Bibliothek

Notizen: Abstract  An ultrastructural and autoradiographic analysis of changes in 188 biopsy specimens of bronchial mucosa of the large bronchi from 76 patients with chronic inflammatory lung diseses was carried out. Fibrosis results in an apparent reduction of metabolic activity in endothelial cells, affecting the proliferation of basal cells with changes in cell differentiation. Endobronchial laser therapy with an helium-neon laser induces proliferative and metabolic processes in the lamina propria of the bronchial mucosa with hyperaemia, intensive diapedesis of leucocytes and formation of leucocytic infiltrations and granulation tissue. The proliferative and metabolic activity of endothelial and stromal cells increases, and delicate fibrous connective tissue is formed.

Notizen: Abstract Magnesium ions added to fixatives for processing to Transmission Electron Microscopy (TEM) have been claimed to cause relaxation of detrusor smooth muscle cells [1]. This should facilitate the morphologic evaluation of the tissue. However, magnesium ions are osmotically active and their addition may cause the fixative to become hypertonic to the tissue. To ascertain whether the presence of magnesium ions causes significant changes compared to those found where the osmolarity is raised without the presence of magnesium, human detrusor specimens were fixed in glutaraldehyde to which increasing amounts of MgCl2 or NaCl were added in different concentrations. With the addition of increasing amounts of MgCl2 and NaCl, the osmolarity of the fixative increased, causing significant changes in the morphology and morphometry of the tissue. The intercellular distances increased, the cells shrank and the shape of the cells changed from smooth and rounded to spiky and angulated. With regard to its muscle-relaxing effect, it was not possible to distinguish the specimens fixed in magnesium-containing fixatives from those without. In this study it was not possible to prove any relaxing effect of magnesium ions added to the fixative. On the contrary the magnesium ions caused an increase in the osmolarity, with significant changes in both the morphometry and the morphology of the human detrusor smooth muscle cells.

Notizen: Abstract We report the presence of round eosinophilic intranuclear inclusions in a patient with sporadic amyotrophic lateral sclerosis (ALS). The inclusions were limited to the hippocampal pyramidal neurons; they were frequently encountered in the CA1 and CA2 regions and much less frequently in the CA3 and CA4 regions and in the subiculum. Ultrastructurally, they consisted of randomly oriented straight filaments, each about 8–14 nm in diameter, some of which had a tubular appearance in cross-section. Electron-dense, granular material was intermingled with the filaments. Immunohistochemically, all the inclusions were positive for ubiquitin, but were negative for several kinds of cytoskeletal protein, including actin, glial fibrillary acidic protein, vimentin, neurofilament polypeptides, keratin, tubulin, tau protein and microtubule-associated protein 2. To our knowledge, this type of neuronal intranuclear inclusion has not so far been reported in ALS, and its distribution limited to the hippocampal formation is of great interest.

Notizen: Abstract Spectrin, a major component of the erythrocyte membrane skeleton, has previously been shown to form a two-dimensional lattice in erythrocytes, and in avian or chicken skeletal muscle. Those results were mainly obtained with antibodies against α-spectrin. Using immunofluorescence of semithin cryosections and single muscle fiber preparations, we show here that β-spectrin forms a costameric network which covers the plasma membrane of human skeletal muscle. These spectrin costameres are correlated with the Z-bands. They are longitudinally connected by fine strands and interrupted by myonuclear lacunae. Under mechanical stretching, the costameres retained their correlation to the Z-bands in normal and dystrophin-deficient muscle, up to the point at which the sarcolemma was disrupted. In stretched muscle, in some regions of the stretched fibers in which the costameres seemed to form double strands, the usually 1:1 correlation of spectrin to the Z-bands changed to a 2:1 relation. In dystrophin-deficient muscle, the costameric scaffold of spectrin in the well-preserved fibers appeared normal, indicating that spectrin can be correctly localized in the absence of dystrophin and that the subcellular spectrin organization does not primarily depend on dystrophin expression. The regular organization and the correlation of spectrin costameres to the Z-bands was notable even in stretched Duchenne muscular dystrophy (DMD) muscle. On the other hand, single teased muscle fibers of DMD muscle showed various degrees of morphological alterations of the costameric network, ranging from a focal disarray to complete loss of costameric organization. Because these findings indicate that the costameric spectrin scaffold undergoes secondary changes during the course of the dystrophic process in dystrophin-deficient muscle, spectrin staining of isolated muscle fibers may also serve as a tool to monitor the effect of gene therapy experiments at the single fiber level.

Notizen: Abstract The reinnervation pattern of crural pacinian corpuscles was examined by light and electron microscopy in eight adult cats of both sexes 3–18 months after sciatic nerve crush. Normal pacinian corpuscles are each supplied with a single myelinated axon and a single cylindrical axon terminal which may branch in the distal part of the inner core. Reinnervation of these vibroreceptors was very satisfactory after sciatic nerve crush: in a sample of 68 corpuscles examined 3–18 months after the operation, 92.6% were found reinnervated, while only 7.4% remained denervated. At the nerve entry, 84.2% of the reinnervated corpuscles were supplied with a single myelinated axon, while 15.8% received two myelinated axons; some of the axons branched before or after entering the inner core. Near the mid-level of the inner core, 60.3% of 63 reinnervated corpuscles were innervated with a single axon terminal, 22.2% were biterminal, while 17.5% had three or more terminals. Regenerated axon terminals induced the formation of thin lamellar layers in the axial region of the original core and, exceptionally, also at the outer aspect of the original core. In monoterminal corpuscles, the shape and ultrastructure of regenerated endings resembled those of normal controls, whereas in multiterminal corpuscles their shape and profiles were variable. In contrast to previous reports, reinnervated corpuscles did not ultimately become monoterminal. On the contrary, the mean number of 1.3 terminals found in reinnervated crural corpuscles at 3–5 months increased to 1.9 terminals per corpuscle 6–18 months after axotomy.

Notizen: Abstract Traumatic brain injury in animals and humans is well known to cause axonal damage diffusely scattered throughout the brain without evidence of other brain parenchymal change. This observation has prompted some to posit that such damaged axons are well positioned to mount a regenerative attempt. The present study uses an immunocytochemical marker specific for regenerating neurites to explore this issue. Further, in an attempt to expedite and enhance any potential regenerative effort, this study evaluates the efficacy of intrathecally applied nerve growth factor. Three sets of experiments were performed in adult cats. One group of animals was subjected to moderate fluid percussion brain injury and followed for 7 or 14 days post injury, with the continuous intraventricular infusion of nerve growth factor delivered by implanted osmotic pumps. These animals were compared to a second group of time-matched, sham-operated animals receiving artificial cerebrospinal fluid infusion. To assess axonal damage immunohistochemical staining for the low molecular weight neurofilament subunit (NF-L) was carried out using an NR4 monoclonal antibody. To localize axons exhibiting a regenerative response immunohistochemical staining for the growth associated protein GAP43 was employed. In sham controls, at the light microscopic level NF-L-immunoreactive axonal swellings were numerous at 7 days, but by 14 days post injury their frequency declined markedly. In contrast, GAP43-immunoreactive, disconnected reactive axonal swellings were rarely observed at 7 days but were numerous at 14 days. Ultrastructural analysis at 14 days post injury of carefully matched sections revealed reactive axons demonstrating sprouting consistent with a regenerative effort. Analysis of tissue from animals of 14 days of survival indicated that supplementation with nerve growth factor did not appear to enhance the capacity of damaged brain axons to mount a regenerative attempt. Rather, it appears that regenerative efforts seen reflect a spontaneous response. A third group of adult cats, subjected to the same injury but not subjected to osmotic pump implantation, was allowed to survive for 22–28 days. Animals in this group also demonstrated GAP43 immunoreactivity in reactive axonal swellings in the brain stem. This study demonstrates that diffusely injured axons can mount a sustained regenerative attempt that is associated with a reorganization of their cytoskeleton and accompanied by an up-regulation of growth-associated proteins.

Notizen: Abstract  Human natural killer (NK) cells contain unique granules with parallel tubular arrays (PTA granules) of approximately 30 nm diameter that can be seen only by electron microscopy. In order to clarify the role of PTA granules in NK cell-mediated cytolysis we examined these structures with regard to frequency and expression of lytic proteins (perforin, granzymes). NK cells (CD3−, CD16+, CD56+) were obtained from heparinized blood of healthy donors and enriched by double-step negative selection using mAb coupled to magnetic beads. PTA granules were found in 31.3% of freshly separated NK cells. When NK cells were cultivated, even in the presence of various stimulating agents (rhIL-2, rhIL-4, rhIL-6, rhIL-12, GM-CSF, rhIFNα, anti-CD16 mAb, dexamethasone), PTA granules disappeared and transformed into conventional primary lysosomes. By immune electron microscopy using antibodies directed against perforin and granzyme B we observed distinct immuno-reactivity in the tubules and in the tubule-associated faintly electron-dense matrix of PTA granules. Immuno-labelling for perforin and granzyme B was also found in the fine granular matrix of primary lysosomes. Finally, we tested the distribution of chondroitin 4-sulfate which is suggested to inactivate lytic proteins. Immuno-reactivity for chondroitin 4-sulfate was detected only in the moderately electron-dense matrix but not in the tubules of PTA granules. These observations indicate that perforin and granzyme B are stored in an inactive form in PTA tubules due to highly ordered paracrystalline protein folding. As soon as the tubules decay the lytic proteins are kept in an environment of chondroitin 4-sulfate for inactivation.

Notizen: Abstract Intracytoplasmic inclusion bodies are rarely found in meningiomas. A 74-year-old woman had an anaplastic meningioma with intracytoplasmic chromophobe inclusion bodies (CIB) histologically. These CIB were various shapes, e.g. round, teardrop-like, fusiform, horseshoe-like, crescentic and perinuclear. The size of CIB ranged from 7 to 14 μm and the nuclei of the tumor cells with CIB were often eccentric. Most CIB were immunopositive only for vimentin, staining more intensely than surrounding cytoplasm in a comparative study using adjacent sections stained with hematoxylin-eosin and vimentin. CIB showed loosely textured filamentous structures which were in parallel and entangled arrangements ultrastructurally. The diameter of the filaments was 13–14 nm and they were thicker than normal intermediate filaments. Moreover, these filaments appeared to be studded with granular and fuzzy substances. These findings suggest that CIB are mainly composed of abnormally synthesized and arranged vimentin filaments.

Notizen: Abstract Prolonged hypoxia results in structural and functional adaptive responses to improve tissue oxygen delivery. Structural changes within the brain include vascular proliferation and elongation. The aim of the current study was to investigate whether ultrastructural changes in capillary walls also occur as part of the adaptive response. Adult rats were exposed to 2 or 3 weeks of moderate hypobaric hypoxia at 0.5 atmospheres and their cerebral microvasculature examined using quantitative ultrastructural methods. We found that hypoxic rats had an 18% increase in their brain capillary diameter but no change in endothelial wall thickness, basement membrane thickness, or coverage of the endothelial wall by pericytes. The increased diameter of cerebral capillaries may play an important role in decreasing the resistance to capillary perfusion which is brought about by the increased erythrocyte fraction in the blood of hypoxic rats. Ultrastructural features relevant to the blood-brain barrier were maintained in hypoxic rats. Pericytes, that are thought to form a second line of defense in the blood-brain barrier, maintained their numerical and size relationships to the endothelial cells. Endothelial junctions were unchanged and endothelial vesicles were somewhat lower in density than normal at 2 weeks of hypoxia, but had regained their normal density by 3 weeks. Mitochondria of the brain capillary endothelial cells maintained normal numerical and volume densities in hypoxia, but the mitochondria of the surrounding neuropil were decreased significantly by about 30%.

