-2307
    Keywords: Key words Melanin ; Oncocytic metaplasia ; Nasopharynx ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  A 64-year-old man presented with a history of discomfort of the throat of a few weeks’ duration. Nasoscopic examination revealed multiple small, brown pigmentations at the left suprapharynx, the base of the left nasal cavity and the pharyngeal openings of the auditory tube on both sides. Microscopically, the lesion showed a glandular pattern of oncocytic epithelium with abundant pigmented granules and melanophages in the surrounding stroma. Immunohistochemically, the dendritic cells in the basal layer were positive for S-100 protein. Electron microscopic study revealed numerous fully melanized melanosomes and hypertrophied mitochondria in the oncocytic cells. Oncocytic cells do not produce melanin for themselves, melanin granules apparently being transferred from the adjacent dendritic cells to the oncocytic cells.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 200 (1999), S. 551-558 
    ISSN: 1432-0568
    Keywords: Key words Retina ; Development ; Retinal pigment epithelium ; Microglia ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Blood-borne mononuclear cells invade the developing retina via the hyaloid vasculature at the optic nerve head. Following removal of apoptotic cell debris they give rise to the network of resident microglia. The population of cells recently described in the peripheral subretinal space of developing human eyes may represent a further population of macrophages destined to become microglia. The aim of the present study was to confirm the presence of subretinal macrophages in the developing eye in other mammalian species and perform preliminary immunophenotypic analysis in rat tissues. The range of species chosen included eutherian mammals (rat and rabbit) and marsupials (wallaby and opossum). Ocular tissues from a range of developmental stages were studied by scanning electron microscopy and transmission electron microscopy. Distinctive networks of dendriform and pleomorphic macrophages were observed by scanning electron microscopy in the peripheral subretinal space of D2 rabbits, newborn and D2 rats and D75 wallaby. Transmission electron microscopic studies of D2 rabbit, newborn and D2 rat and all ages of North American opossum revealed cells with the ultrastructural features of macrophages in the peripheral subretinal space, cilio-retinal junction and between ciliary epithelial cells. Preliminary immunoperoxidase studies using a panel of anti-leukocyte monoclonal antibodies on frozen sections of rat ocular tissues (newborn, D2 and D4) revealed ED1+ Ox42+ ED2+ but Ox6– cells in the peripheral subretinal space, peripheral retina and ciliary body epithelia. The data confirms that subretinal macrophages are a feature of the developing eye in a broad range of mammalian species and immunophenotypic evidence leads the author to postulate that these cells arise from the ciliary body vasculature and may migrate into peripheral neural retina and mature into resident microglia.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 98 (1999), S. 645-650 
    ISSN: 1432-0533
    Keywords: Key words Amyotrophic lateral sclerosis ; Autopsy ; Electron microscopy ; Immunocytochemistry ; Motor ; neuron disease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report two autopsy cases of motor neuron disease (MND) patients with an unusual type of muscular atrophy predominantly affecting the shoulder girdle and the upper extremities with proximal dominance. Both patients are considered to be clinically categorized into the El Escorial suspected form of amyotrophic lateral sclerosis (ALS). At autopsy, they showed marked loss of spinal anterior horn cells accompanied by astrogliosis positively immunostained with anti-glial fibrillary acidic protein antibody at the cervical level. At the lumbosacral level, anterior horn neurons were relatively well preserved and Bunina bodies, ubiquitin-positive skein-like inclusions and Lewy body-like inclusions were observed in the remaining neurons. In one patient, brain stem motor neurons (nerves V, VII, XII) and motor cortex, including Betz cells, were also affected and the corticospinal tracts were degenerated at the level of the thoracic and lumbar spinal cord. Pathological findings of this patient are consistent with those of ALS. In the other patient, the motor cortex, brain stem motor nuclei and the corticospinal tracts were well preserved, which is pathologically compatible with progressive spinal muscular atrophy. These patients with such a peculiar pattern of progressive muscular atrophy should be placed in a subgroup of ALS.
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  • 9
    ISSN: 1432-0568
    Keywords: Key words Vomeronasal organ ; Microvilli ; Monoclonal antibody ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The development of the rat vomeronasal organ was studied morphologically and immunocytochemically, using the monoclonal antibodies (MAbs) VOBM1, VOBM2 and VOM2 that react with the luminal surface of the vomeronasal sensory epithelium. Postnatal day (P) 7, 14, 21, 28, 35 and adult animals were examined. The vomeronasal organ and the blood vessel of the organ markedly increased in size and the vomeronasal glands increased in number between P7 and P14. At P35, the shape of the vomeronasal organ was similar to that of the adult but its size was slightly smaller. Electron microscopy showed that only a few scattered microvilli were present on supporting cells, and receptor cells were immature at P7. At P21, well-branched microvilli of the receptor cells and many microvilli of the supporting cells were observed on the luminal surface of the sensory epithelium. At P35, most apical endings of supporting cells and receptor cells were covered with numerous microvilli. Less developed areas were also present at the luminal surface of the epithelium at P35. At P7, immunoreactivities of the three antibodies were observed as discontinuous thin-layered bands only on the luminal surface of the sensory epithelium and no immunoreactivity was observed in other regions of the vomeronasal organ. Immunoreactivities of the VOBM1, VOBM2 and VOM2 increased with age and were observed as continuous thin-layered bands on the luminal surface of the epithelium by P35. These finding suggest that the development of the vomeronasal organ continues after birth and that the organ may reach maturity just before puberty (P42–49).
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  • 10
    ISSN: 1432-0533
    Keywords: Key words Unmyelinated fibers ; Peripheral nerve ; Electron microscopy ; Histochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Quantitation of unmyelinated fibers (UF) in peripheral nerves has classically relied upon ultrastructural morphometry. Because this method is time-consuming, it is not typically performed in routine analysis of nerve biopsies. We applied the Bodian-Luxol technique to detect unmyelinated axons by light microscopy on semithin sections from resin-embedded nerve tissue. Estimates were compared to ultrastructural counts. The staining appeared highly specific for axons. Excellent correlation was found between optic densities and the population of UF larger than 0.5 μm. The smallest profiles detected by light microscopy had a diameter close to 0.6 μm. This new technique is not a substitute for ultrastructural quantitative morphometry of UF, as very small unmyelinated axons, especially regenerating ones, can not be reliably visualized. However, it provides a valuable light microscopic method for evaluating axonal loss among UF.
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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    Acta neuropathologica 98 (1999), S. 330-336 
    ISSN: 1432-0533
    Keywords: Key words Micropolygyria ; Electron microscopy ; Frontal lobe ; Perivascular space ; Dot-like structure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have reported breaches of glia limitans (GL)-basal lamina (BL) complex with protruding neuroglial tissue in Fukuyama-type congenital muscular dystrophy (FCMD) fetus brain and suggested that some basic deficits in the GL-BL complex may have a pivotal role in formation of micropolygyria in FCMD. We therefore investigated the cerebral GL-BL complex in seven FCMD cases (12–27 years of age), in three cases of Duchenne muscular dystrophy (17–25 years of age) and in two non-neurological controls (28 and 33 years of age). The frontal lobe cortex was examined immunocytochemically using antibodies against collagen type IV and laminin in each case, and ultrastructurally in an adult case of FCMD. In FCMD, the BL of the cortical surface was frequently breached with protruding neural tissue that ultrastructurally showed frequent synapses, neurites that had parallel arranged microtubules, and astrocytic processes. The outermost surface of this tissue was only partly lined by a BL. In the region of the gyral adhesion of micropolygyria, the perivascular space of the apparently entrapped meningeal blood vessels was occupied by neuroglial tissue, which is assumed to have invaded through the occasionally seen breaches of the perivascular GL-BL complex. Electron microscopy of the intruding tissue showed frequent synapses, microtubule-containing neurites and astrocytic processes. No breached GL-BL complex was found in any of the non-FCMD cases. These findings indicate that in FCMD, the cerebral GL-BL complex continues to have a crucial deficit with resulting breaches through which neuroglial tissue protrudes, promoting adhesion of the adjacent cerebral gyri during brain development before and after birth.
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  • 12
    Electronic Resource
    Electronic Resource
    Springer
    Anatomy and embryology 199 (1999), S. 459-469 
    ISSN: 1432-0568
    Keywords: Key words Differentiation ; Electron microscopy ; Histochemistry ; Microtubules ; Neurofilaments
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The organization of the cytoskeleton has been studied during mouse differentiation in cells of the myenteric neuronal lineage. The entire gut was examined starting from day 12.5 of embryonic life (E12.5) until birth (P0). Immunocytochemistry was performed to evaluate the expression of five of the most represented neurofilaments proteins (the low, NF-L, medium, NF-M, and heavy, NF-H, molecular weight subunits, α-internexin and peripherin) and of two of the microtubule-associated proteins (MAP1 and MAP2a+2b). In parallel, the appearance in the differentiating myenteric neurons of filamentous and microtubular structures and their intracytoplasmatic distribution were observed under the electron microscope. A differential immunohistochemical expression of the structural proteins was found. Immature cells expressed α-internexin, peripherin, NF-M and MAP1 by day E12.5; α-internexin expression was strong in these cells, but gradually decreased with age and was practically absent in adulthood. Conversely, the expression of the other three proteins increased with cell differentiation and was still present in adulthood. NF-L and NF-H expression appeared later, by day E16.5, and was weak for the entire pre- and postnatal life. MAP2a+2b was never expressed. Under the electron microscope, at day E12.5 the cytoskeleton was already organized in filamentous and microtubular structures. At this age neurofilaments were few and mainly located in the cell processes, and microtubules were numerous and mainly assembled in the neuritic growth cones, together with synaptic vesicles. With ageing, neurofilaments and microtubules were ubiquitous in the neuron. Data obtained demonstrate that cytoskeletal proteins gradually accumulate in the cells of the neuronal lineage in parallel with the organization of the cytoskeletal structures, which in turn mediate important neural events by the earliest stages of murine embryonic life, including growth of nerve processes and initiation of axonal transport.
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Experimental brain research 125 (1999), S. 19-27 
    ISSN: 1432-1106
    Keywords: Key words Nerve injury ; Nerve repair ; Retrograde reaction ; Regeneration ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Peripheral axotomy of adult cat spinal motoneurons induces a marked loss of synaptic boutons from the cell bodies and dendritic trees. The aim of the present study was to analyze the recovery of synaptic contacts in axotomized motoneurons following reinnervation into muscle. Adult cat spinal motoneurons were first deprived of their muscular contacts for 12 weeks and, then, allowed to reinnervate their target muscle. Two years later, regenerated motoneurons were labeled with horseradish peroxidase to allow quantitative ultrastructural analyses of the synaptic covering of the cell bodies and dendrites. Presynaptic boutons were classified according to their size and the shape of their synaptic vesicles. Results show that a recovery of synaptic covering occurs in the axotomized neurons after muscle reinnervation, but it affects various bouton types to different degrees. The number of S-type boutons synapsing with the soma was 70% higher after reinnervation than at 12 weeks after axotomy, while the number of F-type boutons had increased by only 13%. Compared with the normal situation, the number of S-type boutons synapsing with the proximal dendrites increased from 82% at 12 weeks after axotomy to 180% in the reinnervated state. In conclusion, in adult cat spinal motoneurons, the reestablishment of muscular contact is followed by a normalization of some of the synaptological changes induced by a prolonged state of axotomy. In certain respects restitution is incomplete, but in others it results in overcompensation.
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  • 14
    Electronic Resource
    Electronic Resource
    Springer
    European biophysics journal 28 (1999), S. 263-267 
    ISSN: 1432-1017
    Keywords: Key words Human liver ; Human brain ; Ferritin ; Electron microscopy ; Mössbauer spectroscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Human brain (globus pallidus) and liver tissues were investigated by means of electron microscopy (EM), Mössbauer spectroscopy (MS) and SQUID magnetometry techniques. Based on MS measurements, the iron present was identified to be in the ferritin-like form (61–88%) and in the form of a low-spin iron species (the balance). Its overall concentration was estimated as 1.5(3) mg in the brain and 2.4(5) mg in the liver, per gram of lyophilized tissue. The average core diameter was determined by EM measurements to be equal to 7.5(1.3) nm for the liver and 3.3(5) nm for the brain. Magnetization measurements carried out between 5 and 300 K yielded an estimation of an average blocking temperature, KT BL, as equal to 6.7 K and 8.5 K for the liver and the brain, respectively. From the dependence of KT BL on the external magnetic field it was concluded that the ferritin-like cores in the studied samples can be regarded as non-interacting particles. Finally, the uniaxial magnetic anisotropy constant was determined to be 6×103 J/m3 for the liver and 4×104 J/m3 for the brain.
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  • 15
    ISSN: 1432-1459
    Keywords: Key words CADASIL ; Cerebrovascular disease ; Skin biopsy ; Muscle biopsy ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a microangiopathic syndrome. Although the defective gene has been identified, genetic analysis may be effort some due to its large size and various mutations. Providing a reliable diagnostic marker would therefore be helpful. Electron microscopy has revealed characteristic electron-dense granular deposits in the basal lamina of vessels of patients with CADASIL. We investigated the sensitivity of skin and muscle biopsies for diagnosing CADASIL. We examined 30 family members of three unrelated German families affected by CADASIL. In 14 of the 21 affected individuals we performed skin and muscle biopsies; two patients were clinically asymptomatic. Under electron microscopy all muscle and skin biopsy specimens showed patches of granular and electron-dense material in the basal layer of both arterioles and capillaries. These findings confirm that general microangiopathy is a typical feature of this syndrome and is present in the early phase of the disease with or without clinical manifestation. Thus, as electron microscopy of skin biopsy specimens can establish the diagnosis of CADASIL with high certainty, it may be considered the method of first choice.
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  • 16
    ISSN: 1432-2307
    Keywords: Key words Rhabdomyosarcoma ; Aorta ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  We report an intimal sarcoma presenting as an aortic aneurysm. A 68-year-old man suffered from chest pain and speech disturbance. Computed tomography showed a sacciform aneurysm of the aorta, which was resected, revealing a polypoid tumour measuring 1.5×2×2.5 cm projecting into the lumen. This proved to be a poorly differentiated high-grade sarcoma having morphological, immunophenotypic and ultrastructural features consistent with rhabdomyosarcomatous differentiation. Primary sarcomas of the aorta are extremely rare. Many cases have been diagnosed as ”intimal” on the basis of their site of origin, and they are not easy to classify from their histological pattern. Electron microscopy and the use of a more comprehensive panel of immunohistochemical markers should be applied in the histological classification of ”intimal” sarcoma.
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  • 17
    ISSN: 1432-0878
    Keywords: Key words Descending contralateral movement detector (DCMD) ; Identified neuron ; Vesicles ; Electron microscopy ; 3-D reconstruction ; Locust ; Schistocercagregaria (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The descending contralateral movement detector (DCMD), an identified descending interneuron in the brain of the locust Schistocerca gregaria has been investigated by using light and electron microscopy. We describe the fine structure, distribution and numbers of synapes that it receives from another identified brain neuron, the lobular giant movement detector (LGMD), and from unidentified neurons. The DCMD dendrites emerging from the integrative segment vary in form and number between individuals and sexes but always form a flattened dendritic domain. The arborizations and the integrative segment appear to be exclusively postsynaptic. Two types of synaptic contacts (Type 1 and 2) onto the DCMD can be discerned as having either round (Type 1) or pleiomorphic synaptic vesicles (Type 2) and by large (Type 1) or small (Type 2) subsynaptic appositions. Contact zones of Type 1 synapses are smaller than those of Type 2. LGMD-synapses are of Type 1 and occur intermingled with presynaptic sites of unidentified units. Some branches of the DCMD receiving input from unidentified units are devoid of contacting LGMD processes. Synapses of both types are randomly distributed over the DCMD integrative segment and at fibres with similar sizes.Type 1 synapses are much more frequent than Type 2 synapses and their number is negatively correlated with fibre diameter. For a whole DCMD dendritic arborization, a total of 8500 active zones of chemical synapses has been calculated, including a mininum of 2250 LGMD-synapses and about 1000 Type 2 synapses. The DCMD may thus receive a considerable amount of input from as yet unidentified neurons.
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  • 18
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 296 (1999), S. 359-369 
    ISSN: 1432-0878
    Keywords: Key words Apoptosis ; Electron microscopy ; Meiosis ; Spermatocytes ; Spermatogenesis ; Testis ; TUNEL ; Mouse (10 strains)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Apoptosis of male germ cells is a widespread but little-understood phenomenon in many animal species. The elucidation of its mechanisms could be useful in the understanding of male infertility. We have examined the distribution of dying cells with the terminal transferase-mediated nick-end labeling (TUNEL) method and by an electron-microscopic procedure in the testes of 10 mouse strains, viz., C57BL/10 (B10), SL/NiA (SL), C57BL/6 (B6), C3H/He (C3H), BALB/c (BALB), DBA2 (DBA), CBA/J (CBA), MRL/MpJ-+/+ (M+), MRL/MpJ-lpr/lpr (lpr), and wild-type NJL mice (Mus musculus musculus). In the testes of the B10, NJL, SL, B6, C3H, BALB, DBA, and CBA mice, very few TUNEL-positive cells are distributed in the seminiferous tubules, whereas in the testes of the M+ and lpr mice, many TUNEL-positive cells, which are restricted to stage XII seminiferous tubules, have been identified. The most important finding is that many metaphases of meiotic spermatocytes show a marked TUNEL-positive reaction. Some metaphases show apoptotic morphology electron-microscopically. These results suggest that the testes of MRL strains will provide a useful model for the study of the mechanism of metaphase-specific apoptosis in meiotic spermatocytes.
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  • 19
    ISSN: 1432-0878
    Keywords: Key words Neuromelanin ; Neuron ; Peroxidase ; Oxygen metabolism ; High-definition light microscopy ; Electron microscopy ; Ultrastructure ; Cytochemistry ; Substantia nigra ; Lumbricusterrestris (Annelida)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Histochemical examination of 1-μm tissue sections from the dorsal nerve plexus of the earthworm, Lumbricus terrestris, reveals multiple brown intraneuronal granules. These granules contain material morphologically and histochemically consistent with neuromelanin. When viewed with transmission electron microscopy, these were seen as single membrane-enclosed biphasic granules with diameters of 370–730 nm. Exposure of L. terrestris to high-level environmental oxygen resulted in an increase in the number of neuromelanin-like pigment granules within the neurons of the circular muscle layer. As measured by ortho-phenylenediamine hydrochloride, the endogenous peroxidase activity of extracts from worms incubated in high-level environmental oxygen was 51% more than controls. The endogenous peroxidase activity was localized in situ with 3,3-diaminobenzidine (DAB) and was found to increase in and around the neuromelanin-like pigment-containing neurons within the circular muscle layer. These studies suggest that the nerve net of L. terrestris may serve as a model to study the role of neuromelanin production in oxidative stress and its relationship to endogenous peroxidases.
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  • 20
    ISSN: 1432-0878
    Keywords: Key words Immunohistochemistry ; Electron microscopy ; Interstitial cells of Cajal ; F4/80 ; CSF-1 ; Kit-receptor ; Mouse (op/op)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The osteopetrotic (op/op) mutant mouse possesses an inactivating mutation in the colony-stimulating factor-1 (CSF-1) gene, which results in the absence of certain macrophages and in osteopetrosis, following a lack of osteoclasts. Studies of the op/op mouse indicate that CSF-1-dependent tissue macrophages may belong to a trophic and/or scavenger subpopulation, which through their effect on other cell types can significantly affect tissue functions, and that cells which are CSF-1 independent have antigen presentation and immunological functions.We have previously identified a cell system of regularly distributed macrophages in the muscularis externa of the small intestine and wanted to extend these studies to the op/op mouse.The present investigations with light- and electron-microscopic methods using fluorescent dextran, methylene blue and immunohistochemistry (F4/80, anti-kit receptor, anti-CD3, anti-CD45R/B220) show that macrophages are absent from the muscle layers, with only an occasional macrophage present in the subserosa. In the lamina propria and submucosa, macrophage numbers are reduced. In all other respects the muscularis externa appears normal, including normal organization and number of interstitial cells of Cajal. Control and op/op mice both lack cells expressing CD3 (T lymphocytes), CD45R/B220 (B lymphocytes) and mast cells in the muscularis externa. This leaves the muscularis externa macrophages as the most likely source of local cytokine production under such conditions as postoperative ileus and intussusception in infants, where the muscularis externa appears to be one target of cytokines. We conclude that the lack of macrophages, combined with the preservation of otherwise normal structure, will make the op/op mouse a valuable model by which to assess the functions and relative importance of the muscularis externa macrophages in relation to intestinal motility under normal and pathological conditions.
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  • 21
    ISSN: 1432-0878
    Keywords: Key words Caveolin ; Caveolae ; Lung ; Alveolar epithelial type I cell ; Immunocytochemistry ; Electron microscopy ; Confocal laser scanning microscopy ; Rat (CD)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.
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  • 22
    ISSN: 1435-1536
    Keywords: Key words Double chain surfactants ; Aggregates ; Phase diagrams ; Lamellar phases ; Electron microscopy ; SANS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: Abstract We present the phase diagrams and the properties of newly synthesised double-chain cationic N-alkyl-N-alkyl′-N,N-dimethylammonium bromide surfactants [C x C y DMABr (x = 12, 14 and 16; y = 10, 11, 12, 14 and 16)]. All the systems studied form liquid-crystalline lamellar phases but with different morphologies: unilamellar vesicles at low surfactant concentrations, multilamellar vesicles and tubular aggregates for surfactant concentrations between 2 and 10 wt% and at even higher concentrations planar bilayers of surfactant molecules in the classical Lα phase. The phase diagrams were determined with macroscopic and microscopic methods (polarisation microscopy, freeze-fracture transmission electron microscopy, scanning electron microscopy and differential interference contrast microscopy). The properties of the surfactant solutions were determined with differential scanning calorimetry measurements for Krafft point determination and small-angle neutron scattering measurements for interlamellar spacing and bilayer thickness. Finally, conductivity and viscosity measurements for phase characterisation were carried out.
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  • 23
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    European journal of epidemiology 15 (1999), S. 175-180 
    ISSN: 1573-7284
    Keywords: Electron microscopy ; Epidemiology ; Non-bacterial Gastroenteritis ; Oyster ; Small round structured viruses (SRSVs)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract From 1987 to 1992, 18 outbreaks of acute non-bacterial gastroenteritis occurred in Kyushu district. The most common symptoms were diarrhea, vomiting, nausea and abdominal cramp. Small round structured viruses (SRSVs) were detected in 52 (44.8%) of 116 stool samples from 17 outbreaks by the electron microscopy (EM) method, and a significant increase in the antibody level was noted in 42 (80.7%) of 52 paired serum samples from 12 outbreaks by the immune electron microscopy (IEM) method and in 18 (51.4%) of 35 samples from 8 outbreaks by the western blot (WB) method. However, according to the WB method, antigen-antibody reaction was not observed to reference antigen strips (SRSV-9/Tokyo 86-510, 63kDa) in three of the 8 outbreaks. The detected virus was regarded as an etiologic agent for these outbreaks. In four of 5 outbreaks which appeared associated with eating raw oysters, there was a close relation between SRSV infection and consumption of raw oysters.
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  • 24
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    Colloid & polymer science 277 (1999), S. 164-173 
    ISSN: 1435-1536
    Keywords: Keywords Nanostructures ; Thin films ; Vapor deposition ; Electron microscopy ; Optical properties
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: Abstract Colloidal dispersions of Yb, Er and Pr have been prepared by chemical liquid deposition. The metals were cocondensed at 77 K with 2-methoxyethanol and ethanol to produce solvated metal atoms. The particle size of the dispersions was determined by transmission electron microscopy to range from 52 to 1080 Å; the particles had spherical shapes. After solvent evaporation under vacuum, active solids and amorphous powder were deposited over Cu and Al metal. Dispersion stability, particle size, UV/Vis absorption and zeta potential were studied. The solids prepared by solvent evaporation were characterized by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The films prepared on Al were studied by scanning electron microscopy. The most stable colloid was obtained using 2-methoxyethanol: several concentrations were stable for several months and the zeta potential indicated that this colloid stability is mainly due to solvation effects. FTIR spectroscopy of the solids indicated solvent incorporation in the film. This observation was corroborated by thermal analysis. Information on the thermal stability of the films was obtained by TGA. The UV/Vis absorption spectrum was measured at several concentrations under different conditions.
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  • 25
    ISSN: 1434-4726
    Keywords: Key words Laryngeal cancer ; p53 ; Oncoprotein ; Ki67 ; Proliferating cell nuclear antigen (PCNA) ; DNA ploidy ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The difficult and complicated mechanism of cancer development with little knowledge about the biology of existing cancers can lead to a permanent search for new examination techniques to improve the precision of life expectancy in patients and the selection of the most efficient methods of treatment. The aim of this study was to analyze certain prognostic factors, i.e., p53, Ki67, proliferating cell nuclear antigen (PCNA), DNA ploidy and cell proliferating activity, as well as the degree of morphological differentiation and cell maturity evaluated on an ultrastructural level in patients with laryngeal cancers in connection with data obtained from follow-up examinations and the clinical course of the disease. Neoplastic tissue was taken from 120 patients with laryngeal cancers. All underwent surgical treatment, radiotherapy and combined treatment in the Department of Otolaryngology, Karol Marcinkowski University School of Medical Sciences, Poznań, Poland, and the Department of Otolaryngology – Head and Neck Surgery, Haukeland University, Bergen, Norway. Before beginning treatment all patients underwent histological verification of their neoplastic tissues. Histopathological examination proved that the majority of cases (95%) had a squamous cell carcinoma. The occurrence of changes within the lymph nodes of the neck (N) was significantly correlated with T, S, Ki67, metastases to lymph nodes, DNA ploidy, site and surgery performed. The degree of clinical progression (S) was intercorrelated with T, N, p53, Ki67, PCNA, DNA ploidy, site and laryngectomy. The occurrence of oncoprotein p53 in neoplastic cells was measured by the staining degree of their nuclei and was correlated with T, S, DNA ploidy, metastases to lymph nodes, PCNA and site. The degree of staining of neoplastic cells for the nuclear antigen Ki67 was correlated to T, N, G, S, DNA ploidy, metastases to lymph nodes and surgical treatment. The proliferative antigen PCNA in the examined population of patients was intercorrelated with T, p53, Ki67, metastases to lymph nodes and surgical treatment. The results obtained from DNA flow cytometry could be associated with N, G, p53, Ki67 and metastases to lymph nodes. On the basis of the results obtained, the techniques suggested for the morphological and biological evaluation of neoplastic cells in cancer of the larynx should include TNM classification + G + DNA + p53 + Ki67.
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  • 26
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    Journal of molecular medicine 76 (1998), S. 413-420 
    ISSN: 1432-1440
    Keywords: Key words Endocrine tumors ; Histochemistry ; Electron microscopy ; Pathology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Recent developments in the field of endocrine cell biology and pathology at both morphological and molecular levels are briefly outlined and discussed as a basis for endocrine tumor characterization. The main tools available for identifying the endocrine nature of the tumors, their pathogenetic interpretation, and experimental reproduction with special emphasis on tumor antecedents are reported. Based on this, classifications of endocrine tumors of the pancreas and gastrointestinal tract are developed, covering most clinical (hyperfunctional syndromes included), pathological, and biological patterns, with special emphasis on tumor prognosis.