Notizen: Abstract To clarify the histogenesis and differentiation potential of central neurocytoma, a pathological investigation of seven tumors from three patients was conducted using immunohistochemistry and ultrastructural analysis in addition to systematic in vitro studies. Six tumors were studied immunohistochemically and five were examined ultrastructurally. All cases that were immunostained were positive for synaptophysin in nuclear-free neuropil islands. In five tumors, a few tumor cells, in addition to reactive astrocytes, were positive for glial fibrillary acidic protein (GFAP). Vimentin staining was also positive in a few tumor cells of five specimens. Neurofilament staining was always negative. All cases for which ultrastructure was examined showed various synaptic abnormalities. Cultured cells were subdivided into three distinct tumor cell types: neuronal cells which stained for neurofilament proteins with neurosecretory granules; small flat undifferentiated cells with a high nuclear-cytoplasmic ratio and scant cytoplasmic organelles; and small round or multipolar astrocytic cells with 10-nm intermediate filaments which stained for GFAP. Our tissue culture studies disclosed that cultured neurocytoma cells form a cellular mosaic similar to subependymal plate layers that are composed of mitotically active cells, neurons and glia.

Notizen: Abstract Mucopolysaccharidosis IIID (MPS-IIID) is the rarest of the MPS-III syndromes. It is caused by deficient activity of lysosomal N-acetylglucosamine-6-sulfatase (G6S). To date, the clinical and biochemical features of seven patients with MPS-IIID have been reported, but no biopsy or autopsy findings have been described. The purpose of this report is to define the ultrastructure of affected cells seen in a skin biopsy from a 14-year-old boy. The child presented with progressive mental deterioration, hyperactivity and mild to moderate dysmorphism. The diagnosis of a mucopolysaccharidosis was suggested, but the initial urine analyses were negative for elevated mucopolysaccharides, and only the third analysis showed abnormal excretion of heparan sulfate. Because of the diagnostic difficulties posed by this case, a skin biopsy was performed for morphological and biochemical studies. Numerous vacuoles were noted in Schwann cells, fibroblasts, smooth muscle cells, eccrine gland and ductal epithelium in resin-embedded sections stained with toluidine blue. Ultrastructurally, many lysosomes were distended with abundant, fibrillar material. Occasionally, lamellated membranous structures were present within the same lysosomes. These findings are consistent with those seen in other forms of MPS, in which the lysosomal storage occurs predominantly, but not exclusively, in mesenchymal cells. Furthermore, deficient activity of G6S was confirmed in cultured skin fibroblasts. This study demonstrates that electron microscopy of skin biopsies is a useful method for identification of patients with clinical features of MPS-IIID whether or not heparan sulfaturia is present.

Notizen: Abstract We studied a recurrent meningioma located in the right frontal lobe. The tumor showed high cellularity and the cells had plump, hyalinous cytoplasm. Immunohistochemically, almost all the tumor cells were positive for epithelial membrane antigen and vimentin, and unexpectedly, glial fibrillary acidic protein (GFAP). Ultrastructural investigation revealed abundant 8- to 10-nm filaments in the cytoplasm. Conspicuous interdigitations with numerous desmosomes were present. Frequently, intracellular and intercellular lumina lined by microvilli were also found. We considered the present case to be an unusual variant of meningioma with GFAP expression. A few cases of meningioma with triple expression of GFAP, vimentin and cytokeratin have been reported previously. However, the present case showed obvious pathological differences from these, and had no immunoreactivity for cytokeratin.

Notizen: Abstract The locus coeruleus (LC) is a major noradrenergic nucleus in the brain. The activity of the LC neurons is chronically regulated by opioids. So far, very little is known about the morphological changes induced by chronic treatment with opioids. In the present study, the effects of chronic treatment with morphine and dihydroetorphine, a new narcotic analgesic with lower physical dependence potential than morphine, were investigated on the ultrastructure of the rat LC. Rats received saline or increasing doses of morphine or dihydroetorphine for 5 days by twice daily subcutaneous injections. Withdrawal was precipitated in half of the opioid-treated rats by a single intraperitoneal injection of naloxone 4 h after the last injections of opioids. The ultrastructure of the LC was examined by electron microscopy. Results showed that chronic morphine treatment induced a marked injury to the LC neurons. The primary changes in the cell body were the indentation of nuclei, the fragmentation and degranulation of rough endoplasmic reticulum, as well as the disaggregation of polyribosomes. Myelinoid bodies were seen in the processes. An accumulation of presynaptic vesicles was observed in some of the terminals which formed synaptic junctions with the LC neurons as compared to the normal controls. Naloxone-precipitated withdrawal from morphine did not stop the morphine-induced injury on the LC neurons except that less accumulation of presynaptic vesicles occurred. Chronic dihydroetorphine treatment only induced a slight change in the ultrastructure of the LC neurons. These results indicate that the LC neurons are more vulnerable to chronic treatment with morphine than to that with dihydroetorphine.

Notizen: Abstract This report concerns a comparative immunocytochemical and ultrastructural investigation on pericapillary rosettes (PR) in the lumbar spinal cords of 21 patients with amyotrophic lateral sclerosis (ALS) and 18 age-matched neurologically normal individuals. The purpose of the study was to determine the alteration of PR in relation to the neuronal loss in ALS. The PR were almost always positively immunostained for phosphorylated neurofilament, and some PR immunoreacted with antibodies to synaptophysin and β-amyloid precursor protein. This finding suggests that axonal transport, whether fast or slow, is impaired in the terminal portion of the axon that reaches the capillaries. Some PR were also positively immunostained by the antibody against ubiquitin, anti-calbindin-D 28 K antibody, anti-parvalbumin antibody and the antibody to superoxide dismutase 1. Morphometrically, the number of PR in the anterior horns and lateral column was markedly diminished in ALS compared with controls. At the ultrastructural level, the PR consisted mostly of unmyelinated degenerated axons, and were frequently found outside the basal laminae of the endothelial cell and of the astrocytic foot processes on the opposite side of the capillary, and less often in the space between the two basal laminae. The data indicate that the fate of PR is intimately associated with the neuronal loss of the anterior horn cells and with degenerative change of nerve fibers extending from their mother neurons to the capillaries.

Notizen: Abstract  The purpose of this study was to compare the biomineralization of circumpulpal dentine with that of mantle dentine by ultrastructural and element-analytical techniques. Forty upper second molar germs of 10-day-old albino rats were cryofixed in liquid nitrogen-cooled propane and embedded in resin after freeze drying. Semithin dry sections were cut for analyzing the calcium and phosphorus concentration in initial mantle dentine, at the mineralization front of circumpulpal dentine, in the middle region of circumpulpal dentine and in mantle dentine peripheral to circumpulpal dentine. For the morphological evaluation of mineral deposits we compared ultrathin and unstained sections of cryofixed molars with chemically fixed molars. For both dentine types it was found that they develop via identical steps of mineral formation at collagen fibrils and non-collagenous matrix molecules. In circumpulpal dentine no globular mineral protrusions along the mineralization front (i.e. calcospherites) and no indications of interglobular dentine at the transition from circumpulpal dentine to mantle dentine were present. The von Korff fibres were not only visible in mantle dentine but also in circumpulpal dentine. Matrix vesicles were present only during the formation of an initial coherent layer of mantle dentine and could not be observed during successive formation of mantle dentine and circumpulpal dentine. The element-analytical data did not demonstrate any difference in the mineral content between the two dentine types. Therefore, we conclude that mantle dentine and circumpulpal dentine in the rat molar possess a high degree of structural and chemical similarity and that only the extent of terminal branching of the odontoblast processes gives an approximate estimation of the thickness of mantle dentine.

Notizen: Abstract  Herbst corpuscles were studied in the crural region of perinatal and adult chicken and quail in order to find out their number and dimensions and to learn more about their structure, especially in relation to size. Crural corpuscles are arrayed in an encapsulated string between tibia and fibula. They are closely packed together; a small number of corpuscles is found apart from the string, often attached to the periost. The strings of corpuscles are approximately 40 mm long in adult chicken and 20 mm long in the quail. The crural region of the chicken contains 382.8 ± 90.9 (mean ± SD) corpuscles, the numbers ranging from 301 to 582; in the quail, the mean number is 119.2 ± 27.9, with a range from 83 to 167 corpuscles. In the chicken, one axon supplies an average of 1.60 corpuscles; in the quail, the relation of axons to corpuscles is approximately 0.92. In both species, final numbers of crural corpuscles are already attained before hatching and no difference is found in the mean number and range of corpuscles between perinatal and adult birds. In both chicken and quail, individual strings contain corpuscles of various sizes, from large to very small. The chicken corpuscles are generally twice as large in diameter and often longer than those of the quail. The corpuscles are composed of an axon terminal that projects two rows of axonal spines into the clefts of the inner core and ends with an ultraterminal bulb; the terminal is surrounded with a bilaterally symmetrical inner core, amorphous inner space containing collagen fibrils of various thickness, and a capsule. Large chicken corpuscles contain inner cores composed of up to 100 lamellae, while quail inner cores have half that number at the most. The capsules are usually composed of 8 to 10 lamellar layers in both species, but they are thicker in the chicken than in the quail. The possible functional significance of individual structural components of Herbst corpuscles is discussed.

Notizen: Abstract Electron microscopy combined with morphometry was used to establish values for 24 parameters in normal skin. These results were compared with those similarly obtained from samples of epidermolysis bullosa with a view to facilitating classification of the disease. Six of the eight subtypes of epidermolysis bullosa investigated could be differentiated. Four subtypes showed values for structural components in intact skin which were outside the normal range: (1) epidermolysis bullosa simplex generalisata gravis (hemidesmosomes); (2) epidermolysis bullosa dystrophica Pasini and (3) Cockayne-Touraine (anchoring fibrils); and (4) epidermolysis bullosa acquisita (anchoring fibrils, hemidesmosomes, and lamina lucida and lamina densa aspects of the dermoepidermal junction). Two subtypes revealed specific features which could be assessed qualitatively: distinctive, circumscribed, clumped tonofilament bodies were present in basal keratinocytes from epidermolysis bullosa herpetiformis Dowling-Meara and thick (30 nm diameter) cross-striated anchoring fibrils were absent in epidermolysis bullosa dystrophica generalisata gravis. Epidermolysis bullosa simplex Köbner and Weber-Cockayne forms could not be distinguished.

Notizen: Abstract  The effect of waxy corn starch (WCS) on the texture, water-holding capacity and microstructure of sardine (Sardina pilchardus) surimi gels in two different systems was studied. In the type A system, increasing amounts of WCS (2, 4, 6 or 8 g/100 g surimi) were added to surimi while maintaining the gel moisture constant at 78%; in the type B system, WCS was added without correcting the gel moisture. Gels were made using two different heat treatments [heat-induced setting (HS) and direct cooking (DC)]. When starch was replaced by surimi (type A) and a heat treatment was applied that favoured formation of a preliminary actomyosin (AM) network (i.e. HS), gel strength (GS) was lower than in the control and decreased as more starch was added, despite an increase in the amount of water held by the gel. Scanning electron microscopy (SEM) showed that the matrix network was fibrillar with a globular surface. Starch appeared to be totally gelatinized and surrounded by a continuous matrix. When the amount of dry matter in gels was increased (type B), in no case did starch have a reinforcing effect, despite an increasing water-holding capacity; SEM showed a denser continuous matrix surrounding the gelatinized starch. Both types of gel made using the heat treatment that allows simultaneous gelling of surimi and gelatinization of starch (i.e. DC) exhibited much poorer GS than did HS gels, while addition of starch made practically no difference to gel texture. The findings suggest that the effect of starch is related to the type of gel matrix that forms upon addition of ingredients. Although such gels contained more water or dry matter, their texture parameters were lower, possibly because of the type of network formed by sardine surimi. Nonetheless, gels of acceptable quality were successfully made with added starch by incorporating less surimi.