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  • 27
    ISSN: 1433-0474
    Keywords: Schlüsselwörter Selbstheilendes Kollodiumbaby ; Lamelläre Ichthyose ; Elektronenmikroskopie ; Key words Self-healing collodion baby ; Lamellar ichthyosis ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Collodion baby is a clinical description for a transient condition of the newborn which is caused by various disorders of cornification. The majority of cases are due to autosomal recessive lamellar ichthyosis. In up to 10% of cases „self-healing” of the skin occurs. We report about a mature male baby who at birth was covered entirely with a shining, collodion-like membrane. There also were marked ectropion of the eyelids and eversion of the lips. After shedding of the membrane almost complete clearing of the skin occurred until the age of three months. In the case reported here skin biopsies taken on the 20th day of life showed discrete and non-specific ultrastructural changes, with no features of lamellar ichthyosis or other cornification disorders. Discussion: From these findings and those reported in the literature we conclude that the prognosis of a collodion baby is unpredictable on clinical evaluation. However an ultrastructural examination can contribute to early assessment of prognosis in this heterogeneous neonatal condition.
    Notes: Zusammenfassung Das klinische Bild des Kollodiumbabys wird durch verschiedene Verhornungsstörungen hervorgerufen; in der Regel entwickelt sich später eine lamelläre Ichthyose. Eine Selbstheilung wird in bis zu 10% der Fälle beobachtet. Wir berichten über ein männliches Reifgeborenes, welches bei der Geburt vollständig in eine glänzende, kollodiumähnliche Membran gehüllt war. Weiterhin fanden sich ektropionierte Augenlider und evertierte Lippen. In den ersten Lebenswochen löste sich die Membran gänzlich ab und bis zum Alter von 3 Monaten war die Haut des Patienten weitgehend erscheinungsfrei. Die elektronenmikroskopische Untersuchung einer am 20. Lebenstag entnommenen Hautbiopsie zeigte bei unserem Patienten keinen auf eine lamelläre Ichthyose oder eine andere Verhornungsstörung hinweisenden Strukturdefekt der Keratinozyten. Es fanden sich lediglich unspezifische Veränderungen im Sinn einer Abräumreaktion. Diskussion: Unter Berücksichtigung der Literatur läßt sich folgern, daß das klinische Bild eines Kollodiumbabys keine Rückschlüsse auf den weiteren Verlauf zuläßt. Dagegen kann die Elektronenmikroskopie prognostisch richtungsweisende Aussagen beim ätiologisch heterogenen Erscheinungsbild des Kollodiumbabys erlauben.
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  • 28
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    Pediatric surgery international 13 (1998), S. 400-405 
    ISSN: 1437-9813
    Keywords: Key words Biofilms ; Aetiology ; Bacteria ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Biofilms form on all implanted foreign materials. In venous access ports (VAPs), the biofilm with entrapped organisms may be the source of recurring bacteraemia. At present, little is known of the development of biofilms in VAPs. In this study light, scanning, and transmission electron microscopy were used to investigate the evolution of biofilms in VAPs implanted in 15 African children with Wilms' tumour. The VAPs were removed either emergently because of infection (n = 6) or blockage (n = 3), or electively at the end of chemotherapy (n = 6). Intact biofilms were obtained from lengths of the catheter attached to ports that had been in place for 11 days to 3 years. Each was prepared for light and electron microscopy. In infected ports, shortly after implantation biofilms were thin and comprised of apparently healthy erythrocytes (RBCs) and occasional platelets, leucocytes (WBCs), and bacteria enmeshed in a network of fibrin. Three weeks after implantation, RBCs had autolysed and large numbers of WBCs and bacterial colonies were present within and on the luminal surface. In 1 instance, the lumen of a VAP had been occluded by a “plug” of WBCs. In non-infected patients, the biofilms in long-standing VAPs were of varying thickness and primarily composed of an amorphous granular material. In most cases, healthy and necrotic bacteria were present both within the core and on the surface of the biofilms. The results suggest that while bacteria, per se, are an important factor, the presence and degradation of blood components may be an equally important factor in the development of biofilms in VAP catheters.
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  • 29
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    Virchows Archiv 432 (1998), S. 293-298 
    ISSN: 1432-2307
    Keywords: Key words Follicular dendritic cell tumour ; Nasopharynx ; Immunohistochemistry ; Electron microscopy ; PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  We report the first case of an extranodal follicular dendritic cell (FDC) tumour localized in the nasopharynx of a 44-year-old male patient. The tumour cells were characterized immunohistochemically by strong expression of CD21, HLA-DR and vimentin and focal expression of CD68 and cytokeratin. Electron microscopic examination revealed desmosomal cell junctions between adjacent cell processes. Molecular genetic analysis using polymerase chain reaction (PCR) showed germline configuration of immunoglobulin and T-cell receptor genes. Epstein-Barr virus (EBV) genomes were detectable by PCR. After complete surgical tumour removal and radiotherapy the patient is disease-free 20 months after the initial diagnosis.
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  • 30
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    Virchows Archiv 432 (1998), S. 363-370 
    ISSN: 1432-2307
    Keywords: Key words Atopic dermatitis ; Pruritus ; Cutaneous nerve ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Although pruritus is the cardinal symptom of atopic dermatitis, its mechanism is not well understood. Free nerve endings in the skin are involved in pruritus as itching receptors. We studied the cutaneous nerve fibres in lichenified lesions of 16 patients with adult atopic dermatitis. On immunohistochemistry, fibres immunoreactive for neurofilament, neuron-specific enolase, and protein gene product 9.5 were observed in the papillary dermis and dermoepidermal junctions as well as in the epidermis. In these areas, no fibres stained positively for substance P, neuropeptide Y, vasoactive intestinal peptide, beta endorphin, somatostatin or serotonin. On electron microscopy, the ultrastructure of subepidermal and intraepidermal free nerve endings appeared to be essentially normal. However, the distribution density of the cutaneous nerve fibres was much higher than in normal controls, and the diameter of these fibres was much larger, because of the large number of axons in each nerve fibre. Degranulation of mast cells was not seen. These findings suggest that pruritus in lichenified atopic skin is probably not caused by damage to the cutaneous free nerve endings. In such lesions, the number of the cutaneous free nerve endings is greatly increased, but they may have a normal function.
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  • 31
    ISSN: 1432-2307
    Keywords: Key words Apoptosis ; Cell surface ; Cell nucleus ; Blebs ; TNF-α ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Tumour necrosis factor (TNF)-α-induced apoptosis is associated with several nuclear and cell surface alterations, in particular with the condensation of chromatin and the fragmentation of the cell nucleus, formation of blebs on the cell surface and breakdown of the plasma membrane. However, there is little information about the relationship between the cell surface alterations and the nuclear changes during apoptosis. To study this, cultured WEHI cells were exposed to TNF-α over different time periods. The cytological changes were studied using a correlative approach, which allowed observation of the same cell consecutively under light, scanning and transmission electron microscopy. The earliest sign of cell alteration was a reduction of the number of microvilli after 15 min of TNF-α exposure. This reaction was reversible (reappearance of microvilli) and took place during the first hour, in which neither nuclear alterations nor plasma membrane breakdown were observed. The changes in the nucleus began with condensation of chromatin after approximately 1 h of TNF-α-exposure. After 4–5 h the microvilli disappeared again, particularly in areas where the formation of blebs (blebbing) was observed. Strikingly, cell surface alterations (bleb formation) were detected only in those cells that presented with condensed chromatin, and not in cells with a normal chromatin pattern, proving at least a close correlation between nuclear and cell surface changes during the process of apoptosis.
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  • 32
    ISSN: 1432-0533
    Keywords: Key words Adult ceroid lipofuscinosis ; Kufs’ disease ; Electron microscopy ; Extracerebral biopsies
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In almost all of the earlier reported cases of Kufs’ disease, the adult form of ceroid lipofuscinosis, the diagnosis was ascertained by cerebral tissue examination, while peripheral biopsy examination revealed an apparent poor diffusion of specific lipofuscinic deposits, the finger print profiles (FPs). We report the ultrastructural data from skin, muscle and rectal biopsy specimens from two siblings, both still living, who present clinical features of Kufs’ disease. We observed the presence of FPs in locations that differ from the previous classic reports. Our results emphasize the value of extracerebral biopsies for the diagnosis of Kufs’ disease in vivo, and suggest some physiopathological assumptions based on vascular wall involvement.
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  • 33
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    Anatomy and embryology 197 (1998), S. 359-367 
    ISSN: 1432-0568
    Keywords: Key words Guanylin ; Mucus secretion ; Goblet cells ; Small intestine ; Edema ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Guanylin, structurally related to the heat-stable enterotoxin of E. coli, is a 15-amino-acid peptide isolated from rat small intestine. We investigated the morphological effects of an intravenous injection of rat and human guanylin upon the rat intestine. Various doses of rat guanylin were injected intravenously in anesthetized rats. After 5, 10 and 30 min, rats were killed by intracardiac perfusion with aldehyde fixative, and specimens of the intestine were then prepared for light and electron microscopy. Intravenously injected rat guanylin rapidly induced mucus secretion from crypt goblet cells in the duodenum. About half of the crypt goblet cells secreted mucous granules by compound exocytosis within 5 min. The villus goblet cells, in contrast, were not sensitive to guanylin. Goblet cells in the jejunum were less responsive than those in the duodenum. This secretory response was rare in the ileum and colon. Human guanylin produced similar results. The mucus secretion induced by guanylin was inhibited by a prior-injection of atropine, but not hexamethonium. Moreover, guanylin induced intense edema in the mucosa and submucosa of the small intestine 5 min after the injection, which disappeared after 30 min. A prior-injection of atropine did not block the appearance of edema. In conclusion, the intravenous injection of guanylin induces two phenomena related to water movement: (1) compound exocytosis of mucous granules from crypt goblet cells in the rat duodenum and jejunum; (2) perineural, inter-epithelial and intra-epithelial edema in the rat small intestine.
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  • 34
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    Acta neuropathologica 96 (1998), S. 151-156 
    ISSN: 1432-0533
    Keywords: Key words Neuropathy ; Hypoglycemia ; Insulin ; implant ; Rat ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Insulin-dependent diabetes mellitus is a chronic metabolic disease that causes long-term secondary complications such as neuropathy. The occurrence of diabetic neuropathy has generally been thought of as being associated with hyperglycaemia. However, in a previous light microscopic examination of plantar nerves in diabetic BB/Wor rats treated with insulin implants we found that eu-/hyperglycaemic rats present a normal picture, whereas eu-/hypoglycaemic rats show severe changes. The aim of the present work is to supplement our previous light microscopic report with electron microsocpic data from the lateral plantar nerve of normal, eu-/hyperglycaemic and eu-/hypoglycaemic BB/Wor rats. Under the electron microscope lateral plantar nerves collected from eu-/hyperglycaemic rats presented a qualitatively normal picture. In addition, the fibre numbers and the size distribution of the myelinated fibres were normal. In contrast, specimens from eu-/hypoglycaemic BB/Wor rats showed severe qualitative changes, interpreted as signs of axonal de- and regeneration. The total number of axons was somewhat subnormal and the sizes of the myelinated fibres were strongly shifted towards smaller diameters. These data confirm our previous light microscopic observations. We conclude that eu-/hypoglycaemic BB/Wor rats treated with insulin implants, but not similarly treated eu-/hyperglycaemic animals, develop a neuropathy in their plantar nerves.
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  • 35
    ISSN: 1432-0533
    Keywords: Key words Cat ; Spinocerebellar degeneration ; Purkinje cell ; Distal dendrite ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Human spinocerebellar degeneration is one of the intractable diseases. We studied the detailed neuropathology of cats with hereditary cerebellar degeneration obtained from the experimental breeding. The findings included almost total loss of Purkinje cells with an increase in Bergmann’s glia in the cerebellar hemisphere, preservation of some Purkinje cells in the vermis and moderate neuronal depletion of the olive nucleus. Cerebellar and pontine nuclei were normal. The cerebrum and spinal cord as well as the peripheral nervous system appeared normal. Electron microscopic examination revealed swelling of the distal dendrites of Purkinje cells in the less-affected nodule of the vermis, and clusters of presynaptic boutons without any synaptic contact in the severely affected folia where Purkinje cell bodies and dendrites disappeared. Prolonged existence of presynapses in the molecular and Purkinje cell layers was confirmed by positive immunoreactivity to anti-synaptophysin. Quantitative analysis using electron microscopy demonstrated an apparent increase in the density and mean size of presynapses in the molecular layer of the severely affected folia. These findings indicate that degeneration of Purkinje cells started at the most distal part of the dendrite in this animal model of cerebellar degeneration, and that presynapses, axon terminals of the granular cells and basket cells can exist for a long time even after complete degeneration of the Purkinje cells. Further investigation of this novel animal model may promote a better understanding of pathogenesis of human hereditary cerebellar degeneration.
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  • 36
    ISSN: 1432-0568
    Keywords: Key words Sensory neurons ; Autonomic neurons ; Neuropeptides ; Immunohistochemistry ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The innervation of the knee joint synovial membrane of the guinea pig, i.e., the synoviocyte layer, the subjacent connective tissue and the connective tissue region beneath, was analyzed with immunohistofluorescence and electron microscopy. A screening of the innervation with antibodies against the general axon marker – protein gene product (PGP) 9,5 – revealed the presence of nerve fibers distributed in various regions of the knee joint synovial membrane. Confirmating previous studies, some of these nerve fibers stained with antibodies to tyrosine hydroxylase (TH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP). In addition, dynorphin (DYN)-containing fibers were detected, which have not been reported previously in normal joints. In general, the immunoreactive fibers were observed close to the synoviocytes and at blood vessels. Fibers with colocalization of NPY- and TH-like immunoreactivities (LIs), as well as of DYN- and TH-LIs were demonstrated. In the electron microscope, bundles of unmyelinated fibers as well as single fibers were found in the connective tissue region below the synoviocytes. Varicose parts of the nerve fibers contained mainly small, clear vesicles. Small and large dense-cored vesicles were also seen, but less frequently. Denser portions of the plasma membranes of some axons were observed in these regions, facing the extracellular space. Myelinated fibers were also observed in some nerve bundles. These findings emphasize the complex innervation of the synovial membrane, with nerve fibers containing a host of neuroactive substances. Altogether, these fibers are probably involved in many functions such as vasoregulation and control of synovial secretion in addition to being a source of mediators in joint inflammation.
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  • 37
    ISSN: 1432-1106
    Keywords: Key words Glutamate receptors ; Immunocytochemistry ; Electron microscopy ; Human cortex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Specimens of human cerebral cortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the metabotropic glutamate receptor subunit mGluR1a and the ionotropic glutamate receptor GluR2/3. A small number of non-pyramidal neuronal cell bodies were labelled for mGluR1a. Double immunolabelling with mGluR1a and GluR2/3 showed that most pyramidal cell bodies were labelled for GluR2/3 but not for mGluR1a. Despite the non-colocalisation of these two receptor subtypes in cell bodies, however, many dendrites and dendritic spines were double-labelled for mGluR1a and GluR2/3 at electron microscopy. As there is evidence that most neurons positive for GluR2/3 are pyramidal cells, this suggests that mGluR1a is present in dendrites of pyramidal neurons, despite absent or low levels of immunoreactivity in their cell bodies.
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  • 38
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    Experimental brain research 118 (1998), S. 1-13 
    ISSN: 1432-1106
    Keywords: Key words Nerve injury ; Retrograde reaction ; Spinal cord ; Electron microscopy ; Cat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The aim of this electron-microscopic study was to analyze the distribution of synaptic contacts on the cell bodies and dendrites of permanently axotomized adult cat spinal α-motoneurons. Following transection and ligation of the medial gastrocnemius nerve, the synaptic covering of the cell bodies and three different dendritic compartments of homonymous α-motoneurons was analyzed quantitatively at 3, 6, and 12 weeks postoperatively. The synaptic boutons were classified according to their size and the shape of their synaptic vesicles. On the soma, a transient increase in the number of boutons was noted at 3 weeks and 6 weeks postoperatively, while after 12 weeks the bouton number had decreased to half of its normal value. The transient increase was mainly due to an increase in the number of F-type boutons. At 12 weeks postoperatively, the synaptic covering was reduced by 83% on the soma and by 57% on the proximal dendrites. In the distal dendritic regions, the values for synaptic covering remained largely unchanged. In summary, axotomized motoneurons exhibit a reduction in synaptic covering which is maximal on the cell body and becomes less pronounced centrifugally along the dendrites. However, if also taking into account the loss of distal dendritic branches that occurs in axotomized motoneurons, the total loss of boutons is several times larger in the dendrites than on the soma.
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  • 39
    ISSN: 1432-072X
    Keywords: Key wordsV. cholerae O139 ; Lipopolysaccharide ; Electron microscopy ; Freeze-substitution technique ; Capsule
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The morphological and physical characteristics of the capsule of Vibrio cholerae O139 were examined. An electron microscopic study using the freeze-substitution technique showed that all of the V. cholerae strains of the O139 serogroup examined have a very thin fibrous layer on the outside of the outer membrane. In contrast, the mutants of strain O139, strain MO10T4 (which lacks capsule synthesis), and strain Bengal-2R1 (which fails to synthesize both the capsule and the O-antigen of lipopolysaccharide) were all found to have lost the surface layer. In addition, the capsule layer could also not be observed on the surface of V. cholerae strain O1. To determine the biological characteristics of the capsule of strains of the O139 serogroup, we investigated the serum killing activity and bacterial phagocytosis by polymorphonuclear leukocytes. The O139 strains were more resistant to the serum killing activity than were the V. cholerae O1 strain and the O139 mutant strains, thus suggesting that the existence of the capsule gave a serum-resistant character to the O139 strains. The surface character of the O139 strains had the same hydrophobic character as did that of the O139 mutant strains and the O1 strain. In addition, all the V. cholerae O1 and O139 strains examined, including the mutant strains, were effectively ingested by the human polymorphonuclear leukocytes. The number of ingested bacteria was not significantly different among the strains, and the ingestion of the acapsular O139 mutants thus showed that the capsule does not play an antiphagocytic role. These data suggest that the capsule of V. cholerae O139 has a physiological function different from that of the ordinal hydrophilic capsule that is found in invasive bacteria such as Klebsiella pneumoniae.
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  • 40
    ISSN: 1432-072X
    Keywords: Key words Magnetic bacteria ; Biomineralization ; Magnetite ; 16S rRNA ; In situ hybridization ; Ultrastructure ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.
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  • 41
    ISSN: 1432-9492
    Keywords: Transformation ; Clay-DNA complexes ; Nucleases ; X-ray diffraction ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geosciences , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract DNA bound on clay minerals, sand, and humic acids has been shown, both in vitro and in situ, to be capable of transforming bacteria and to resist degradation by nucleases, which could result in the crypticity of genes in soil and other natural habitats. To determine where DNA is bound on clay minerals, which may help to explain how bound DNA becomes resistant to degradation by nucleases but retains the ability to transform competent cells, chromosomal DNA from Bacillus subtilis bound on montmorillonite (M) and kaolinite (K) was examined by X-ray diffractometry and transmission and scanning electron microscopy. X-ray diffraction analysis showed that the basal spacings of M and K were not altered, indicating that this DNA did not significantly intercalate the clays. Scanning and transmission electron microscopy showed that the binding of this DNA was primarily on the edges of M and K, although some binding was also apparent on the planar surfaces. Based on the results of these studies, it is postulated that: 1.extension from the edges of the clays enables the unbound end of DNA to interact with receptor sites on competent cells and result in their transformation; and 2.binding on clays alters the electron distribution and/or conformation of DNA, which reduces its hydrolysis by nucleases.
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  • 42
    ISSN: 1432-0878
    Keywords: Key words Prion protein (PrPc) ; Electron microscopy ; Secretory granules ; Membrane ; Extracerebral tissues ; Hamster ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  In transmissible spongiform encephalopathies (TSE), such as scrapie in animals and Creutzfeldt-Jakob disease in humans, the central event is the conversion of a host-encoded amyloidogenic protein (PrPc) into an abnormal isoform (PrPsc) that accumulates as amyloid in TSE brain. PrPc is a membrane sialoglycoprotein synthesized in the central nervous system and elsewhere. We have examined the ultrastructural localization of PrPc in numerous hamster and some human extracerebral tissues, by means of a post-embedding electron-microscopic method combined with immunogold labeling. In stomach, intestine, lung, and kidney from hamsters, and in stomach, kidney, and spleen from humans, immunogold labeling specific for PrPc is observed on various cellular substructures related to secretory pathways: Golgi apparatus, secretory globules, and plasma membrane. In mucous epithelial cells of stomach and intestine, PrPc appears to be concentrated in secretory globules, suggesting a role for PrPc in the secretory function of the digestive tract. The secretory aspect of PrPc may be a key to understanding the physiopathological mechanisms underlying TSE.
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  • 43
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 293 (1998), S. 1-22 
    ISSN: 1432-0878
    Keywords: Key words Piecemeal degranulation ; Histamine ; Charcot-Leyden crystal protein ; Anaphylactic degranulation ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The evidence for vesicular transport as a mechanism for secretion by human basophils is reviewed. Initially, direct electron-microscopic inspection of experimentally produced and sequentially biopsied contact allergy skin lesions revealed a unique form of secretion termed piecemeal degranulation, characterized by the slow emptying of secretory granule contents (with retention of empty containers) in the absence of extrusion of entire granules. Budding of small vesicles to/from secretory granules was observed, and cytoplasmic vesicles were abundant. A generalized degranulation model was proposed to unify classical regulated secretion and this new form of secretion. Investigation of the mechanism(s) of secretion from human basophils required the development of numerous tools and resources. Chief among these were: (a) isolation and purification of circulating basophils; (b) identification of specific growth factors to increase the supply of this rare granulocyte; (c) understanding of secretogogue mechanisms and reliable analyses of secreted basophil products; and (d) development of ultrastructural preparations allowing imaging of small vesicles and quantifiable small electron-dense tags for granule materials in small vesicles. Applications of these tools to well-defined models of basophil secretion have established a role for vesicles as a mechanism for effecting secretion of histamine and the Charcot-Leyden crystal protein from activated human basophils.
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  • 44
    ISSN: 1432-0878
    Keywords: Key words Dendritic cells ; Maturation ; Intrahepatic translocation ; Immunohistochemistry ; Electron microscopy ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Specific populations of hepatic sinusoidal cells were stained with monoclonal antibodies that recognize monocytes/macrophages (ED1), tissue macrophages (Kupffer cells) (ED2), MHC class II (Ia) antigen (MRC OX6), and dendritic cells/γ,δ T-cells (MRC OX62) and analyzed by light and electron microscopy. The majority of ED1+ and/or ED2+ cells were localized to the hepatic parenchyma, whereas OX6+ and/or OX62+ cells were more densely distributed within Glisson’s sheath than in the hepatic parenchyma. Double-immunoperoxidase staining of normal liver for ED1, ED2, and OX6 identified dendritic cells (DC) of two different phenotypes, ED1+ED2–OX6+ and ED1–ED2–OX6+. DC can be classified into three different types based on ultrastructural characteristics. The first type (type I) is characterized by one or more long cytoplasmic processes and a well-developed lysosomal system. The second type (type II) has an inconspicuous lysosomal system, abundant hyaloplasm, and characteristic short cytoplasmic processes. The third type (type I–II) has cytologic features intermediate between those of type I and type II DC. At the electron-microscopic level, these three cell types are found in the sinusoidal lumen, whereas the majority of type II DC are located in the space of Disse and Glisson’s sheath. Furthermore, some OX6-labeled elongated DC appeared to traverse the lumen of sinusoids through endothelial pores to enter the space of Disse. One hour after intravenous injection of latex particles (0.81 μm in diameter), numerous latex-laden dendritic cells (ED1+OX6+, type I and type I–II) were detected in the lumen of hepatic sinusoids, but not in the space of Disse or Glisson’s sheath. These findings suggest that normal rat liver contains resident dendritic cells which downregulate phagocytic activity and mature into potent accessory cells during migration from the portal vein toward the central vein. These DC then traverse the sinusoidal lumen to the hepatic lymph system via the space of Disse.
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  • 45
    ISSN: 1617-4623
    Keywords: Key words Bacteriophage λ ; DnaA-DNA interactions ; Electron microscopy ; DNA-protein complexes ; Escherichia coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage λ DNA demonstrated differential binding of DnaA along the whole λ DNA. Interaction of DnaA with the λ replication region (from the promoter p R to the origin of replication, oriλ) demonstrated a strong binding of DnaA to the region around the p o promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the λ DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.
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  • 46
    ISSN: 1615-6102
    Keywords: Sporopollenin ; Solubilisation ; 2-Aminoethanol ; Reaggregation ; Electron microscopy ; X-ray microscopy ; Thypha angustifolia L
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Exines fromTypha angustifolia L. pollen were dissolved in hot 2-aminoethanol. The solubilisate was successively fractionated and reaggregated via a dialysis cascade with dialysis tubings of different exclusion volumina. Four fractions of reaggregated material with different molecular mass were obtained. Fraction 1 with a molecular mass above 25,000 Da, fraction 2 with a molecular mass between 10,000–25,000 Da, fraction 3 with a molecular mass between 5,000–10,000 Da, and fraction 4 of a molecular mass lower than 5,000 Da. The fractions were comparatively analysed by scanning and transmission electron microscopy and X-ray microscopy. The material of the fractions with a molecular mass above 10,000 Da exhibit high congruence to the initial material. Analysis of the reaggregated material with the lowest molecular mass revealed special distinct substructures which in form and size showed high similarities to substructures of exines described in literature. In detail, spherical substructures consisting of an electron-dense core surrounded by an electron-transparent corona and in addition elongated substructures with a distinctive surface sculpture were detected.
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  • 47
    ISSN: 1615-6102
    Keywords: Callose ; Electron microscopy ; Gasteria verrucosa ; Phenolics ; Phytomelan ; Seed coat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In the seed coat ofGasteria verrucosa the deposition of phytomelan takes place during seed development in three stages. Phytomelan is a black cell wall material which is chemically very inert. First the radial walls and part of the transverse cell wall of the outer epidermis of the outer integument become thickened by exocytosis of dictyosome vesicles. Callose is deposited at the tangential plasma membrane against those walls. After the callose deposition about two thirds of the original cell volume is filled with callose. During the second stage the callose is broken down, probably into glucose monomers or small polymers. At the same time cellulose is deposited at the outer tangential plasma membrane, forming a wall between the dissolving callose and the plasma membrane. In the third phase small granules appear in the solution of dissolved callose. which grow out and finally fuse to form a block of phytomelan, consisting of spherical 15-nm units. Remarkable is the function of the callose: it determines the size of the phytomelan block, and it probably functions as carbohydrate source for the phytomelan synthesis and/or for the cellulose inner layer. In this study transmission electron microscopy and cryo scanning electron microscopy are used to study the three developmental stages of the formation of the phytomelan layer.