Notizen: Abstract  Asplenium trichomanes L. subsp. trichomanes spermatozoids are spirals of about five turns. Keels link the elements of the microtubular ribbon with the plates of the lamellar layer (LL) which are uninterrupted, parallel and curved with an inner angle of about 150°. Electron-opaque filaments connect the microtubules of the multilayered structure (MLS) and the osmiophilic crest, the LL and the MLS-associated mitochondrion and the latter and the plasmalemma. The nucleus occupies the 2.5–3 posterior turns and has an inner honeycomb-shaped chromatin mass and an outer highly condensed chromatin mass with randomly scattered electron-transparent areas. The basal bodies of the ca. 50 flagella are bounded by a reticulum of granular material which forms a plug inside their proximal region; the proximal region of the flagellum has a 9 + 0 pattern. The axoneme has a 9 + 2 pattern.

Notizen: Abstract  A protocol for isolating viable eggs in Plumbago zeylanica by mechanical dissection is reported. The optimum solution for isolation was 0.8 M mannitol + 10 mM MOPS + 10 mM CaCl2, (pH 4.5–5.0) with an osmolality of 860–940 mmol/kg. Eggs retain their viability for at least 24 h. Isolated eggs were true protoplasts without cell walls and could tolerate osmolality of 437 mmol/kg to 965 mmol/kg. Observation of the isolated eggs using transmission electron microscopy indicated that they were well preserved and reflected the ultrastructure of physiologically active cells, displaying features similar to those of in vivo egg cells. Notable differences include the absence of a filiform apparatus and the accumulation of dense particles in the plastids, which was most conspicuous in egg cells that were damaged during isolation.

Notizen: Abstract Female Wistar rats were treated orally for 5 days with 80 mg/kg body weight of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), a microsomal Ca2+ ATPase inhibitor. Motor endplates of the lumbrical muscles were examined by light and electron microscopy. There was a decrease in body weight in the treated rats from the first day after administration, and toxic signs appeared after the third day, such as adoption of a prone position, salivation, lacrymation, and an abnormal gait and/or muscle weakness. No remarkable macroscopic or light microscopic changes were noted in the lumbrical muscles as well as other peripheral nerves of hind legs of the treated rats killed 1 day after the last DTBHQ treatment. Ultrastructurally, neurotoxicity characterized by loss of synaptic vesicles and mitochondria in the motor endplates, and by destruction of the motor terminals was detected in the lumbrical muscles of the treated rats. These results strongly indicate that DTBHQ targets the motor endplates in the rat lumbrical muscles and suggest that the resultant damage is responsible for the appearance of neurological signs, such as an abnormal gait and loss of muscle control.

Notizen: Abstract  Human herpesvirus 7 (HHV-7) was grown in a CD4+ lymphoblastic cell line (SupT1) and in cord blood mononuclear cells (CBMC). Virus infection was demonstrated by immunohistology with positive control sera, with monoclonal antibodies and by in situ hybridization for viral DNA. Cytopathic effects following HHV-7 infection generally resemble those after HHV-6 infection but are less pronounced. The ultrastructural appearance of HHV-7 and the replicative stages were similar to those described by Kramarsky and Sander for HHV-6. There were some minor discrepancies, including quite an extensive and space-filling tegument, a slightly different structure of the nucleoid, the frequent finding of nucleocapsids without any visible core and apparently scarce or delicate spikes on the envelope. These differences may suggest HHV-7 rather than HHV-6, but this finding needs confirmation. Mature HHV-7 particles measured 170 nm in diameter, with nucleocapsids of 90–95 nm and a tegument of about 30 nm.

Notizen: Abstract  The ECL cells are histamine- and peptide hormone-producing endocrine cells in the rat oxyntic mucosa. They are rich in secretory vesicles and also contain microvesicles and electron-dense granules. They operate under the control of circulating gastrin. In the present study, we examined the ECL-cell ultrastructure after long term treatment with omeprazole, which is known to induce hypergastrinemia, and after withdrawal of the drug. Rats received omeprazole (400 µmol/kg per day, orally) for 16 days and were killed 1, 5, 20, or 40 days after the last dose of the drug. Oxyntic mucosal specimens were processed for electron microscopy. Electron micrographs of ECL-cell profiles were analyzed planimetrically. The ECL-cell profile area increased promptly in response to omeprazole, the secretory vesicles and granules were reduced in number and volume density, the microvesicles were unchanged in number but reduced in volume density, and vacuoles appeared. Within a week after stopping the omeprazole treatment, the numbers and volume densities of secretory vesicles and microvesicles returned to pre-stimulation values. Also, the vacuoles disappeared promptly. The ECL-cell profile area decreased below the pre-stimulation level within five days after stopping treatment, while, in contrast, the granules increased in number and volume density. Somewhat surprisingly, the cell size and the granule compartment did not return to normal until 40 days after stopping treatment.

Notizen: Abstract. In order to capture the exocytotic figures of gastrin cells in the pyloric antrum of the stomach, we examined antral cells of the mouse, rat, and guinea pig by electron microscopy following stimulation with the cholinergic secretagogue carbamylcholine. Increased numbers of omega profiles indicative of exocytosis were seen in the basal or lateral cell membrane after stimulation with carbamylcholine. The number of exocytotic figures in stimulated gastrin cells was higher in the guinea pig than in the mouse and rat. Coated and non-coated omega profiles and coated pits in the plasma membrane were smaller than the secretory granules. Omega profiles with or without electron-dense contents were seen. Coated and non-coated vesicles were often visible near the plasma membrane of stimulated gastrin cells in all three species, large cytoplasmic vacuoles also being found in the guinea pig. In the mouse pretreated with horseradish peroxidase, reaction deposits were observed in the omega profiles and in microvesicles near the plasma membrane. These results suggest that, after exocytosis, membrane retrieval and endocytosis occur in the gastrin cells.

Notizen: Abstract. Striated rootlets in ciliated cells are conical banded structures composed of longitudinally aligned filaments. The formation of striated rootlets during ciliognesis in the human oviduct epithelium was studied by electron microscopy. Primitive rootlets appeared at the proximal side of basal bodies before or at the same time as ciliary budding. After the formation of several striations, the tip of the rootlets extended deeply toward the interior of the cell and became differentiated into two distinct parts, viz., the proximal conical part connected to the basal body and the distal fibrillar part. The periodicity of the striations in the fibrillar part was 68.5±2.95 nm, about 5 nm longer than that of the conical part (63.9±2.25 nm). The dark band in the striation was thicker in the fibrillar part than in the conical part. Since the fibrillar part was not observed in the mature cilium, this part was considered as being either degraded or changed into the conical part during ciliogenesis.

Notizen: Abstract. The subcellular localization of PECAM-1 in endothelial cells was examined by using advanced morphological techniques, such as confocal scanning microscopy and immunolabeling procedures for electron microscopy. The localization of PECAM-1 was studied immunohistochemically with five specific monoclonal antibodies and one polyclonal antibody (all anti-human) in human and rabbit myocardium and in isolated endothelial cells. In vivo, PECAM-1 was localized uniformly on the plasma membrane of all vascular endothelial cells, predominantly on the luminal side of vessels. No specific increase in labeling was found at sites of cell-to-cell contact. In vitro, primary isolated cells (human umbilical vein endothelial cells) showed continuous labeling of the entire cell membrane. Cells of higher passages were labeled in a manner similar to freshly isolated cells. Our findings refute the commonly accepted hypothesis that PECAM-1 is localized only at cell-to-cell contacts. Further, we have not been able to confirm the hypothesis regarding the important mechanical role of PECAM-1 in stabilizing the endothelial monolayer. Since PECAM-1 is also expressed on platelets and is known to bind to itself, the way in which PECAM-1-positive endothelial cells are protected against binding of PECAM-1-positive platelets remains unclear. In view of these findings, the role of PECAM-1 in the leukocyte migration cascade needs to be re-evaluated.

Notizen: Abstract . This paper presents the first description of peroxisomes in polyplacophorans. As in other molluscs, the hepatopancreas of chitons is composed of basophilic and digestive cells. In the basophilic cells, the endoplasmic reticulum is abundant and several Golgi stacks can be observed. These cells also possess secretion granules and vacuoles with spherites. The digestive cells are mainly characterized by the presence of many food vacuoles. Several peroxisomes were observed in the basophilic cells of Acanthochiton crinita, most of them almost spherical. The matrix is filled with tubular structures and a crystalline nucleoid is also present in these organelles. In the digestive cells of A. crinita, peroxisomes are also almost spherical and possess two kinds of nucleoids. One of them presents a diamond shape and a bundle of tubular structures forms a second kind of nucleoid, which shows an elongated form. In Lepidochitona cinerea, the peroxisomes of basophilic cells are spherical or oval. Within the matrix, a cluster of dense rods and a prismatic nucleoid were observed. In the digestive cells of this species, almost spherical or oval peroxisomes are common, but they are smaller than the peroxisomes of the preceding cells. Nucleoids were not detected, but a few dense rods could be observed in the matrix. In both cell types of the two species, catalase activity was detected in the peroxisomal matrix. In addition, the elongated nucleoid of A. crinita digestive-cell peroxisomes and the nucleoid of L. cinerea basophilic-cell peroxisomes also present catalase activity.

Notizen: Abstract. The mucosal surface area of the chicken duodenum, jejunum, and ileum was determined during development (from 1-day to 12-week-old animals). The morphometric analysis was performed at three magnification levels. The nominal (serosal) surface area was determined at the macroscopic level, from intestinal length and perimeter. Villus and microvillus amplification factors were estimated at light-microscopic and transmission electron-microscopic levels, respectively. The results show, during the period considered: (1) a similar increase in nominal surface area for the three segments (6.5 to 7.2-fold), (2) a rise followed by a slight decrease in the villus amplification factor in the third week of age in the duodenum, a two-fold increase of this variable in the jejunum and no significant developmental variations in the ileum, (3) an increase in the microvillus amplification factor of 1.5-fold in the duodenum and jejunum and of 1.2-fold in the ileum, although a pronounced decrease in the first week of age was observed in the three segments. In conclusion, total mucosal surface area increased, from 1 day to 12 week, 12- to 13-fold in the duodenum and ileum and 20-fold in the jejunum.

Notizen: Abstract. The apical cytoplasm of several absorbing epithelia contains well-developed apical tubules (AT) which contribute to membrane recycling from endocytic vacuoles to the apical cell membrane. In this study, we examined three-dimensional structures of the AT in rat kidney proximal tubule cells by transmission and scanning electron microscopy. In thin sections, the AT appeared as straight tubules with a rather constant diameter (70–90 nm), but others were curved and, occasionally, branching. No AT were labeled with the marker for the external cell surface (ruthenium red) or exhibited histochemical enzyme activity for lysosomal hydrolase (acid phosphatase). After intravenous injection of horseradish peroxidase, it was absorbed in the kidney proximal tubule cells and the AT were labeled with HRP reaction products. Stereo-viewing of the labeled AT in thick sections revealed that they formed an interconnected tubular network. Scanning electron microscopy allowed a three-dimensional view of the AT, in which a network of branching and anastomosing tubules was revealed. These observations indicate that the AT are intracellular endosomal compartments which form an extensive tubular network in the apical cytoplasm. The possibility that this apical tubular network serves as a large membrane store for membrane recycling is discussed.

Notizen: Summary Trichodesmium is the first described example of a filamentous cyanobacterium without heterocysts that contains cells specialised for nitrogen fixation. The ultrastructure of cells with and without nitrogenase were compared using primarilyTrichodesmium tenue Wille, but alsoT. thiebautii Gomont andT. erythraeum Ehrenberg et Gomont. Immunohistochemistry demonstrated that the cytoplasm of certain cells was densely labelled with antibodies against Fe-protein (dinitrogenase reductase). Comparative TEM-image analysis revealed that these cells were also distinguished by a denser thylakoid network, dividing the vacuole-like space into smaller units. The nitrogenase-containing cells also exhibited less extensive gas vacuoles as well as fewer and smaller cyanophycin granules compared to cells which lacked nitrogenase. Carboxysomes were present in both cell types in equal proportion. Longitudinal sections showed that cells with nitrogenase were arranged adjacent to each other, and that groups of cells with and without nitrogenase may coexist in the same trichome. The correlation between modifications in ultrastructure and the presence of nitrogenase suggests a new type of cyanobacterial cell specialisation related to nitrogen fixation. The results obtained also question the systematic affiliation of the genusTrichodesmium.

Notizen: Summary Basic proteins ofBlastocystis hominis were detected by the ammoniacal silver and ethanolic phosphotungstic acid techniques using electron microscopy. The central vacuole showed many silver grains when treated with ammoniacal silver and an increased electron density when treated with phosphotungstic acid. The intensity of positive reactions correlated with the electron density of the central vacuole, because cells having an electron-lucent central vacuole showed no silver grain deposits. Since it is known that the concentration of electron-dense materials in the central vacuole increases during log phase of growth, and then decreases in stationary phase, this organelle must accumulate basic proteins during cell growth.

Notizen: Abstract Exposure of postmitotic growing and non-growing cells of the unicellular green alga Micrasterias denticulata to different UV-B cut-off wavelengths together with simulated sunlight in a sun simulator has revealed a marked resistence of the algae against strong irradiation. While down to a cut-off wavelength of 284 nm irradiated during the most sensitive stage of cell development chloroplast ultrastructure remains unaffected, severe changes in arrangement and structure of stroma and grana thylakoids occur only at the lowest cut-off wavelengths of 280 and 275 nm. The structural alterations end up in a more or less complete desintegration of grana and stroma thylakoids with the remaining membraneous structures appearing in negative staining thus indicating drastic changes in membrane composition. Photosynthetic activity determined by chlorophyll fluorescence (ratio of variable to maximal fluorescence) and oxygen evolution responded more sensitively to UV-B irradiation. With decreasing UV cut-off wavelengths and prolonged incubation a decrease of photochemistry of PS II occured reaching its lowest values after 60 min at 275 and 280 nm. Oxygen production was even maintained under strong UV irradiation with a cut-off wavelenght of 275 nm up to 15 min. With prolonged UV-B treatment any activity was lost. HPLC separations of pigments exhibited the appearance of break-down products (mainly derivatives of chl b and chl a) with decreasing cut-off wavelength and increasing exposure time. The xanthophyll cycle pigments seemed to be unaffected at least for an irradiation period of 60 to 90 min at low UV cut-offs. Possible mechanisms of UV stress avoidance or protection are discussed with regard to the varying altitudes of the natural habitats of the algae.

Notizen: Abstract We have examined with transmission electron microscopythe epithelial layer exposed to the rhynchocoel fluidof the proboscis in the heteronemertine Riseriellus occultus. This epithelium is organized asa monociliated, pseudostratified myoepitheliumconsisting of two cell types: apically situatedmonociliated supportive cells and subapical myocyteslacking cilia. The low supportive cells form acontinuous adluminal sheet and reach with numerouscytoplasmic processes into the extracellular matrix;these cells are characterized by numerous, irregularlyshaped, apical folds projecting into the rhynchocoelfluid, delimiting broad extracellular spaces. Theauthors suppose that both apical and basal folds couldaccommodate stretching of the endothelium when theproboscis is everted. The apical folds of thesupportive cells increase the interface of these withthe rhynchocoel fluid; this feature, together with thepresence of pinocytotic vesicles in such cells,suggest that they could be involved in the exchange ofsubstances between the rhynchocoel fluid and theproboscis. The myocytes are scattered singly withinthe monociliated pseudostratified myoepithelium. Theyare situated between the supportive cells and thesubjacent extracellular matrix. Basement membraneseparating both cells types is lacking. Myofibrillarparts protrude basally from the myocyte somata. Themyofibrillar parts lie in direct apposition to theextracellular matrix, and are oriented circular to thelongitudinal axis of the proboscis. We consider themyocytes to be intra-epithelial, myoepithelial cells.

Notizen: Abstract Twenty-eight cases of nasopharyngeal angiofibroma were studied immunohistochemically for cytoskeletal phenotyping of stromal cells. Electron microscopy was also used to study the ultrastructure of five of the tumors. All typical stromal cells showed intensive immunostaining for vimentin, but were negative for smooth muscle actin and desmin. Ultrastructurally, most of these cells appeared to be exclusively fibroblasts. However, in some areas stromal cells were seen that morphologically resembled myofibroblasts by their shapes and arrangement, and were characterized by the coexpression of vimentin and smooth muscle actin. Electron microscopy confirmed their myofibroblastic nature. The present study showed that the typical stromal cells in nasopharyngeal angiofibromas were fibroblasts and not myofibroblasts. In these tumors myofibroblasts occurred only focally, in connection with fibrotic areas and exclusively as a vimentin+/actin+ cytoskeletal phenotype. This indicates that myofibroblasts are not primary stromal tumor cells in nasopharyngeal angiofibromas, but occur due to regressive changes.

Notizen: Abstract The ultrastructural morphology of the interface region between the stria vascularis (SV) and spiral ligament (SL) was examined in the neonatal rat cochlea via transmission electron microscopy. At postnatal day (PND) 3, morphology of both basal cells and fibrocytes was simple and immature. Only a small number of fibrocytes was observed in the SL. Intercellular junctions between basal cells and fibrocytes, and between adjacent fibrocytes, were few. At PND 7, the number of fibrocytes increased, and more organelles appeared within their cytoplasm. From PND 11 to 14, nuclei of the basal cells appeared to be more spindle-shaped and contained more heterochromatin. The cytoplasm of the fibrocytes was pale, and a greater number of cytoplasmic vesicles and mitochondria emerged. More intercellular junctions were observed between basal cells and fibrocytes at the interface region and between fibrocytes in the SL. By PND 21, the morphology of basal cells and fibrocytes and their intercellular junctionsappeared to be adult-like. These morphological observations correlate with previous reports on the functional maturation of the developing rat cochlea.

Notizen: Semipurified lipase of Candida rugosa (CRSL) was subjected to chemical modification, and the activities of the modified lipase, in hydrolysis and esterification reactions, were examined. The esterification reactions were carried out in the absence and presence of isooctane. When the enzyme was modified with polyethylene glycol (PEG), two methodologies were studied. The activation of PEG with p-NO2-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG. The chemical modification with PEG increases the stability of pure lipases in isooctane at 50°C (extreme conditions). The chemically modified enzymes are useful for biotransformations in organic solvents. In addition the nitration of tyrosines with tetranitromethane was also studied. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 252-260, 1997.

Notizen: To investigate the response of hybridoma cells to hypoosmotic stress, S3H5/γ2bA2 and DB9G8 hybridomas were cultivated in the hypoosmolar medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum] resulting from sodium chloride subtraction. Both hybridomas showed similar responses to hypoosmotic stress in regard to cell growth and antibody production. The cell growth and antibody production at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Both cells grew well at 219 mOsm/kg, though their growth and antibody production were slightly decreased. When the osmolality was further decreased to 168 mOsm/kg, the cell growth did not occur. When subjected to hyperosmotic stress, both cells displayed significantly enhanced specific antibody productivity (qAb). However, the cells subjected to hypoosmotic stress did not display enhanced qAb. Taken together, both hyperosmotic and hypoosmotic stresses depressed the growth of S3H5/γ2bA2 and DB9G8 hybridomas. However, their response to hypoosmotic stress in regard to qAb was different from that to hyperosmotic stress. © 1997 John Wiley & Sons, Inc. Biotechnol Biong 55: 565-570, 1997.

Notizen: This study demonstrates a new approach to reduce the amount of fungal cellulase required for the conversion of cellulose into ethanol. Escherichia coli KO11, a biocatalyst developed for the fermentation of hemicellulose syrups, was used to produce recombinant endoglucanase as a co-product with ethanol. Seven different bacterial genes were expressed from plasmids in KO11. All produced cell-associated endoglucanase activity. KO11(pLOI1620) containing Erwinia chrysanthemi celZ (EGZ) produced the highest activity, 3,200 IU endoglucanase/L fermentation broth (assayed at pH 5.2 and 35°C). Recombinant EGZ was solubilized from harvested cells by treatment with dilute sodium dodecyl sulfate (12.5 mg/ml, 10 min, 50°C) and tested in fermentation experiments with commercial fungal cellulase (5 filter paper units/g cellulose) and purified cellulose (100 g/L). Using Klebsiella oxytoca P2 as the biocatalyst, fermentations supplemented with EGZ as a detergent-lysate of KO11(pLOI1620) produced 14%-24% more ethanol than control fermentations supplemented with a detergent-lysate of KO11(pUC18). These results demonstrate that recombinant bacterial endoglucanase can function with fungal cellulase to increase ethanol yield during the simultaneous saccharification and fermentation of cellulose. © 1997 Wiley & Sons, Inc. Biotechnol Bioeng 55: 547-555, 1997.

Notizen: An expression system has been developed for the introduction of DNA cassettes into the region between the transcription and translation start sites of a gene of interest. This cassette system was used to engineer mRNA stability through the introduction of hairpins at the 5′ end. A synthetic DNA cassette was designed so that the resulting mRNA hairpin would be positioned one nucleotide from the 5′ mRNA end. The hairpin-containing mRNA exhibited a half-life 3 times that of the mRNA with no hairpin, resulting in increases in both mRNA and protein levels. These results indicate that it is possible to engineer mRNA stability as an additional means of controlling gene expression. © 1997 John Wiley & Sons Inc. Biotechnol Bioeng 55: 557-580, 1997

Notizen: Preferential transport in adsorptive membranes can be used to selectively remove biochemicals directly from fermentation broths. During preferential transport, an adsorbing solute is selectively transported across the membrane while nonadsorbing solutes and cells are retained by the membrane. This technique was used to separate lysozyme directly from a feed containing lysozyme, myoglobin, and yeast cells. We found that because the oscillatory flows used in preferential transport involve strokes that are close to symmetric, they are very efficient in alleviating cake formation due to cell deposition on the membrane surface. Theoretical results suggest that, by optimizing process variables, preferential transport can lead to a continuous concentrated stream of the adsorbing protein. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 581-591, 1997.