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  • 48
    ISSN: 1615-6102
    Keywords: Algae ; Virus assembly ; DAPI staining ; Electron microscopy ; Hincksia hincksiae ; Immunofluorescence ; Marine double-stranded DNA virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The filamentous brown algaHincksia hincksiae can be infected by a large icosahedral double-stranded DNA virus (HincV-1). The virus shows extended latency and is replicated only in cells homologous to sporangia. Virus formation was studied by transmission electron microscopy, DAPI staining, and β-tubulin immunofluorescence. Inhibition of cytokineses results in multinucleate cells, which are the first indication of virus replication in productive cells; the microtubular cytoskeleton does not seem to be affected by the virus. Replication of viral DNA begins in the nuclei, which increase in size and eventually disintegrate. Virus assembly takes place in a mixed nucleo-/cytoplasm. Capsids bud from cisternae, which are interpreted as modified endoplasmic reticulum aggregated to virus assembly centres. The internal membranous component of the virus is thus derived from the endoplasmic reticulum. The particles are empty (electron translucent) when assembled, and the nucleoprotein core seems to be packaged subsequently through an opening in the capsid. A number of fine structural features not previously reported from brown algae and related to virus formation are described. Our results on Hincksia hincksiae virus are compared with observations made on various other icosahedral DNA viruses infecting eukaryotic algae and animals.
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  • 49
    Electronic Resource
    Electronic Resource
    Springer
    International journal of legal medicine 111 (1998), S. 57-61 
    ISSN: 1437-1596
    Keywords: Key words Experimental drowning ; Tracer study ; Electron microscopy ; X-ray microanalysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Law
    Notes: Abstract In animal experiments, studies on the mechanisms involved in drowning were carried out using latex and gold tracers of defined size and concentration. The tracers were detectable by fluorescence microscopy (latex tracers) and by electron microscopy (gold tracers) in the lungs, kidneys and lymph nodes and were analysed further by X-ray microanalysis using a transmission scanning electron microscope. Tracers with small diameters were shown to penetrate intercellular gaps of the alveolar epithelium and the larger tracers were incorporated into the epithelial and endothelial cells by active pinocytotic mechanisms thus passing through the air-blood barrier. The detection and analysis of tracers in organs of the systemic circulation originating from the immersion fluid can assist in understanding the pathophysiology of drowning and in some selected cases, in making a more definitive diagnosis.
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  • 50
    ISSN: 0006-3525
    Keywords: folding type-specific secondary structure propensities ; amino acids ; α-helical proteins ; β sheet proteins ; α/β proteins ; α+β proteins ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35-49, 1998
    Additional Material: 6 Ill.
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  • 51
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Biopolymers 45 (1998), S. 69-83 
    ISSN: 0006-3525
    Keywords: DNA branched junctions ; branch migration ; superhelical torque ; control of DNA structure ; endonuclease VII ; nanomechanical device ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: DNA branched junctions are analogues of Holliday junction recombination intermediates. Partially mobile junctions contain a limited amount of homology flanking the branch point. A partially mobile DNA branched junction has been incorporated into a synthetic double-stranded circular DNA molecule. The junction is flanked by four homologous nucleotide pairs, so that there are five possible locations for the branch point. Two opposite arms of the branched junction are joined to form the circular molecule, which contains 262 nucleotides to the base of the junction. This molecule represents a system whereby torque applied to the circular molecule can have an impact on the junction, by relocating its branch point. Ligation of the molecule produces two topoisomers; about 87% of the product is a relaxed molecule, and the rest is a molecule with one positive supercoil. The position of the branch point is assayed by cleaving the molecule with endonuclease VII. We find that the major site of the branch point in the relaxed topoisomer is at the maximally extruded position in the relaxed molecule. Upon the addition of ethidium, the major site of the branch point migrates to the minimally extruded position. © 1998 John Wiley & Sons, Inc. Biopoly 45: 69-83, 1998
    Additional Material: 9 Ill.
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  • 52
    ISSN: 0006-3525
    Keywords: conformation ; aggregation ; κ-carrageenan ; flow field-flow fractionation ; multiangle light scattering ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The relatively novel combination of flow field-flow fractionation (FFF) and multiangle light scattering (MALS) was employed to study a nondegraded κ-carrageenan in different 0.1M salt solutions. The applicability of the technique was tested, and the effects of salt type and salt composition on the molar mass and radius of gyration were studied. A conformational ordering was induced at room temperature by switching the solvent from 0.1M NaCl (coil form) to 0.1M NaI (helix form). An approximate doubling of the average molar mass and an increase in radius of gyration was then observed, in agreement with results obtained previously using size exclusion chromatography-MALS. This increase in size was attributed to conformational ordering and to the formation of double helices. Severe aggregation was observed above 40% CsI in the 0.1M mixed salt solution of CsI and NaI. This was ascribed to the association of helices into large aggregates. For these large associates, having molar masses of several millions, a reversal of the elution order in flow FFF was detected. © 1998 John Wiley & Sons, Inc. Biopoly 45: 85-96 1998
    Additional Material: 10 Ill.
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  • 53
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    New York : Wiley-Blackwell
    Biopolymers 45 (1998), S. 119-133 
    ISSN: 0006-3525
    Keywords: conformations of D-alanyl-D-alanine ; β-lactam ; structural overlay ; AMBER force field ; AM1 ; ab initio ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In this article a conformational analysis of the D-alanyl-D-alanine dipeptide, both charged and neutral, has been carried out. The preferred conformations were determined by means of ab initio and semiempirical quantum, together with empirical force field calculations. The AMBER* force field and the 6-31 + G** and 6-31G** ab initio levels give rise to a coincident minimum energy structure, which, on the other hand, differs from that determined by AM1, 3-21 + G, and 3-21G. The solvent effect on the different charged and neutral conformations have been considered through the AMSOL semiempirical method. A quantification regarding the structural similarities between the different dipeptide conformations and the ampicillin has been performed. The results show that the best overlay is attained by the minimum structure energy obtained by using the 6-31 + G** methodology, which presents a planar amidic nitrogen. © 1998 John Wiley & Sons, Inc. Biopoly 45: 119-133, 1998
    Additional Material: 5 Ill.
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  • 54
    ISSN: 0006-3525
    Keywords: chemical oxidation ; cellulose ; conformational transition ; capillary viscosity ; microcalorimetry ; calcium ions ; gels ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The conformational behavior of different molecular weight fractions of a synthetic C6-oxidized derivative of cellulose were investigated by means of capillary viscometry, CD, and microcalorimetric measurements. Experiments were carried out in the presence of either monovalent or divalent counterions.The experimental data indicated that C6-oxidized cellulose can assume an ordered extended conformation at low ionic strength, induced by the intrachain repulsions of negative charges. This conformation was suggested to be very similar to the fully extended structure of cellulose. In addition to this, upon increasing the ionic strength, a conformational transition of the order-to-disorder type occurred. In fact, the screening of the electrostatic repulsions introduced a number of conformational kinks into the cellulosic backbone, which enabled the polymer to assume a more coiled conformation hence producing less viscous aqueous solutions. © 1998 John Wiley & Sons, Inc. Biopoly 45: 157-163, 1998
    Additional Material: 6 Ill.
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  • 55
    ISSN: 0006-3525
    Keywords: conformational stability ; biological polyelectrolytes ; enthalpy ; entropy ; conformational transitions ; carrageenan ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A new method is proposed for the determination of the enthalpy and entropy changes of nonionic origin upon conformational transition of linear biopolyelectrolytes in solution. For all transition midpoints, defined by given temperature and ionic strength, the total free energy change of the system is zero, which means that the nonionic contribution to the free energy change is equal in value and opposite in sign to the polyelectrolytic one. The counterion condensation theory of linear polyelectrolytes provides for the appropriate analytical expression to be used in such calculations. Linear plots of the proper functions of the calculated free energy changes vs the proper functions of temperature allows for the determination of the enthalpic and entropic terms of the nonionic free energy change of transition.The method has been applied to the extensive available data of the ion-induced conformational change of κ-carrageenan, a linear sulfated galactan extracted from seaweeds. The method has proved very successful, with the results showing a remarkable convergency of the enthalpy values for different monovalent counterions. On the other hand, the above approach has made it possible to explain the known effect of counterion specificity on the transition by a small difference in the nonionic entropic contributions. © 1998 John Wiley & Sons, Inc. Biopoly 45: 203-216, 1998
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  • 56
    ISSN: 0006-3525
    Keywords: uv resonance Raman spectroscopy ; Raman cross section ; hypochromism ; DNA ; deoxynucleoside ; protein ; aromatic amino acid ; virus assembly ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Ultraviolet resonance Raman (UVRR) spectra of H2O and D2O solutions of the nucleoside (dA, dG, dC, dT) and aromatic amino acid (Phe, Trp, Tyr) constituents of DNA viruses have been obtained with laser excitation wavelengths of 257, 244, 238, and 229 nm. Using the 981 cm-1 marker of Na2SO4 as an internal standard, Raman frequencies and scattering cross sections were evaluated for all prominent UVRR bands at each excitation wavelength. The results show that UVRR cross sections of both the nucleosides and amino acids are strongly dependent on excitation wavelength and constitute sensitive and selective probes of the residues. The results provide a library of UVRR marker bands for structural analysis of DNA viruses and other nucleoprotein assemblies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 247-256, 1998
    Additional Material: 4 Ill.
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  • 57
    ISSN: 0006-3525
    Keywords: hemoglobin ; hexagonal bilayer ; Lumbricus ; electron microscopy ; three-dimensional reconstruction ; small-angle x-ray scattering ; three-dimensional models ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of cylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm-1 and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h 〉 1 nm-1) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex. © John Wiley & Sons, Inc. Biopoly 45: 289-298, 1998
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  • 58
    ISSN: 0006-3525
    Keywords: conformational changes ; vicinal glycosylation ; branched α-l-Rhap(1-2)[β-d-Galp(1-3)]-β-d-Glc1-OMe trisaccharide ; parent disaccharides ; hydrogen bond ; isotope effect ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Conformations of the α-l-Rhap(1-2)-β-d-Glc1-OMe and β-d-Galp(1-3)-β-d-Glc1-OMe disaccharides and the branched title trisaccharide were examined in DMSO-d6 solution by 1H-nmr. The distance mapping procedure was based on rotating frame nuclear Overhauser effect (NOE) constraints involving C- and O-linked protons, and hydrogen-bond constraints manifested by the splitting of the OH nmr signals for partially deuteriated samples. An “isotopomer-selected NOE” method for the unequivocal identification of mutually hydrogen-bonded hydroxyl groups was suggested. The length of hydrogen bonds thus detected is considered the only one motionally nonaveraged nmr-derived constraint. Molecular mechanics and molecular dynamics methods were used to model the conformational properties of the studied oligosaccharides. Complex conformational search, relying on a regular Φ,Ψ-grid based scanning of the conformational space of the selected glycosidic linkage, combined with simultaneous modeling of different allowed orientations of the pendant groups and the third, neighboring sugar residue, has been carried out. Energy minimizations were performed for each member of the Φ,Ψ grid generated set of conformations. Conformational clustering has been done to group the minimized conformations into families with similar values of glycosidic torsion angles. Several stable syn and anti conformations were found for the 1→2 and 1→3 bonds in the studied disaccharides. Vicinal glycosylation affected strongly the occupancy of conformational states in both branches of the title trisaccharide. The preferred conformational family of the trisaccharide (with average Φ,Ψ values of 38°, 17° for the 1→2 and 48°, 1° for the 1→3 bond, respectively) was shown by nmr to be stabilized by intramolecular hydrogen bonding between the nonbonded Rha and Gal residues. © 1998 John Wiley & Sons, Inc. Biopoly 46: 417-432, 1998
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  • 59
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    New York : Wiley-Blackwell
    Biopolymers 46 (1998), S. 489-492 
    ISSN: 0006-3525
    Keywords: refractive index increment ; proteins ; solvent ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The refractive index increment of a protein solution is a property not only of the protein, but also of the solvent. This is demonstrated theoretically and confirmed experimentally using analytical interferometry. © 1998 John Wiley & Sons, Inc. Biopoly 46: 489-492, 1998
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  • 60
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    New York : Wiley-Blackwell
    Biopolymers 47 (1998), S. 1-1 
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: No abstract.