Notizen: A mathematical model of glycolysis in Saccharomyces cerevisiae is presented. The model is based on rate equations for the individual reactions and aims to predict changes in the levels of intra- and extracellular metabolites after a glucose pulse, as described in part I of this study. Kinetic analysis focuses on a time scale of seconds, thereby neglecting biosynthesis of new enzymes. The model structure and experimental observations are related to the aerobic growth of the yeast. The model is based on material balance equations of the key metabolites in the extracellular environment, the cytoplasm and the mitochondria, and includes mechanistically based, experimentally matched rate equations for the individual enzymes. The model includes removal of metabolites from glycolysis and TCC for biosynthesis, and also compartmentation and translocation of adenine nucleotides. The model was verified by in vivo diagnosis of intracellular enzymes, which includes the decomposition of the network of reactions to reduce the number of parameters to be estimated simultaneously. Additionally, sensitivity analysis guarantees that only those parameters are estimated that contribute to systems trajectory with reasonable sensitivity. The model predictions and experimental observations agree reasonably well for most of the metabolites, except for pyruvate and adenine nucleotides. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 592-608, 1997.

Notizen: The design of a reactor in which two interacting cell populations (microorganisms and plants) could grow under controlled conditions was considered. In this reactor, the cell populations are separated by a membrane which permits semi-in vivo study of induced interaction-specific changes in metabolism. In this paper, the interaction of suspension culture of Nicotiana tabacum (tobacco) and the Oomycete, Phytophthora nicotiana was simulated. The results of the computer simulation show the induced metabolic changes as a consequence of the biological interaction. The paper introduces a novel approach in the strategy for the study of interacting population in suspension cultures. This type of system has potential applications in studies of the regulation of secondary metabolism and for the production of high values pharmaceuticals. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 609-615, 1997.

Notizen: The strength of adhesion and dynamics of detachment of murine 3T3 fibroblasts from self-assembled monolayers were measured in a radial-flow chamber (RFC) by applying models for fluid mechanics, adhesion strength probability distributions, and detachment kinetics. Four models for predicting fluid mechanics in a RFC were compared to evaluate the accuracy of each model and the significance of inlet effects. Analysis of these models indicated an outer region at large radial positions consistent with creeping flow, an intermediate region influenced by inertial dampening, and an inner region dominated by entrance effects from the axially-oriented inlet. In accompanying experiments patterns of the fraction of cells resisting detachment were constructed for individual surfaces as a function of the applied shear stress and evaluated by comparison with integrals of both a normal and a log-normal distribution function. The two functions were equally appropriate, yielding similar estimates of the mean strength of adhesion. Further, varying the Reynolds number in the inlet, Red, between 630 and 1480 (corresponding to volumetric flow rates between 0.9 and 2.1 mL/s) did not affect the mean strength of adhesion. For these same experiments, analysis of the dynamics of detachment revealed three temporal phases: 1) rapid detachment of cells at the onset of flow, consistent with a first-order homogeneous kinetic model; 2) time-dependent rate of detachment during the first 30 sec. of exposure to hydrodynamic shear, consistent with the first-order heterogeneous kinetic model proposed by Dickinson and Cooper (1995); and 3) negligible detachment, indicative of pseudo-steady state after 60 sec. of flow. Our results provide rigorous guidelines for the measurement of adhesive interactions between mammalian cells and prospective biomaterial surfaces using a RFC. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 616-629, 1997.

Notizen: Glucose and lactate metabolic rates were evaluated for cultures of cord blood (CB) mononuclear cell (MNC), peripheral blood (PB) MNC, and PB CD34+ cell cultures carried out in spinner flasks and in T-flasks in both serum-containing and serum-free media. Specific glucose uptake rates (qgluc, in micromoles per cell per hour) and lactate generation rates (qlac) correlated with the percentage of colony-forming cells (CFC) present in the culture for a broad range of culture conditions. Specifically, the time of maximum CFC percentage in each culture coincided with the time of maximum qgluc and qlac in cultures with different seeding densities and cytokine combinations. A two-population model (Qlac = α[CFC] + β([TC] - [CFC]), where [TC] is total cell concentration; Qlac is volumetric lactate production rate in micromoles per milliliter per hour; α is qlac for an average CFC; and β is qlac for an average non-CFC) was developed to describe lactate production. The model described lactate production well for cultures carried out in both T-flasks and spinner flasks and inoculated with either PB or CB MNC or PB CD34+ cells. The values for α and β that were derived from the model varied with both the inoculum density and the cytokine combination. However, preliminary results indicate that cultures carried out under the same conditions from different samples with similar initial CD34+ cell content have similar values for β and β. These findings suggest that it should be possible to use lactate production data to predict the harvest time that corresponds to the maximum number of CFC in culture. The ability to harvest ex vivo hematopoietic cultures for transplantation when CFC are at a maximum has the potential to speed the rate at which immunocompromised patients recover. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 693-700, 1997.

Notizen: A mathematical model to estimate the solar irradiance profile and average light intensity inside a tubular photobioreactor under outdoor conditions is proposed, requiring only geographic, geometric, and solar position parameters. First, the length of the path into the culture traveled by any direct or disperse ray of light was calculated as the function of three variables: day of year, solar hour, and geographic latitude. Then, the phenomenon of light attenuation by biomass was studied considering Lambert-Beer's law (only considering absorption) and the monodimensional model of Cornet et al. (1900) (considering absorption and scattering phenomena). Due to the existence of differential wavelength absorption, none of the literature models are useful for explaining light attenuation by the biomass. Therefore, an empirical hyperbolic expression is proposed. The equations to calculate light path length were substituted in the proposed hyperbolic expression, reproducing light intensity data obtained in the center of the loop tubes. The proposed model was also likely to estimate the irradiance accurately at any point inside the culture. Calculation of the local intensity was thus extended to the full culture volume in order to obtain the average irradiance, showing how the higher biomass productivities in a Phaeodactylum tricornutum UTEX 640 outdoor chemostat culture could be maintained by delaying light limitation. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 701-714, 1997.

Notizen: Both parallel fermentations with Aspergillus awamori (CBS 115.52) and a literature study on several fungi have been carried out to determine a relation between fungal morphology and agitation intensity. The studied parameters include hyphal length, pellet size, surface structure or so-called hairy length of pellets, and dry mass per-wet-pellet volume at different specific energy dissipation rates. The literature data from different strains, different fermenters, and different cultivation conditions can be summarized to say that the main mean hyphal length is proportional to the specific energy dissipation rate according to a power function with an exponent of -0.25 ± 0.08. Fermentations with identical inocula showed that pellet size was also a function of the specific energy dissipation rate and proportional to the specific energy dissipation rate to an exponent of -0.16 ± 0.03. Based on the experimental observations, we propose the following mechanism of pellet damage during submerged cultivation in stirred fermenters. Interaction between mechanical forces and pellets results in the hyphal chip-off from the pellet outer zone instead of the breakup of pellets. By this mechanism, the extension of the hyphae or hair from pellets is restricted so that the size of pellets is related to the specific energy dissipation rate. Hyphae chipped off from pellets contribute free filamentous mycelia and reseed their growth. So the fraction of filamentous mycelial mass in the total biomass is related to the specific energy dissipation rate as well.To describe the surface morphology of pellets, the hyphal length in the outer zone of pellets or the so-called hairy length was measured in this study. A theoretical relation of the hairy length with the specific energy dissipation rate was derived. This relation matched the measured data well. It was found that the porosity of pellets showed an inverse relationship with the specific energy dissipation rate and that the dry biomass per-wet-pellet volume increased with the specific energy dissipation rates. This means that the tensile strength of pellets increased with the increase of specific energy dissipation rate. The assumption of a constant tensile strength, which is often used in literature, is then not valid for the derivation of the relation between pellet size and specific energy dissipation rate. The fraction of free filamentous mycelia in the total biomass appeared to be a function of the specific energy dissipation in stirred bioreactors. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 715-726, 1997.

Notizen: We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:921-926, 1997.

Notizen: Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five™) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five™ cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five™ cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five™ cell cultures, but not in Sf-9 cell cultures. In addition, High Five™ cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five™ cell cultures. It was also found that the medium had a significant effect on High Five™ cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.

Notizen: No abstract.

Notizen: Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (aw = 0.2, aw = 0.4 and aw = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from aw = 0.2 to aw = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1-8, 1997.

Notizen: Arabinose, fructose, galactose, myo-inositol, lyxose, mannose, ribose, and xylose were incubated individually and with glucose in the presence of Aspergillus niger glucoamylase at pH 4.5 and 45°C. Glucoamylase condenses galactose, glucose, and mannose individually into disaccharides. It also produces mixed disaccharides when each of the eight carbohydrates is incubated with glucose. Many products were identified by gas chromatography of the derivatized reaction mixtures followed by mass spectroscopy of the individual chromatographic peaks. Galacto-, gluco-, or mannopyranosyl rings appear to be present at the nonreducing ends of all the disaccharides produced. Molecules linked through primary hydroxyl groups have the highest equilibrium constants of all products formed, since these bonds are thermodynamically favored. However, glucoamylase is capable of forming bonds with many available hydroxyl groups, as previously demonstrated when it was incubated with glucose alone. Formation rates of different bonds linking different residues vary widely. These results demonstrate that glucoamylase has a wide selectivity toward residues it will condense into disaccharides and toward bonds it will form between them. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 9-22, 1997.

Notizen: We discuss how methods based on hidden Markov models performed in the fold-recognition section of the CASP2 experiment. Hidden Markov models were built for a representative set of just over 1,000 structures from the Protein Data Bank (PDB). Each CASP2 target sequence was scored against this library of HMMs. In addition, an HMM was built for each of the target sequences and all of the sequences in PDB were scored against that target model, with a good score on both methods indicating a high probability that the target sequence is homologous to the structure. The method worked well in comparison to other methods used at CASP2 for targets of moderate difficulty, where the closest structure in PDB could be aligned to the target with at least 15% residue identity. Proteins, Suppl. 1:134-139, 1997. © 1998 Wiley-Liss, Inc.

Notizen: Structure predictions for two targets from the CASP2 meeting are presented and compared with the experimental structure. These predictions were made using a novel simplified flexible geometry representation of protein structure. The method uses potentials which mimic the physical forces involved in protein folding in a simplified representation of protein structure, and not by directly using data derived from statistical analyses of known protein structures. Additionally, the method is designed to work with a single protein sequence. The method was successful in generating reasonable protein-like structures, with mainly buried hydrophobic residues, exposed charges, and a good fraction of secondary structure. Specific details of the structure were remarkably close to the experimental structure. However, the overall fold in both cases was totally wrong. Some specific causes of this incorrect folding are suggested and a major improvement to the algorithm proposed. Proteins, Suppl. 1:172-178, 1997. © 1998 Wiley-Liss, Inc.

Notizen: The docking section of CASP2 is reviewed. Seven small molecule ligand-protein targets and one protein-protein target were available for predictions. Many of the small molecule ligand complexes involved serine proteases. Overall results for the small molecule targets were good, with at least one prediction for each target being within 3 Å root-mean-square deviation (RMSD) for nearly all targets and within 2 Å RMSD for over half the targets. However, no single docking method seemed to consistently perform best. In addition, the predictions closest to the experimental results were not always those ranked the highest, pointing out that the evaluation (scoring) of potential solutions is still an area that needs improvement. The protein-protein target proved more difficult. None of the predictions did well in reproducing the geometry of the complex, although in many cases the interacting surfaces of the two proteins were predicted with reasonable accuracy. This target consisted of two large proteins and, therefore was a demanding target for docking methods. Proteins, Suppl. 1:198-204, 1997. © 1998 Wiley-Liss, Inc.