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  • 61
    ISSN: 0006-3525
    Keywords: hepatitis A ; synthetic peptides ; CD ; liposomes ; computational study ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110-121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic β-structure of the peptide.To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the β-structure in the presence of liposomes. © 1998 John Wiley & Sons, Inc. Biopoly 45: 479-492, 1998
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  • 62
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    Biopolymers 46 (1998), S. 31-37 
    ISSN: 0006-3525
    Keywords: DNA liquid crystals ; DNA fragments ; screened Coulomb interactions ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The critical volume fractions pertaining to the formation of DNA liquid crystals were obtained from polarization microscopy, 31P-nmr, and phase separation experiments. The DNA length (approximately one to two times the persistence length 50 nm), ionic strength, and counterion variety dependencies are reported. The cholesteric-isotropic transition is interpreted in terms of the coexistence equations, which are derived from the solution free energy including orientational entropy and excluded volume effects. With the wormlike chain as reference system, the electrostatic contribution to the free energy is evaluated as a thermodynamic perturbation in the second virial approximation with a Debye-Hückel potential of mean force. The hard core contribution has been evaluated with scaled particle theory and/or a simple generalization of the Carnahan-Starling equation of state for hard spheres. For sufficiently high ionic strengths, the agreement is almost quantitative. At lower amounts of added salt deviations are observed, which are tentatively attributed to counterion screening effects. The contour length dependence agrees with a DNA persistence length 50 nm. © 1998 John Wiley & Sons, Inc. Biopoly 46: 31-37, 1998
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  • 63
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    Biopolymers 46 (1998), S. 245-252 
    ISSN: 0006-3525
    Keywords: gelatin ; gelation ; atomic force microscopy ; interfacial rheology ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Gelation of gelatin under various conditions has been followed by atomic force microscopy (AFM) with the objective of understanding more fully the structure formed during the gelation process. AFM images were obtained of the structures formed from both the bulk sol and in surface films during the onset of gelation. While gelation occurred in the bulk sol, the extent of helix formation was monitored by measurements of optical rotation, and the molecular aggregation was imaged by AFM. Interfacial gelatin films formed at the air-water interface were also studied. Measurements of surface tension and surface rheology were made periodically and Langmuir-Blodgett films were drawn from the interface to allow AFM imaging of the structure of the interfacial layer as a function of time. Structural studies reveal that at low levels of helical content the gelatin molecules assemble into aggregates containing short segments of dimensions comparable to those expected for gelatin triple helices. With time larger fibrous structures appear whose dimensions suggest that they are bundles of triple helices. As gelation proceeds, the number density of fibers increases at the expense of the smaller aggregates, eventually assembling into a fibrous network. The gel structure appears to be sensitive to the thermal history, and this is particularly important in determining the structure and properties of the interfacial films. © 1998 John Wiley & Sons, Inc. Biopoly 46: 245-252, 1998
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  • 64
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    Biopolymers 48 (1998), S. 65-81 
    ISSN: 0006-3525
    Keywords: nucleotide analogue interference mapping ; phosphorothioate ; group I intron ; interference suppression ; RNA ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In this review I will outline several chemogenetic approaches used to determine the chemical basis of large ribozyme function and structure. The term chemogenetics was first used to describe site-specific functional group modification experiments in the analysis of DNA-protein interactions. Within the past few years equivalent experiments have been performed on large catalytic RNAs using both single-site substitution and interference mapping techniques with nucleotide analogues. While functional group mutagenesis is an important aspect of a chemogenetic approach, chemical correlates to genetic revertants and suppressors must also be realized for the genetic analogy to be intellectually valid and experimentally useful. Several examples of functional group revertants and suppressors have now been obtained within the Tetrahymena group I ribozyme. These experiments define an ensemble of tertiary hydrogen bonds that have made it possible to construct a detailed model of the ribozyme catalytic core. The model includes a functionally important monovalent metal ion binding site, a wobble-wobble receptor motif for helix-helix packing interactions, and a minor groove triple helix. © 1998 John Wiley & Sons, Inc. Biopoly 48: 65-81, 1998
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  • 65
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    Biopolymers 48 (1998), S. 83-96 
    ISSN: 0006-3525
    Keywords: nucleic acid ; disulfide cross-link ; structure ; dynamics ; stability ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In this review I discuss straightforward and general methods to modify nucleic acid structure with disulfide cross-links. A motivating factor in developing this chemistry was the notion that disulfide bonds would be excellent tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross-links have been used to study proteins. The chemistry described has been used to synthesize disulfide cross-linked hairpins and duplexes, higher order structures like triplexes, nonground-state conformations, and tRNAs. Since the cross-links form quantitatively by mild air oxidation and do not perturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids. © 1998 John Wiley & Sons, Inc. Biopoly 48: 83-96, 1998
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  • 66
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    Biopolymers 48 (1998), S. 113-135 
    ISSN: 0006-3525
    Keywords: divalent cations ; magnesium ; RNA ; ion binding ; RNA folding ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Divalent cations, like magnesium, are crucial for the structural integrity and biological activity of RNA. In this article, we present a picture of how magnesium stabilizes a particular folded form of RNA. The overall stabilization of RNA by Mg2+ is given by the free energy of transferring RNA from a reference univalent salt solution to a mixed salt solution. This term has favorable energetic contributions from two distinct modes of binding: diffuse binding and site binding. In diffuse binding, fully hydrated Mg ions interact with the RNA via nonspecific long-range electrostatic interactions. In site binding, dehydrated Mg2+ interacts with anionic ligands specifically arranged by the RNA fold to act as coordinating ligands for the metal ion. Each of these modes has a strong coulombic contribution to binding; however, site binding is also characterized by substantial changes in ion solvation and other nonelectrostatic contributions. We will show how these energetic differences can be exploited to experimentally distinguish between these two classes of ions using analyses of binding polynomials. We survey a number of specific systems in which Mg2+-RNA interactions have been studied. In well-characterized systems such as certain tRNAs and some rRNA fragments these studies show that site-bound ions can play an important role in RNA stability. However, the crucial role of diffusely bound ions is also evident. We emphasize that diffuse binding can only be described rigorously by a model that accounts for long-range electrostatic forces. To fully understand the role of magnesium ions in RNA stability, theoretical models describing electrostatic forces in systems with complicated structures must be developed. © 1999 John Wiley & Sons, Inc. Biopoly 48: 113-135, 1998
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  • 67
    ISSN: 0006-3525
    Keywords: 1H-nmr ; molecular modeling ; peptaibol ; peptide-lipid interaction ; sodium dodecyl sulfate micelles ; trichorzianin TA VII ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the α,α-dialkylated amino acids, α-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignments have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHCαH coupling constants, temperature coefficients of amide protons (Δδ/ΔTNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances and six Φ dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 Å for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a β-turn that leads to an angle about 90°-100° between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 75-88, 1998
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  • 68
    ISSN: 0006-3525
    Keywords: 9-hydroxyellipticine ; DNA ; CD ; linear dichroism ; resonance light scattering ; intercalation ; drug-drug interactions ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The binding of 9-hydroxyellipticine to calf thymus DNA, poly[d(A-T)]2, and poly-[d(G-C)]2 has been studied in detail by means of CD, linear dichroism, resonance light scattering, and molecular dynamics. The transition moment polarizations of 9-hydroxyelliptiycine were determined in polyvinyl alcohol stretched film. Spectroscopic solution studies of the DNA/drug complex are combined with theoretical CD calculations using the final 50 ps of a series of molecular dynamics simulations as input. The spectroscopic data shows 9-hydroxyellipticine to adopt two main binding modes, one intercalative and the other a stacked binding mode involving the formation of drug oligomers in the DNA major groove. Analysis of the intercalated binding mode in poly[d(A-T)]2 suggests the 9-hydroxyellipticine hydroxyl group lies in the minor groove and hydrogen bonds to water with the pyridine ring protruding into the major groove. The stacked binding mode was examined using resonance light scattering and it was concluded that the drug was forming small oligomer stacks rather than extended aggregates. Reduced linear dichroism measurements suggested a binding geometry that precluded a minor groove binding mode where the plane of the drug makes a 45° angle with the plane of the bases. Thus it was concluded that the drug stacks in the major groove. No obvious differences in the mode of binding of 9-hydroxyellipticine were observed between different DNA sequences; however, the stacked binding mode appeared to be more favorable for calf thymus DNA and poly[d(G-C)]2 than for poly[d(A-T)]2, an observation that could be explained by the slightly greater steric hindrance of the poly[d(A-T)]2 major groove. A strong concentration dependence was observed for the two binding modes where intercalation is favored at very low drug load, with stacking interactions becoming more prominent as the drug concentration is increased. Even at DNA : drug mixing ratios of 70:1 the stacked binding mode was still important for GC-rich DNAs. © 1998 John Wiley & Sons, Inc. Biopoly 46: 127-143, 1998
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  • 69
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    Biopolymers 46 (1998), S. 169-179 
    ISSN: 0006-3525
    Keywords: macromolecular carriers ; drug targeting and delivery ; branched chain synthetic polypeptides ; membrane-synthetic polypeptide interaction ; lipid monolayers/bilayers ; polymer therapeutics ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The surface properties at the air/water interface and the interaction of branched chain polymeric polypeptides with a general formula poly[Lys-(DL-Alam-X1)], where X = Π (AK), Ser (SAK), or Glu (EAK), with phospholipids were investigated. Polylysine derivatives with polycationic (SAK, AK) or amphoteric (EAK) were capable to spread and form stable monomolecular layers. The stability of monolayers at the air/water interface was dependent on the side-chain terminal amino acid residue of polymers and can be described by SAK 〈 AK 〈 EAK order. The area per amino acid residue values calculated from compression isotherms were in the same range as compared to those of linear poly-α-amino acids and proteins. Moreover, these polymers interact with phospholipid monomolecular layers composed of dipalmitoyl phosphatidyl choline (DPPC) or DPPC/PG (PG: phosphatidyl glycerol; 95/5, mol/mol). Data obtained from compression isotherms of phospholipids spread on aqueous polymer solutions at different initial surface pressure indicated that insertion into lipid monolayers for SAK or AK is more pronounced than for EAK. The interaction between branched polypeptides and phospholipid membranes was further investigated using lipid bilayers with DPPC/PG and fluorescent probes located either at the polar surface [1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) sodium anilino naphthalene sulfonate (ANS)] or within the hydrophobic core (DPH) of the liposome. Changes in fluorescence intensity and in polarization were observed when TMA-DPH or ANS, but not DPH were used. Comparative data also indicate that all three polymers interact only with the outer surface of the bilayer, but even the most marked penetration of polycationic polypeptide (SAK) did not result in alteration of the ordered state of the alkyl chains in the bilayer. Taken together, data obtained from mono- or bilayer experiments suggest that the interaction between branched polymers and phospholipids are highly dependent on the charge properties (Ser vs Glu) and on the identity (Ser vs Ala) of side-chain terminating amino acids. The binding of polymers to the model membranes could be mainly driven by electrostatic forces, but the significant role of hydrophilic properties in case of SAK cannot be excluded. © 1998 John Wiley & Sons, Inc. Biopoly 46: 169-179, 1998
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  • 70
    ISSN: 0006-3525
    Keywords: Cα,α-dialkylated glycines ; molecular dynamics ; geometry and conformation ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The relationship between the local backbone conformation and bond angles at Cα of symmetrically substituted Cα,α-dialkylated glycines (Cα,α-dimethylglycine or α-aminoisobutyric acid, Aib; Cα,α-diethylglycine, Deg; Cα,α-di-n-propylglycine, Dpg) has been investigated by molecular dynamics (MD) simulation adopting flat bottom harmonic potentials, instead of the usual harmonic restraints, for the Cα bond angles. The MD simulations show that the Cα bond angles are related to the local backbone conformation, irrespectively of the side-chain length of Aib, Deg, and Dpg residues. Moreover, the N-Cα-C′ (τ) angle is the most sensitive conformational parameter and, in the folded form, is always larger and more flexible than in the extended one. © 1998 John Wiley & Sons, Inc. Biopoly 46: 239-244, 1998
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  • 71
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    Biopolymers 46 (1998), S. 319-327 
    ISSN: 0006-3525
    Keywords: methionine ligation ; parathyroid hormones ; biomimetic ligation ; S-methylation ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319-327, 1998
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  • 72
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    Biopolymers 46 (1998), S. 359-373 
    ISSN: 0006-3525
    Keywords: boundary element method ; DNA electrophoresis ; electrophoretic mobility of DNA ; free solution electrophoretic mobility of DNA ; ion relaxation, DNA electrophoresis ; modeling electrophoresis of polyions ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Boundary element methods are used to model the free solution electrophoretic mobility of short DNA fragments. The Stern surfaces of the DNA fragments are modeled as plated cylinders that reproduce translational and rotational diffusion constants. The solvent-accessible and ion-accessible surfaces are taken to be coincident with the Stern surface. The mobilities are computed by solving simultaneously the coupled Navier-Stokes, Poisson, and ion-transport equations. The equilibrium electrostatics are treated at the level of the full Poisson-Boltzmann equation and ion relaxation is included. For polyions as highly charged as short DNA fragments, ion relaxation is substantial. At .11 M KCl, the simulated mobilities of a 20 base pair DNA fragment are in excellent agreement with experiment. At .04 M Tris acetate, pH = 8.0, the simulated mobilities are about 10-15% higher than experimental values and this discrepancy is attributed to the relatively large size of the Tris counterion. The length dependence of the mobility at .11 M KCl is also investigated. Earlier mobility studies on lysozyme are reexamined in view of the present findings. In addition to electrophoretic mobilities, the effective polyion charge measured in steady state electrophoresis and its relationship to the preferential interaction parameter γgG is briefly considered. © 1998 John Wiley & Sons, Inc. Biopoly 46: 359-373, 1998
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  • 73
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    Biopolymers 45 (1998), S. 341-346 
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: No abstract.
    Additional Material: 5 Ill.