Notizen: We have applied our docking program FLEXX to all eight CASP2 targets involving protein complexes with small ligands. Of the seven targets that were kept in the CASP2 experiment, we could solve two. We found important parts of the solution in four other examples, and were unsuccessful on the remaining example. This paper discusses all predictions in detail. Each of our prediction runs took just a few minutes of computer time on a standard workstation and could thus be demonstrated in real time at the CASP meeting. We believe that this speed is the prime strength of our program FLEXX. In quality, our predictions are competitive with those produced by other predictors. The experiment showed that possible objectives of improvement of the FLEXX program are to incorporate relevant aspects of receptor flexibility, deal with water molecules in the receptor pocket, allow for a postoptimization to refine favorable complexes, and improve the scoring function. Proteins, Suppl. 1:221-225, 1997. © 1998 Wiley-Liss, Inc.

Notizen: We report the crystal structure of MalE-B133, a recombinant form of the maltodextrin-binding protein (MBP) of Escherichia coli carrying an inserted amino-acid sequence of a B-cell epitope from the preS2 region of the hepatitis B virus (HBV). The structure was determined by molecular replacement methods and refined to 2.7 Å resolution. MalE-B133 is an insertion/deletion mutant of MBP in which residues from positions 134 to 142, an external α helix in the wild-type structure, are replaced by a foreign peptide segment of 19 amino acids. The inserted residues correspond to the preS2 sequence from positions 132 to 145 and five flanking residues that arise from the creation of restriction sites. The conformation of the recombinant protein, excluding the inserted segment, closely resembles that of wild-type MBP in the closed maltose-bound form. MalE-B133 was shown by previous studies to display certain immunogenic and antigenic properties of the hepatitis B surface antigen (HBsAg), which contains the preS2 region. The crystal structure reveals the conformation of the first nine epitope residues (preS2 positions 132 to 140) exposed on the surface of the molecule. The remaining five epitope residues (preS2 positions 141 to 145) are not visible in electron density maps. The path of the polypeptide chain in the visible portion of the insert differs from that of the deleted segment in the structure of wild-type MBP, displaying a helical conformation at positions 134 to 140 (preS2 sequence numbering). A tripeptide (Asp-Pro-Arg) at the N terminus of the helix forms a stable structural motif that may be implicated in the cross-reactivity of anti-HBsAg antibodies with the hybrid protein. Proteins 27:1-8 © 1997 Wiley-Liss, Inc.

Notizen: We propose a binary word encoding to improve the protein secondary structure prediction. A binary word encoding encodes a local amino acid sequence to a binary word, which consists of 0 or 1. We use an encoding function to map an amino acid to 0 or 1. Using the binary word encoding, we can statistically extract the multiresidue information, which depends on more than one residue. We combine the binary word encoding with the GOR method, its modified version, which shows better accuracy, and the neural network method. The binary word encoding improves the accuracy of GOR by 2.8%. We obtain similar improvement when we combine this with the modified GOR method and the neural network method. When we use multiple sequence alignment data, the binary word encoding similarly improves the accuracy. The accuracy of our best combined method is 68.2%. In this paper, we only show improvement of the GOR and neural network method, we cannot say that the encoding improves the other methods. But the improvement by the encoding suggests that the multiresidue interaction affects the formation of secondary structure. In addition, we find that the optimal encoding function obtained by the simulated annealing method relates to non-polarity. This means that nonpolarity is important to the multiresidue interaction. Proteins 27:36-46 © 1997 Wiley-Liss, Inc.

Notizen: The interaction between the human B cell receptor CD40 and its ligand on T cells is critical for B cell proliferation and the regulation of humoral immune responses. CD40 is a member of the tumor necrosis factor receptor (TNFR) family. We report here the construction and analysis of a detailed three-dimensional model of the TNFR-homologous extracellular region of CD40. This study provides an example for structure-based model building in the presence of low sequence similarity. The assessment of model quality and sequence-structure compatibility is emphasized, and limitations of the model are discussed. The current CD40 model predicts structural details beyond the backbone level. Features of the CD40 ligand binding site are discussed in conjunction with the results of a previous mutagenesis study. Proteins 27:59-70 © 1997 Wiley-Liss, Inc.

Notizen: The conformational dynamics of β-glycosidase from Sulfolobus solfataricus was investigated by following the emission decay arising from the large number of tryptophanyl residues that are homogeneously dispersed in the primary structure. The fluorescence emission is characterized by a bimodal lifetime distribution, suggesting that the enzyme structure contains rigid and flexible regions, properly located in the macromolecule. The enzyme activity and thermostability appear to be related to the dynamic properties of these regions as evidenced by perturbation studies of the enzyme structure at alkaline pH and by addition of detergents such as SDS. The pH increase affects the protein dynamics with a remarkable loss of thermal stability and activity; these changes occur without any significant variation in the secondary structure as revealed by far-UV dichroic measurements. In the presence of 0.02% (w/v) SDS at alkaline pH, the enzymatic activity and thermostability are recovered. Under these conditions, the conformational dynamics appear to be similar to that evidenced at neutral pH. Further increases in SDS concentration, at alkaline pH, render the activity and thermostability of β-glycosidase similar to those observed in the absence of detergent. Proteins 27:71-79 © 1997 Wiley-Liss, Inc.

Notizen: A tertiary model of the human GSTT2 Theta class glutathione transferase is presented based on the recently solved crystal structure of a related thetalike isoenzyme from Lucilia cuprina. Although the N-terminal domains are quite homologous, the C-terminal domains share less than about 20% identity. The model is used to consolidate the role of Ser 11 in the active site of the enzyme as well as to identify other residues and mechanisms of likely catalytic importance. The T2 subfamily of theta class enzymes have been shown to inactivate reactive sulfate esters arising from arylmethanols. A possible reaction pathway involving the conjugation of glutathione with one such sulfate ester, 1-menaphthyl-sulfate, is described. It is also proposed that the C-terminal region of the enzyme plays an important role in allowing substrate access to the active site. Proteins 27:118-130 © 1997 Wiley-Liss, Inc.

Notizen: The cytokines IL-6, LIF, CNTF, OSM, IL-11, and CT-1 have been grouped into the family of IL-6-type cytokines, since they all require gp130 for signal transduction. Interestingly, gp130 binds directly to OSM, whereas complex formation with the other cytokines depends on additional receptor subunits. Only limited structural information on these cytokines and their receptors is available. X-ray structures have been solved for the cytokines LIF and CNTF, whose up-up-down-down four-helix bundle is common to all of these cytokines, and for the receptors of hGH and prolactin, which contain two domains with a fibronectin III-like fold. Since cocrystallization and x-ray analysis of the up to four different proteins forming the receptor complexes of the IL-6-type cytokines is unlikely to be achieved in the near future, model building based on the existing structural information is the only approach for the time being. Here we present model structures of the complexes of human and murine IL-6 with their receptors. Their validity can be deduced from the fact that published mutagenesis data and the different receptor specificity of human and murine IL-6 can be understood. It is now possible to predict the relative positions and contacts for all molecules in their respective complexes. Such information can be used for the rational design of cytokine and receptor antagonists, which may have a valuable therapeutic perspective. Proteins 27:96-109 © 1997 Wiley-Liss, Inc.

Notizen: Acyl carrier proteins (ACPs) from spinach and from Escherichia coli have been used to demonstrate the utility of proton NMR for comparison of homologous structures. The structure of E. coli ACP had been previously determined and modeled as a rapid equilibrium among multiple conformational forms (Kim and Prestegard, Biochemistry 28:8792-8797, 1989). Spinach ACP showed two slowly exchanging forms and could be manipulated into one form for structural study. Here we compare this single form to postulated multiple forms of E. coli ACP using the limited amount of NOE data available for the spinach protein. A number of long-range NOE contacts were present between homologous residues in both spinach and E. coli ACP, suggesting tertiary structural homology. To allow a more definitive structural comparison, a method was developed to use spinach ACP NOE constraints to search for regions of structural divergence from two postulated forms of E. coli ACP. The homologous regions of the two protein sequences were aligned, additional distance constraints were extracted from the E. coli structure, and these were mapped onto the spinach sequence. These distance constraints were combined with experimental NOE constraints and a distance geometry simulated annealing protocol was used to test for compatibility of the constraints. All of the experimental spinach NOE constraints could be successfully combined with the E. coli data, confirming the general hypothesis of structural homology. A better fit was obtained with one form, suggesting a preferential stabilization of that form in the spinach case. Proteins 27:131-143 © 1997 Wiley-Liss, Inc.

Notizen: Plants adapt to various stresses by developmental alterations that render them less easily damaged. Expression of the TCH2 gene of Arabidopsis is strongly induced by stimuli such as touch and wind. The gene product, TCH2, belongs to the calmodulin (CaM) family of proteins and contains four highly conserved Ca2+-binding EF-hands. We describe here the structure of TCH2 in the fully Ca2+-saturated form, constructed using comparative molecular modeling, based on the x-ray structure of paramecium CaM. Like known CaMs, the overall structure consists of two globular domains separated by a linker helix. However, the linker region has added flexibility due to the presence of 5 glycines within a span of 6 residues. In addition, TCH2 is enriched in Lys and Arg residues relative to other CaMs, suggesting a preference for targets which are more negatively charged. Finally, a pair of Cys residues in the C-terminal domain, Cys126 and Cys131, are sufficiently close in space to form a disulfide bridge. These predictions serve to direct future biochemical and structural studies with the overall aim of understanding the role of TCH2 in the cellular response of Arabidopsis to environmental stimuli. Proteins 27:144-153 © 1997 Wiley-Liss, Inc.

Notizen: A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenease), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a= 64.5 Å b= 84.9 Å c= 146.1 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 Å3/Da. The crystals diffract to at least 2.8 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. A 2.56 Å resolution native data set has been collected at beamline X12-C at the NSLS. © 1997 Wiley-Liss, Inc.

Notizen: A study was undertaken to investigate whether diverse carbonic anhydrase (CA) isozymes (both native Zn as well as cobalt-substituted) are able to catalyze the hydrolysis of anions such as cyanide, cyanate, and thiocyanate. A controversy exists between the crystallographic and spectroscopic data of CA II-anion adducts. In the former case it has been shown that “metal poisons” such as CN-and CNO-are not directly coordinated to the active site Zn(II) ion whereas spectroscopic studies indicate otherwise. A theoretical study in the above systems did not resolve this controversy, since it was calculated that all three anions can act as CA substrates. In this paper we prove experimentally that none of them may act as substrates of CA and propose an explanation to the above controversy, discussing the mode of binding of small molecules within the enzyme active site. © 1997 Wiley-Liss, Inc.

Notizen: The free energy difference between folded and unfolded state is about the same for most proteins and it is not more than the energy of a few noncovalent interactions. In addition to the numerous noncovalent interactions, some proteins contain one or more disulfide bonds, which, as covalent crosslinks, significantly stabilize their tertiary structure. Correlation between the presence of disulfide bond(s), and the number noncovalent interresidue interactions of various kinds is analyzed here. The number of interactions per residue is almost the same for all protein. Also the number of long-range interactions per residue is the same in all proteins. Proteins with S(SINGLE BOND)S bond(s) (extracellular proteins) have more medium-range and fewer short-range interactions than those without S(SINGLE BOND)S bonds. However, the difference is independent of the number of these covalent crosslinks. We concluded that the different distributions of the various kinds of noncovalent interaction reflect the needs of proteins in the different environments, the extracellular and the intracellular ones, rather than the presence of the disulfide bond(s). We also pointed out that the observed differences in the distributions of short- and medium-range interactions are in good agreement with different secondary structure compositions of extracellular and intracellular proteins. Proteins 27:360-366, 1997. © 1997 Wiley-Liss, Inc.

Notizen: To investigate how the properties of individual amino acids result in proteins with particular structures and functions, we have examined the correlations between previously derived structure-dependent mutation rates and changes in various physical-chemical properties of the amino acids such as volume, charge, α-helical and β-sheet propensity, and hydrophobicity. In most cases we found the ΔG of transfer from octanol to water to be the best model for evolutionary constraints, in contrast to the much weaker correlation with the ΔG of transfer from cyclohexane to water, a property found to be highly correlated to changes in stability in site-directed mutagenesis studies. This suggests that natural evolution may follow different rules than those suggested by results obtained in the laboratory. A high degree of conservation of a surface residue's relative hydrophobicity was also observed, a fact that cannot be explained by constraints on protein stability but that may reflect the consequences of the reverse-hydrophobic effect. Local propensity, especially α-helical propensity, is rather poorly conserved during evolution, indicating that non-local interactions dominate protein structure formation. We found that changes in volume were important in specific cases, most significantly in transitions among the hydrophobic residues in buried locations. To demonstrate how these techniques could be used to understand particular protein families, we derived and analyzed mutation matrices for the hypervariable and framework regions of antibody light chain V regions. We found a surprisingly high conservation of hydrophobicity in the hypervariable region, possibly indicating an important role for hydrophobicity in antigen recognition. Proteins 27:336-344, 1997. © 1997 Wiley-Liss, Inc.

Notizen: Alphaviruses are enveloped, insect-borne viruses, which contain a positive-sense RNA genome. The protein capsid is surrounded by a lipid membrane, which is penetrated by glycoprotein spikes. The structure of the Sindbis virus (SINV) (the type virus) core protein (SCP) was previously determined and found to have a chymotrypsin-like structure. SCP is a serine proteinase which cleaves itself from a polyprotein. Semliki Forest virus (SFV) is among the most distantly related alphaviruses to SINV. Similar to SCP, autocatalysis is inhibited in SFCP after cleavage of the polyprotein by leaving the carboxy-terminal tryptophan in the specificity pocket. The structures of two different crystal forms (I and II) of SFV core protein (SFCP) have been determined to 3.0 Å and 3.3 Å resolution, respectively. The SFCP monomer backbone structure is very similar to that of SCP. The dimeric association between monomers, A and B, found in two different crystal forms of SCP is also present in both crystal forms of SFCP. However, a third monomer, C, occurs in SFCP crystal form I. While monomers A and B make a tail-to-tail dimer contact, monomers B and C make a head-to-head dimer contact. A hydrophobic pocket on the surface of the capsid protein, the proposed site of binding of the E2 glycoprotein, has large conformational differences with respect to SCP and, in contrast to SCP, is found devoid of bound peptide. In particular, Tyr184 is pointing out of the hydrophobic pocket in SFCP, whereas the equivalent tyrosine in SCP is pointing into the pocket. The conformation of Tyr184, found in SFCP, is consistent with its availability for iodination, as observed in the homologous SINV cores. This suggests, by comparison with SCP, that E2 binding to cores causes major conformational changes, including the burial of Tyr184, which would stabilize the intact virus on budding from an infected cell. The head-to-tail contacts found in the pentameric and hexameric associations within the virion utilize the same monomer surface regions as found in the crystalline dimer interfaces. Proteins 27:345-359, 1997. © 1997 Wiley-Liss, Inc.

Notizen: No abstract.

Notizen: We present a statistical mechanical description of biomolecular hydration that accurately describes the hydrophobic and hydrophilic hydration of a model α-helical peptide. The local density of water molecules around a biomolecule is obtained by means of a potential-of-mean-force (PMF) expansion in terms of pair- and triplet-correlation functions of bulk water and dilute solutions of nonpolar atoms. The accuracy of the method is verified by comparing PMF results with the local density and site-site correlation functions obtained by molecular dynamics simulations of a model α-helix in solution. The PMF approach quantitatively reproduces all features of the peptide hydration determined from the molecular dynamics simulation. Regions of hydrophobic hydration near the Cα and Cβ atoms along the helix are well reproduced. The hydration of exposed polar groups at the N- and C-termini of the helix are also well described by the theory. A detailed comparison of the local hydration by means of site-site radial distribution functions evaluated with the PMF theory shows agreement with the molecular dynamics simulations. The formulation of this theory is general and can be applied to any biomolecular system. The accuracy, speed of computation, and local character of this theory make it especially suitable for studying large biomolecular systems. © 1997 Wiley-Liss Inc.

Notizen: The crystal structure of despentapeptide insulin, a monomeric insulin, has been refined at 1.3 Å spacing and subsequently used to predict and model the organization in the insulin fibril. The model makes use of the contacts in the densely packed despentapeptide insulin crystal, and takes into account other experimental evidence, including binding studies with Congo red. The dimensions of this model fibril correspond well with those measured experimentally, and the monomer-monomer contacts within the fibril are in accordance with the known physical chemistry of insulin fibrils. Using this model, it may be possible to predict mutations in insulin that might alleviate problems associated with fibril formation during insulin therapy. © 1997 Wiley-Liss Inc.

Notizen: An α/β barrel is predicted for the three-dimensional (3D) structure of Bacillus subtilis ferrochelatase. To arrive at this structure, the THREADER program was used to find possible homologous 3D structures and to predict the secondary structure for the ferrochelatase sequence. The secondary structure was fit by hand to the selected homologous 3D structure then the MODELLER program was used to predict the fold of ferrochelatase. Molecular biological information about the conserved residues of ferrochelatase was used as the criteria to help select the homologous 3D structure used to predict the fold of ferrochelatase. Based on the predicted structure possible, ligands binding to the iron and protoporphyrin IX are discussed. The structure has been deposited in the Brookhaven database as ID 1FJI. © 1997 Wiley-Liss Inc.

Notizen: The characteristic CXC chemokine disulfide core of interleukin-8 (IL-8) has been rearranged in a variant replacing the 9 - 50 disulfide with a 9 - 38 disulfide. The new variant has been characterized by its binding affinity to IL-8 receptors A and B and the erythrocyte receptor DARC. This variant binds the three receptors with affinities between 500- and 2,500-fold lower than wild-type IL-8. Binding affinity results are also reported for the variant with alanine substituted for both cysteines 9 and 50. The Glu38 → Cys/Cys50 → Ala IL-8 crystallizes in space group P212121 with cell parameters a = 46.4, b = 49.2, and c = 69.5 Å, and has been refined to an R-value of 19.4% for data from 10 to 2 Å resolution. Analysis of the structure confirms the new disulfide arrangement and suggests that changes at Ile10 may be the principal cause of the lowered affinities. © 1997 Wiley-Liss Inc.

Notizen: Basic amino acid pairs in polypeptides represent important markers for processing enzymes to produce biologically active products. Such enzymes related to the serine peptidase subtilisin have recently been identified in eukaryotes. Herein is described and kinetically characterized a new type of processing enzyme, oligopeptidase B, which is encountered in the prokaryote Escherichia coli, and belongs to the prolyl oligopeptidase family of serine peptidases. The enzyme hydrolyzes the peptides at the carboxy end of dibasic sites by two orders of magnitude faster with respect to monobasic substrates. The kcat/Km is extremely high, 63 μM-1 s-1, for the substrate benzyloxycarbonyl-L-arginyl-L-arginyl-7-(4- methylcoumaryl)amide. The bell-shaped pH dependence of the rate constant is perturbed by some ionizing group(s). This effect is abolished at 1 M NaCl. In addition, high ionic strength inhibits the reaction considerably by increasing Km, which is indicative of an electrostatic interaction between the arginyl residues and the enzymatic carboxy groups. In distinction from that found with most serine endopeptidases, kinetic deuterium isotope measurements with oligopeptidase B indicate that the rate-limiting step of the reaction is a physical step rather than a chemical one characterized by general acid/base catalysis. The present result will contribute to our understanding of the processing phenomena in prokaryotes, as well as in higher organisms. Proteins 28:375-379, 1997. © 1997 Wiley-Liss, Inc.

Notizen: Quasielastic neutron scattering experiments performed on yeast phosphoglycerate kinase in the native form and denatured in 1.5 M guanidinium chloride reveal a change in the fast (picosecond time scale) diffusive internal dynamics of the protein. The momentum and energy transfer dependences of the scattering for both states are fitted by an analytical model in which, on the experimentally accessible picosecond time scale and angstrom length scale, the dynamics of a fraction of the nonexchangeable hydrogens in the protein is described as a superposition of vibrations with uniform diffusion in a sphere, the rest of the hydrogens undergoing only vibrational motion. The fraction diffusing changes, from ≈60% in the native protein to ≈82% in the denatured protein. The radius of the sphere also changes slightly, from ≈1.8 Å in the native protein to ≈2.2 Å in the denatured protein. Possible implications of these results for the general protein folding problem are discussed. Proteins 28:380-387, 1997 © 1997 Wiley-Liss, Inc.

Notizen: A novel helix-coil transition theory has been developed. This new theory contains more types of interactions than similar theories developed earlier. The parameters of the models were obtained from a database of 351 nonhomologous proteins. No manual adjustment of the parameters was performed. The interaction parameters obtained in this manner were found to be physically meaningful, consistent with current understanding of helix stabilizing/destabilizing interactions. Novel insights into helix stabilizing/destabilizing interactions have also emerged from this analysis. The theory developed here worked well in sorting out helical residues from amino acid sequences. If the theory was forced to make prediction on every residue of a given amino acid sequence, its performance was the best among ten other secondary structural prediction algorithms in distinguishing helical residues from nonhelical ones. The theory worked even better if one only required it to make prediction on residues that were “predictable” (identifiable by the theory); 〉90% predictive reliability could be achieved. The helical residues or segments identified by the helix-coil transition theory can be used as secondary structural contraints to speed up the prediction of the three-dimensional structure of a protein by reducing the dimension of a computational protein folding problem. Possible further improvements of this helix-coil transition theory are also discussed. Proteins 28:344-359, 1997. © 1997 Wiley-Liss, Inc.

Notizen: A computational strategy for homology modeling, using several protein structures comparison, is described. This strategy implies a formalized definition of structural blocks common to several protein structures, a new program to compare these structures simultaneously, and the use of consensus matrices to improve sequence alignment between the structurally known and target proteins. Applying this method to cytochromes P450 led to the definition of 15 substructures common to P450cam, P450BM3, and P450terp, and to proposing a 3D model of P450eryF. Proteins 28:388-404, 1997 © 1997 Wiley-Liss, Inc.

Notizen: Computational structure prediction of streptavidin-peptide complexes for known recognition sequences and a number of random di-, tri-, and tetrapeptides has been conducted, and mechanisms of peptide recognition with streptavidin have been investigated by a new computational protocol. The structural consensus criterion, which is computed from multiple docking simulations and measures the accessibility of the dominant binding mode, identifies recognition motifs from a set of random peptide sequences, whereas energetic analysis is less discriminatory. The predicted conformations of recognition tripeptide and tetrapeptide sequences are also in structural harmony and composed of peptide fragments that are individually unfrustrated in their bound conformation, resulting in a minimally frustrated energy landscape for recognition peptides. Proteins 28:421-433, 1997. © 1997 Wiley-Liss, Inc.