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  • 74
    ISSN: 0006-3525
    Keywords: diffusional encounter ; Brownian dynamics ; average Boltzmann factor ; acetylcholinesterase ; Poisson-Boltzmann ; electrostatics ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The utility of the average Boltzmann factor around the active site of an enzyme as the predictor of the electrostatic enhancement of the substrate binding rate is tested on a set of data on wild-type acetylcholinesterase and 18 charge mutants recently obtained by Brownian dynamics simulations. A good correlation between the average Boltzmann factors and the substrate binding rate constants is found. The effects of single charge mutations on both the Boltzmann factor and the substrate binding rate constant are modest, i.e., 〈5 fold increase or decrease. This is consistent with the experimental results of Shafferman et al. but does not support their suggestion that the overall rate of the catalytic reaction is not limited by the diffusional encounter of acetylcholinesterase and its substrate. © 1998 John Wiley & Sons, Inc. Biopoly 45: 355-360, 1998
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  • 75
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    Biopolymers 45 (1998), S. 469-478 
    ISSN: 0006-3525
    Keywords: molecular dynamics ; hydrated proteins ; crystal structures ; density distributions ; globular proteins ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Using molecular dynamics simulations of fully hydrated proteins and analysis of crystal structures contained in the Protein Data Bank, we develop a transferable set of perpendicular radial distribution functions for water molecules around globular proteins. These universal functions may be used to reconstruct the unique three-dimensional solvent density distribution around every individual protein with a modest error. We discuss potential applications of this solvent treatment in protein x-ray crystallographic refinements and in theoretical modeling. We also present a fast, grid-based algorithm for construction of the perpendicular solvent density distributions. © 1998 John Wiley & Sons, Inc. Biopoly 45: 469-478, 1998
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  • 76
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    Biopolymers 47 (1998), S. 23-29 
    ISSN: 0006-3525
    Keywords: independently folded polypeptide motifs ; miniproteins ; natural target domains ; BBA motif ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In this paper we present a redesign strategy for the development of uniquely folded polypeptide motifs of less than 40 residues. These mini proteins are based on natural target domains, including the zinc finger domains (BBA motif) Nomenclature corresponds to the defined elements of secondary structure, beginning at the N-terminus of the peptide. Roman lettering refers to a specific motif while Greek characters correspond to the elements of secondary structure within that motif. and the disulfide-rich snake and scorpion toxins (BBB motif). These motifs are designed to act as the molecular framework for the construction of novel functional polypeptides. We will explore the structural determinants of the folded BBA motif, inspired by the zinc finger peptides, in relation to the redesign process. © 1998 John Wiley & Sons, Inc. Biopoly 47: 23-29, 1998
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  • 77
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    Biopolymers 47 (1998), S. 75-81 
    ISSN: 0006-3525
    Keywords: ion channel ; synthetic peptide ; de novo design ; template-assembled synthetic proteins ; supramolecular assembly ; membrane protein ; planer lipid bilayers ; amphiphilic peptide ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: To create ion channel function by synthetic peptides is a challenge in the de novo design of artificial membrane proteins. Amphiphilic α-helical motifs of ∼ 20 amino acid residues to span lipid bilayers are most often used for the creation of peptide ion channels. Template molecules to tether helical peptides have been employed to obtain more organized pore structures. Approaches to form molecular assembly of peptides in the membranes by hydrogen bonding have been also investigated. We have developed approaches to assemble helices with individual amino acid sequences to construct artificial helical proteins. Using one of these approaches, four helices corresponding to the voltage sensor segments (S4 in repeat I-IV) of the sodium channel were assembled on a peptide template to give a protein having ion channel activity with rectification. © 1998 John Wiley & Sons, Inc. Biopoly 47: 75-81, 1998
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  • 78
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    Biopolymers 47 (1998), S. 127-142 
    ISSN: 0006-3525
    Keywords: collagen mimetics ; triple helix ; peptoid ; template ; biophysics ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Collagen peptidomimetics have been synthesized as an alternative to natural collagen. The incorporation of unnatural residues such as peptoids in the collagen sequences can demonstrate potent and specific biological activity and enhance the biostability against enzymatic degradation. Furthermore, the use of achiral peptoids simplifies synthetic strategies by reducing racemization problems. The peptoid residue N-isobutylglycine (Nleu) has been successfully incorporated into a series of collagen mimetics composed of Gly-Pro-Nleu, Gly-Nleu-Pro, and Gly-Nleu-Nleu. The discovery of template-assembled collagen mimetics and metal binding ability has laid the foundation for new opportunities in the design of novel collagen mimetic complexes. This review summarizes the synthesis and integrated biophysical analyses of the structures of these collagen mimetics. Solid phase segment condensation techniques have been utilized for the synthesis of the single chain and template-assembled analogues. The characterization of the collagen-like structures has been established by temperature-dependent optical rotation measurements, CD, NMR spectroscopy, and molecular modelling simulations. © 1998 John Wiley & Sons, Inc. Biopoly 47: 127-142, 1998
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  • 79
    ISSN: 0006-3525
    Keywords: non-natural amino acid ; peptide ; squarylium dye ; thin film ; poly(3-methylthiophene) ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: We designed a polypeptide that behaves as a photodevice by using a non-natural amino acid with replacement of an α-hydrogen by a squarylium dye and succeeded in syntheses of the non-natural amino acid derivative containing a squarylium and its peptide with trialanine Ala-Ala-Ala. Strong dye-dye interactions were confirmed by absorption and CD spectra for the peptide in 2,2,2-trifluoroethanol solution and in water suspension. The non-natural amino acid derivative could be deposited onto a PMeT/Au electrode by the micelle disruption method. © 1998 John Wiley & Sons, Inc. Biopoly 47: 179-183, 1998
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  • 80
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    Biopolymers 47 (1998), S. 263-263 
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: No abstract.
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  • 81
    ISSN: 0006-3525
    Keywords: electrostatically driven Monte Carlo method ; cluster analysis ; global energy minimum ; perturbed conformations ; conformational space ; lowest energy conformations ; polypeptide chain ; melittin, membrane-bound portion ; Empirical Conformational Energy Program for Peptides 3 ; annealing methods ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The electrostatically driven Monte Carlo (EDMC) method has been greatly improved by adding a series of new features, including a procedure for cluster analysis of the accepted conformations. This information is used to guide the search for the global energy minimum. Alternative procedures for generating perturbed conformations to sample the conformational space were also included. These procedures enhance the efficiency of the method by generating a larger number of low-energy conformations.The improved EDMC method has been used to explore the conformational space of a 20-residue polypeptide chain whose sequence corresponds to the membrane-bound portion of melittin. The ECEPP/3 (Empirical Conformational Energy Program for Peptides) algorithm was used to describe the conformational energy of the chain. After an exhaustive search involving 14 independent runs, the lowest energy conformation (LEC) (-91.0 kcal/mol) of the entire study was encountered in four of the runs, while conformations higher in energy by no more than 1.8 kcal/mol were found in the remaining runs with the exception of one of them (run 8). The LEC is identical to the conformation found recently by J. Lee, H.A. Scheraga, and S. Rackovsky [(1998) “Conformational Analysis of the 20-Residue Membrane-Bound Portion of Melittin by Conformational Space Annealing,” Biopolymers, Vol. 46, pp. 103-115] as the lowest energy conformation obtained in their study using the conformational space annealing method. These results suggest that this conformation corresponds to the global energy minimum of the ECEPP/3 potential function for this specific sequence; it also appears to be the conformation of lowest free energy. © 1998 John Wiley & Sons, Inc. Biopoly 46: 117-126, 1998
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  • 82
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    Biopolymers 46 (1998), S. 195-200 
    ISSN: 0006-3525
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: No abstract.
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  • 83
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    Biopolymers 46 (1998), S. 201-214 
    ISSN: 0006-3525
    Keywords: band broadening ; dispersion ; DNA ; gels ; electrophoresis ; fluorescence recovery ; photobleaching ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: We determined quantitatively the band broadening effect during gel electrophoresis by measuring the longitudinal dispersion coefficient Dx, with a fluorescence recovery after photobleaching setup, coupled to an electrophoretic cell. We carried out measurements as a function of the electric field, the average pore size, and the molecular length of DNA fragments. Our results are in good agreement with the predictions of the biased reptation model with fluctuations described by T. A. Duke et al. [(1992) Physics Review Letters, vol. 69, pp. 3260-3263]. This agreement is observed on single-stranded DNA [persistence length ≅ 4 nm; B. Tinland et al. (1997) Macromolecules, vol. 30, pp. 5763-5765] in polyacrylamide gels and on double-stranded DNA (persistence length ≅ 50 nm) in agarose gels, two systems where the ratio between the average pore size and the Kuhn length is larger than 1. © 1998 John Wiley & Sons, Inc. Biopoly 46: 201-214, 1998
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  • 84
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    Biopolymers 46 (1998), S. 403-415 
    ISSN: 0006-3525
    Keywords: molecular dynamics ; DNA curvature ; DNA flexibility ; TATA box functionality ; TATA box binding protein (TBP) ; TBP recognition ; TBP binding ; TBP transcriptional activation ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Four 1.5 ns molecular dynamics (MD) simulations were performed on the d(GCTATAAAAGGG) · d(CCCTTTTATAGC) double helix dodecamer bearing the Adenovirus major late promoter TATA element and three iso-composition mutants for which physical and biochemical data are available from the same laboratory. Three of these DNA sequences experimentally induce tight binding with the TATA box binding protein (TBP) and induce high transcription rates; the other DNA sequence induces much lower TBP binding and transcription. The x-ray crystal structures have previously shown that the duplex DNA in DNA-TBP complexes are highly bent. We performed and analyzed MD simulations for these four DNAs, whose experimental structures are not available, in order to address the issue of whether inherent DNA structure and flexibility play a role in establishing these observed preferences. A comparison of the experimental and simulated results demonstrated that DNA duplex sequence-dependent curvature and flexibility play a significant role in TBP recognition, binding, and transcriptional activation. © 1998 John Wiley & Sons, Inc. Biopoly 46: 403-415, 1998
    Additional Material: 6 Ill.
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  • 85
    ISSN: 0006-3525
    Keywords: vibrational CD ; solution conformation ; alanine oligopeptides ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The solution conformation of a number of small, linear alanine oligomers was investigated via ir (or vibrational) CD (VCD). We find that these oligopeptides assume distinct solution conformations that depend primarily on chain lengths, and to a lesser degree on temperature, ionic strength, and pH. As expected, the longer chain oligomers exhibit more distinct VCD features and, presumably, more stable solution structures. At the level of the hexamer, however, aggregation of the peptide occurs. The fast time scale of VCD allows solution structures to be detected that may not be observable using slower techniques such as various forms of nmr spectroscopy. The VCD results reported here confirm that it is generally possible to obtain conformational information for small, linear homo- and heterooligopeptides via VCD spectroscopy. In this respect, the sensitivity of VCD is similar to that of electronic CD. Furthermore, the temperature dependence of the VCD results indicate that at elevated temperatures, the increasing number of conformational states results in a loss of discernible conformers, and consequently, a broadening and weakening of the VCD features. © 1998 John Wiley & Sons, Inc. Biopoly 46: 455-463, 1998
    Additional Material: 7 Ill.
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  • 86
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    Biopolymers 47 (1998), S. 5-22 
    ISSN: 0006-3525
    Keywords: hemoproteins ; model systems ; miniaturized proteins ; mimochromes ; helix structures ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The present paper highlights and reviews current research in the field of hemoprotein models. Hemoproteins have been extensively studied in order to understand structure-function relationships, and to design new molecules with desired functions. A wide number of synthetic analogues have been developed, using quite different approaches. They differ in molecular structures, ranging from simple meso-substituted tetraaryl-metalloporphyrins and peptide-porphyrin conjugates.In this paper we summarize the state of the art on peptide based hemoprotein models. We also report here the approach used by us to develop a new class of molecules, named mimochromes. They can be regarded as miniaturized hemoproteins, because mimochromes are low molecular weight compounds with some structural and functional properties common to those of the parent high molecular weight protein. The basic structure of mimochromes is a deuteroporphyrin ring covalently linked to two helical peptide chains. Two molecules of this series have been fully characterized. All the information derived from their structural analysis has been applied to the design of new analogues with additional functions. © 1998 John Wiley & Sons, Inc. Biopoly 47: 5-22, 1998
    Additional Material: 15 Ill.
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  • 87
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    Biopolymers 46 (1998), S. 503-516 
    ISSN: 0006-3525
    Keywords: discrete charge model of DNA ; dielectric cylinder in water ; effective dielectric constant ; salt effects ; Debye shielding factor ; potential variations in DNA surface ; Boltzmann averaged bending angles ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: We have studied electrostatic properties of DNA with a discrete charge model consisting of a cylindrical dielectric core with a radius of 8 Å and a dielectric constant Di = 4, surrounded by two helical strings of phosphate point charges at 10 Å from the axis, immersed in an aqueous medium with dielectric constant Dw = 78.54. Eliminating the dielectric core makes potentials in the phosphate surface less negative by about 0.5 kT/e. Salt effects are evaluated for the model without a dielectric core, using the shielded Coulomb potential. Smearing the phosphate charges increases their potential by about 2.5 kT/e, due mostly to the self-potential of the smeared charge. Potentials in the center of the minor and major grooves vary less than 0.02 kT/e along their helical path. The potential in the center of the minor groove is from 1.0 to 1.7 kT/e, more negative than in the center of the major groove, depending on dielectric core and salt concentration. So multivalent cations and also larger cationic ligands, such as some antibiotics, are likely to adsorb in the minor groove, in agreement with earlier computations by A. and B. Pullman. Dielectric effects on the surface potential and the local potential variations are found to be relatively small. Bending of DNA is studied by placing a multivalent cation, MZ+, in the center of the minor or major groove, curving DNA around it for a certain length, and calculating the free energy difference between the bent and the straight configuration. Boltzmann averaged bending angles, 〈β〉, are found to be maximal in 0.03M monovalent salt, for a length of about 50 or 25 Å of curved DNA when an MZ+ ion is adsorbed in the minor or the major groove, respectively. When the dielectric constant of water is used throughout the calculation, we find maximal bends of 〈β〉 = 11° for M2+ and 〈β〉 = 16° for M3+ in the minor groove, 〈β〉 = 13° for M3+ in the major groove. The absence of bends in DNA adsorbed to mica in the presence of Mg salts supports the role of Mg2+ in “ion bridging” between DNA and mica. The treatment of the effective dielectric constant between two points outside a dielectric cylinder in water is appended. © 1998 John Wiley & Sons, Inc. Biopoly 46: 503-516, 1998
    Additional Material: 14 Ill.
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  • 88
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    Biopolymers 47 (1998), S. 63-73 
    ISSN: 0006-3525
    Keywords: protein de novo design ; novel macromolecules ; topological templates ; Template Assembled Synthetic Proteins (TASP) ; biosensors ; protein folding ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The ultimate goal in protein de novo design is the creation of novel macromolecules with tailor-made receptor, sensory, and catalytic functions. Despite considerable progress in understanding basic rules of secondary structure formation and protein stability, the well-known protein folding problem is still far from being solved and, in general, only a limited number of designed proteins are folded uniquely. In this article the state-of-the-art in protein design is demonstrated on some selected examples, indicating that the construction of protein-like macromolecules mimicking some essential features of natural proteins seems to be within reach. Thus, protein design and mimicry has become an interdisciplinary challenge with most intriguing perspectives. © 1998 John Wiley & Sons, Inc. Biopoly 47: 63-73, 1998
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  • 89
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    Biopolymers 47 (1998), S. 451-463 
    ISSN: 0006-3525
    Keywords: bacteria ; antibiotics ; linear amphipathic α-helical antimicrobial peptides ; peptide-lipid interactions ; membrane permeation ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The increasing resistance of bacteria to conventional antibiotics resulted in a strong effort to develop antimicrobial compounds with new mechanisms of action. Antimicrobial peptides seem to be a promising solution to this problem. Many studies aimed at understanding their mode of action were described in the past few years. The most studied group includes the linear, mostly α-helical peptides. Although the exact mechanism by which they kill bacteria is not clearly understood, it has been shown that peptide-lipid interactions leading to membrane permeation play a role in their activity. Membrane permeation by amphipathic α-helical peptides can proceed via either one of the two mechanisms: (a) transmembrane pore formation via a “barrel-stave” mechanism; and (b) membrane destruction/solubilization via a “carpet-like” mechanism. The purpose of this review is to summarize recent studies aimed at understanding the mode of action of linear α-helical antimicrobial peptides. This review, which is focused on magainins, cecropins, and dermaseptins as representatives of the amphipathic α-helical antimicrobial peptides, supports the carpet-like rather the barrel-stave mechanism. That these peptides vary with regard to their length, amino acid composition, and net positive charge, but act via a common mechanism, may imply that other linear antimicrobial peptides that share the same properties also share the same mechanism. © 1999 John Wiley & Sons, Inc. Biopoly 47: 451-463, 1998
    Additional Material: 2 Ill.
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  • 90
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    Biopolymers 48 (1998), S. 39-55 
    ISSN: 0006-3525
    Keywords: modified nucleotides ; site-specific probes ; RNA structure ; RNA function ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Modified nucleotides can be incorporated site specifically into RNA by the use of total chemical synthesis as well as by use of a variety of recombinant RNA techniques. The range of nucleotide analogues includes modifications to base, sugar, and phosphate for structure-function analysis and for cross-linking studies as well as to answer specific mechanistic questions in RNA catalysis. We describe how RNA containing site-specific modifications are prepared, concentrating in particular on routes involving chemically synthesized oligoribonucleotides, and give examples of their application in studies of the hammerhead and hairpin ribozymes. © 1998 John Wiley & Sons, Inc. Biopoly 48: 39-55, 1998
    Additional Material: 9 Ill.
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  • 91
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    Biopolymers 48 (1998), S. 29-37 
    ISSN: 0006-3525
    Keywords: amide synthase ; catalytic antibodies ; Diels-Alderase ; ideal catalyst platform ; in vitro selection ; Lewis acid ; modified uridines ; modified RNA ; ribozymes ; SELEX ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In this manuscript the catalytic ability of RNA is examined and compared to other biopolymers. Despite having considerably fewer catalytically enabling properties when compared to proteins, the power of in vitro selection has allowed for RNA and DNA catalysts to be isolated. RNA catalysis has been expanded by incorporating modified bases to enrich the structural and functional diversity of RNA. Successful examples of new RNA chemistry using base modifications include carbon-carbon bond forming reactions and creation of highly specific active sites that are capable of recognizing small organic molecules without the need for nucleic acid templating or intercalation. In fact, the scope of functional modifications available for use in the RNA platform may eventually surpass those that are found in proteins and there are already hints that well chosen modifications allow nucleic acid catalysts to take advantage of mechanisms not available to selected protein catalysts for similar reactions. The chemical versatility of RNA is just emerging and future research directions will likely entail more creative methods for functional modification that will lead to new catalysts. © 1998 John Wiley & Sons, Inc. Biopoly 48: 29-37, 1998
    Additional Material: 6 Ill.
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  • 92
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    Biopolymers 48 (1998), S. 137-153 
    ISSN: 0006-3525
    Keywords: RNA ; pseudoknot ; Turnip Yellow Mosaic Virus ; Mouse Mammary Tumor Virus ; Beet Western Yellows Virus ; Simian Retrovirus type-1 ; Hepatitis Delta Virus ; translational frameshifting ; ribozyme ; nmr ; x-ray diffraction ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Recently, several high-resolution structures of RNA pseudoknots have become available. Here we review the progress in this area. The majority of the structures obtained belong to the classical or H-type pseudoknot family. The most complicated pseudoknot structure elucidated so far is the Hepatitis Delta Virus ribozyme, which forms a nested double pseudoknot. In particular, the structure-function relationships of the H-type pseudoknots involved in translational frameshifting have received much attention. All molecules considered show interesting new structural motifs. © 1999 John Wiley & Sons, Inc. Biopoly 48: 137-153, 1998
    Additional Material: 13 Ill.
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  • 93
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    Journal of Polymer Science Part A: Polymer Chemistry 36 (1998), S. 737-748 
    ISSN: 0887-624X
    Keywords: inverse microemulsion polymerization ; MADQUAT ; nucleation ; photopolymerization ; molecular weights ; flocculants ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The polymerization of inverse microemulsions of 2-methacryloyl oxyethyl trimethyl ammonium chloride stabilized by a blend of nonionic emulsifiers (a sorbitan sesquioleate and a sorbitan monooleate) and initiated by UV light in the presence of Azobis(isobutyronitrile) (AIBN) was investigated. The effect of initiator concentration, light intensity, emulsifier concentration, and dispersed phase weight fraction on the polymerization rate (Rp), number of polymer particles (Np), and polymer molecular weight (Mw) was studied. The application of this process to tubular reactors is discussed. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 737-748, 1998
    Additional Material: 15 Ill.
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  • 94
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    Journal of Polymer Science Part A: Polymer Chemistry 36 (1998), S. 1217-1226 
    ISSN: 0887-624X
    Keywords: polypropylene ; pyrene ; two-solute system ; pulse radiolysis ; solute ionic species ; solute excited states ; low-temperature radiolysis ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A pulse radiolysis study of isotactic polypropylene (PP) film has been carried out with the main aims of investigating charge trapping in an undoped system and solute radical ion generation in an pyrene (Py) doped matrix. In PP, pulse radiolysis gives electron-positive hole pairs. The electron can be stabilized in the undoped system as a trapped electron, et-. The transient absorption spectrum of et- in the near-IR (up to 1800 nm) was observed in the temperature range 30-100 K. This IR absorption was not detected in the case of oxidized PP. In such a matrix electrons can be scavenged by oxidation products generating respective radical-anions (absorption in the UV RANGE, λ 〈 350 nm). In a doped matrix transient absorption bands centered at 450 and 500 nm were observed which can be assigned to the Py radical cation and anion, respectively. The recombination of these ionic species leads to monomer excited-state formation observed during and after the 17 ns pulse. Contrary to the Py-doped polyethylene no excimer emission was detected at room temperature even if Py content in PP was close to 0.02 mol dm-3. The rate of Py radical-ion decay was found to be temperature dependent. Two linear parts of the Arrhenius plot were observed which intersected at ca. 240 K, the glass transition temperature, Tg, for PP. The activation energies calculated for two parts of Arrhenius plot were equal to 111 and ca. 0.78 kJ mol-1 for T 〉 Tg and T 〈 Tg, respectively. Some preliminary results concerning the ionic processes in PP containing two solutes (Py, 3,3′-dimethyldiphenyl) were presented. The mechanism of ionic recombination in PP will be proposed and discussed. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 1217-1226, 1998
    Additional Material: 7 Ill.
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  • 95
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    Bognor Regis [u.a.] : Wiley-Blackwell
    Journal of Polymer Science Part A: Polymer Chemistry 36 (1998), S. 1345-1348 
    ISSN: 0887-624X
    Keywords: acid functional ; telechelic ; benzyl alcohol ; poly(ε-caprolactone) ; polymerization ; living ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Additional Material: 2 Ill.
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  • 96
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    Journal of Polymer Science Part A: Polymer Chemistry 36 (1998), S. 883-888 
    ISSN: 0887-624X
    Keywords: temperature sensitive ; particle ; emulsion polymerization ; morphology ; adsorption ; enzyme ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Two kinds of temperature-sensitive composite polymer particles were prepared by seeded emulsion copolymerizations of (dimethylamino)ethyl methacrylate and ethylene glycol dimethacrylate with 0.14 μm-sized polystyrene and 0.26 μm-sized poly(methylmethacrylate) seed particles. To evaluate the usefulness as a carrier for biomolecules, the enzymatic activities of trypsin adsorbed on these two composite polymer particles were measured at temperatures above and below each lower critical solution temperature (LCST). In both cases, adsorbed trypsin retained its enzymatic activity during repeated adsorption/desorption measurements. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 883-888, 1998
    Additional Material: 7 Ill.
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  • 97
    ISSN: 0887-624X
    Keywords: (1→6)-2,5-anhydro-D-glucitol ; HPLC ; optical resolution ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The cyclopolymerization of 3,4-di-O-allyl-1,2 : 5,6-dianhydro-D-mannitol (1) was carried out using BF3·OEt2 and t-BuOK. The polymer obtained by the polymerization with BF3·OEt2 mainly consisted of (1→6)-bonded 3,4-di-O-allyl-2,5-anhydro-D-glucitol as the five-membered constitutional repeating unit, though it contained a small amount of other cyclic repeating units. On the other hand, during the polymerization using t-BuOK, the stereoregular polymer (1→6)-linked 3,4-di-O-allyl-2,5-anhydro-D-glucitol (2) was synthesized via a regio- and stereoselective mechanism. Cleavage of the allyl ether linkage in polymer 2 occurred to produce the polymer consisting of only 2,5-anhydro-D-glucitol units, i.e., (1→6)-2,5-anhydro-D-glucitol (3). Chromatographic enantioseparation of chloroquine and tröger base has been performed on (3,5-dimethylphenyl)carbamate and 4-methylbenzoate derivatives of 3 as a chiral stationary phase for high-performance liquid chromatography. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 901-909, 1998
    Additional Material: 4 Ill.
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  • 98
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    Journal of Polymer Science Part A: Polymer Chemistry 36 (1998), S. 939-947 
    ISSN: 0887-624X
    Keywords: fluorine ; perfluoropolyether ; polyester ; segmented ; thermoplastic ; interfacial synthesis ; thermal transitions ; dynamic-mechanical properties ; chemical resistance ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Linear segmented polyesters containing soft perfluoropolyether (PFPE) and hard aromatic segments are obtained by an interfacial polycondensation reaction of an acyl chloride-ended fluorinated prepolymer with various aromatic diphenols in the presence of phase transfer catalysts (PTC) as accelerators. Experimental conditions for reaching high molecular weights are discussed. The calorimetric analysis (DSC) of all the polyesters synthesized shows a typical biphasic morphology, where a very low Tg (〈 -110°C) corresponding to the segregated PFPE moiety, is always accompanied by another Tg or a higher melting temperature, depending on the nature of the hard phase. Dynamic-mechanical analysis (DMA) has been carried out confirming the DSC results and suggesting diversified mechanical behaviors at the various temperatures in line with the amorphous or semicrystalline nature of the polymer. Chemical resistance was finally tested by dipping in several solvents and chemicals. The new polyesters show high contact angles, a moderate swelling in many organic solvents and excellent stability in aggressive hydrolytic environments. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 939-947, 1998
    Additional Material: 6 Ill.
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  • 99
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    Bognor Regis [u.a.] : Wiley-Blackwell
    Journal of Polymer Science Part A: Polymer Chemistry 36 (1998), S. 1919-1928 
    ISSN: 0887-624X
    Keywords: emulsion polymerization ; vinylidene chloride ; kinetics ; sodium lauryl sulfate ; potassium persulfate ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Emulsion polymerization of vinylidene chloride was carried out at 50°C using sodium lauryl sulfate as emulsifier and potassium persulfate as initiator, respectively. Contrary to the results so far reported, the stirring rate did not affect the progress of the polymerization and such an abnormal kinetic behavior as the rate of polymerization suddenly drops in the course of polymerization was not observed. The number of polymer particles produced was proportional to the 0.7 power of the concentration of emulsifier forming micelles and to the 0.3 power of the initial initiator concentration, respectively, and was independent of the initial monomer concentration. The rate of polymerization was in proportion to the 0.3 power of the concentration of emulsifier forming micelles, to the 0.5 power of the initial initiator concentration, to the 0.2 power of the initial monomer concentration, and to the 0.45 power of the number of polymer particles, respectively. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 1919-1928, 1998
    Additional Material: 13 Ill.
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  • 100
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    Bognor Regis [u.a.] : Wiley-Blackwell
    Journal of Polymer Science Part A: Polymer Chemistry 36 (1998), S. 1961-1964 
    ISSN: 0887-624X
    Keywords: Poly(4,4′-phenylene-3,3′,4,4′-biphenyltetracarboxyimide) ; poly(4,4′-oxydiphenylenepyromellitimide) ; precursor solution ; aprotic polar solvent ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: No abstract.
    Additional Material: 4 Ill.
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