Notizen: Databases of multiple sequence alignments are a valuable aid to protein sequence classification and analysis. One of the main challenges when constructing such a database is to simultaneously satisfy the conflicting demands of completeness on the one hand and quality of alignment and domain definitions on the other. The latter properties are best dealt with by manual approaches, whereas completeness in practice is only amenable to automatic methods. Herein we present a database based on hidden Markov model profiles (HMMs), which combines high quality and completeness. Our database, Pfam, consists of parts A and B. Pfam-A is curated and contains well-characterized protein domain families with high quality alignments, which are maintained by using manually checked seed alignments and HMMs to find and align all members. Pfam-B contains sequence families that were generated automatically by applying the Domainer algorithm to cluster and align the remaining protein sequences after removal of Pfam-A domains. By using Pfam, a large number of previously unannotated proteins from the Caenorhabditis elegans genome project were classified. We have also identified many novel family memberships in known proteins, including new kazal, Fibronectin type III, and response regulator receiver domains. Pfam-A families have permanent accession numbers and form a library of HMMs available for searching and automatic annotation of new protein sequences. Proteins: 28:405-420, 1997. © 1997 Wiley-Liss, Inc.

Notizen: We have crystallized the N-terminal actin binding domain (ABD1) of human fimbrin, a representative member of the largest class of actin crosslinking proteins. Diffraction from these crystals is consistent with the orthorhombic space group P212121 (a = 50.03 Å, b = 61.24 Å, c = 102.30 Å). These crystals contain one molecule in the asymmetric unit and diffract to at least 1.9 Å resolution. The crystal structure of ABD1 will be the first structure of an actin crosslinking domain. Proteins 28:452-453, 1997. © 1997 Wiley-Liss, Inc.

Notizen: The transitions between the water-equilibrated structures of the GTP and GDP forms of Ha-ras-p21 have been calculated by using the targeted molecular dynamics (TMD) method (Schlitter et al., Mol. Sim. 10:291-309, 1993) both in vacuo and with explicit solvent simulation. These constrained molecular dynamics calculations result in different pathways, depending on the nucleotide bound. Each pathway consists in a sequence of transitions affecting six segments of the protein, four of them forming a hydrophilic cleft around the nucleotide. The transitions are initiated by the removal or introduction of the γ-phosphate of the nucleotide and proceed sequentially, crossing several low-energy transition states. The movements are transmitted either by direct interactions between the segments or through the nucleotide. The GTP to GDP pathway is initiated by the removal of the nucleotide γ-phosphate. This gives some space to Gly12, Gly13, and Val14. Their movement is transmitted to the target recognition domain and the switch II region, forcing these segments to adopt another position. In a second step the target recognition domain and the switch II region undergo conformational transitions to reach an intermediate conformation. Finally, there is a relaxation of the target recognition domain to its final state that forces the switch II region to reach its target conformation. The calculated pathways allow the identification of many residues that play an important role in the conformational changes, explain the altered transformation properties of some, and suggest mutations to alter the pathway. Proteins 28:434-451, 1997. © 1997 Wiley-Liss, Inc.

Notizen: Blind predictions of the local structure of nine CASP2 targets were made using the I-sites library of short sequence - structure motifs, revealing strengths and weaknesses in this new knowledge-based method. Many turns between secondary structural elements were accurately predicted. Estimates of the confidence of prediction correlated well with the accuracy over the whole set. Bias toward structures used to develop the library was minimal, probably because of the extensive use of cross-validation. However, helix positions were better predicted by the PHD program. The method is likely to be sensitive to the quality of the sequence alignment. A general measure for evaluating local structure predictions is suggested. Proteins, Suppl. 1:167-171, 1997. © 1998 Wiley-Liss, Inc.

Notizen: A partly folded state of hen egg-white lysozyme has been characterized in 50% DMSO. Low concentrations of DMSO (〈10%) have little effect on the overall folded conformation of lysozyme as seen from 1H NMR chemical shift dispersion. At increasing DMSO concentrations (〉10%) a cooperative transition of the structure to a new, partially folded state is observed. This transition is essentially complete by ∼50% DMSO. NMR studies show an overall decrease in chemical shift dispersion with marked broadening of many resonances. A substantial number of backbone and side chain-side chain NOEs suggests the presence of secondary and tertiary interactions in the intermediate state. Tertiary organization of the aromatic residues is also demonstrated by enhanced near-UV circular dichroism and limited exposure of tryptophans as monitored by iodide quenching of fluorescence. The intermediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural transition from this state to a largely unfolded conformation is cooperative. H/D exchange rates of several amide protons and four indole protons of tryptophans (W28, W108, W111, and W123), measured by refolding from 50% DMSO at different time intervals reveal that protection factors are high for the helical domain, whereas NH groups in the triple stranded antiparallel β-sheet domain are largely solvent-exposed. An ordered hydrophobic core in the intermediate state comprising of helix A, helix B, and helix D is consistent with the high protection factors observed. The structured intermediate in 50% DMSO resembles the early kinetic intermediate observed in the refolding of hen egg white lysozyme, as well as a molten globule state of equine lysozyme at low pH. The results demonstrate the potential use of nonaqueous structure perturbing solvents like DMSO to stabilize partially folded conformations of proteins. Proteins 29:492-507, 1997. © 1997 Wiley-Liss, Inc.

Notizen: We present the results of lattice Monte Carlo simulations of protein folding in the framework of a model taking into account (i) the dependence of the energy of interaction of amino-acid residues on their orientation and (ii) the rigidity of the polypeptide chain with respect to the formation of kinks. If the chain is flexible, the final protein structures are predicted to be compact. Increasing the energy cost of creation of kinks is found to favor the formation of flat structures mimicking an ideal antiparallel β sheet. For compact structures, the kinetics of folding exhibit the standard two-phase regime (a rapid collapse to one of the metastable state, followed by slow reconfiguration of the chain to the native structure). For flat structures, the transition to the native state is often gradual. Proteins 29:508-516, 1997. © 1997 Wiley-Liss, Inc.

Notizen: The cross-species specificity of the cohesin-dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin-containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin-containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium-dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin-dockerin interaction seems to be species-specific. Based on intra- and cross-species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino-acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium-binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface. Proteins 29:517-527, 1997. © 1997 Wiley-Liss, Inc.

Notizen: X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B-cell differentiation; this incapacitates antibody production in XLA patients, who suffer from recurrent, sometimes lethal, bacterial infections. BTK plays an important role in B-cell development; it interacts with several proteins in the context of signal transduction. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in a patient family. To understand the role of BTK, we studied binding of BTK SH3 domain (aa 216-273, 58 residues) and truncated SH3 domain (216-259, 44 residues) with proline-rich peptides; the first peptide constitutes the SH3 domain of BTK, while the latter peptide lacks 14 amino acid residues of the C terminal. Proline-rich peptides selected from TH domain of BTK and p120cbl were studied. It is known that BTK TH domain binds to SH3 domains of various proteins. We found that BTK SH3 domain binds to peptides of BTK TH domain. This suggests that BTK SH3 and TH domains may associate in inter- or intramolecular fashion, which raises the possibility that the kinase may be regulating its own activity by restricting the availability of both its ligand-binding modules. We also found that truncated SH3 domain binds to BTK TH domain peptide less avidly than does normal SH3 domain. Also, we show that the SH3 and truncated SH3 domains bind to peptide of p120cbl, but the latter domain binds weakly. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context, hence the weaker binding. These results delineate the importance of C terminal in binding of SH3 domains and indicate also that improper folding and the altered binding behavior of mutant BTK SH3 domain likely leads to XLA. Proteins 29:545-552, 1997. © 1997 Wiley-Liss, Inc.

Notizen: Interleukin-6 (IL-6) is a multifunctional cytokine that regulates cell growth, differentiation, and cellular functions in many cell lineages. Recently, evidences for the formation of an active hexameric complex with an IL-6:IL-6Rα:gp130 stoichiometry of 2:2:2 have been obtained by different experimental approaches. Analysis of the electrostatic potential complementarity between IL-6 and its receptors has been used, in this study, to guide the assembly of homology-based 3D models of the components. The results strongly support a mechanism whereby the active cytokine (IL-6:IL-6Rα) associates with the signal transducing gp130 protein, and the trimeric complex formed further dimerizes to form the hexameric species. Furthermore, computational simulations of the multiprotein complexes provide a rationalization of data from mutation experiments and highlight some key protein-protein interactions which have not yet been the subject of mutagenesis studies. Proteins 29:528-544, 1997. © 1997 Wiley-Liss, Inc.

Notizen: Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had 〈10-5 of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon. Proteins 29:553-561, 1997. © 1997 Wiley-Liss, Inc.

Notizen: The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemoglobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca. 400 kDa), and one in the coelomic fluid, C1 (ca. 400 kDa). V1 Hb consists of four heme-containing, globin chains (b-e) and four linker chains (L1-L4). V2 and C1 Hbs are exclusively built from globin chains, six for V2 (a-f) and five for C1 (a-e). The complete amino acid sequence of the isolated monomeric globin chain b, common to all Riftia Hbs, has been determined by automated Edman degradation sequencing of the peptides derived by digestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polypeptide chain is composed of 144 amino acid residues, providing a Mr of 16, 135.0 Da. Moreover, the primary sequence of chain b revealed 3 Cys residues at position 4, 75, and 134. Cys-4 and Cys-134 are located at positions where an intra-chain disulfide bridge is formed in all annelid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lamellibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosynthetic process of the symbiotic bacteria they harbor. Sulfide-binding experiments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) suggest that free Cys residues on globin chains, and the numerous Cys found in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites. Blocking the free Cys by N-ethylmaleimide, we confirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermore, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vetimentiferan, and pogonophoran extracellular Hbs to clarify the systematic position of tubeworms. Riftia chain b clearly belongs to the “strain A” family with 30 to 80% identity with the other sequences analyzed. Its position in the tree confirmed a close relationship between vestimentiferan, pogonophoran, and annelid Hbs. Proteins 29:562-574, 1997. © 1997 Wiley-Liss, Inc.

Notizen: No abstract.

Notizen: No abstract.

Notizen: Computer simulations have been used to design a polypeptide with a 310 helix conformation. The study has been been performed taking advantage of the intrinsic helix forming tendency of α-Aminoisobutyric acid. In order to avoid the formation of the α helix, which is the other common helical conformation adopted by α-Aminoisobutyric acid-based peptides, retropeptide bonds have been included in the sequence. Thus, retropeptides are not able to form the intramolecular hydrogen bonding interactions characteristic of the α helix. The influences of both the peptide length and the solvent have been examined and compared with those of the polypeptide without retropeptide bonds. Proteins 29:575-582,1997. © 1997 Wiley-Liss, Inc.

Notizen: Five models by homology containing insertions and deletions and ranging from 33% to 48% sequence identity to the known homologue, and one high sequence identity (85%) model were built for the CASP2 meeting. For all five low identity targets: (i) our starting models were improved by the Internal Coordinate Mechanics (ICM) energy optimization, (ii) the refined models were consistently better than those built with the automatic SWISS-MODEL program, and (iii) the refined models differed by less than 2% from the best model submitted, as judged by the residue contact area difference (CAD) measure [Abagyan, R.A., Totrov, M.J. Mol. Biol. 268:678-685, 1997]. The CAD measure is proposed for ranking models built by homology instead of global root-mean-square deviation, which is frequently dominated by insignificant yet large contributions from incorrectly predicted fragments or side chains. We demonstrate that the precise identification of regions of local backbone deviation is an independent and crucial step in the homology modeling procedure after alignment, since aligned fragments can strongly deviate from the template at various distances from the alignment gap or even in the ungapped parts of the alignment. We show that a local alignment score can be used as an indicator of such local deviation. While four short loops of the meeting targets were predicted by database search, the best loop 1 from target T0028, for which the correct database fragment was not found, was predicted by Internal Coordinate Mechanics global energy optimization at 1.2 Å accuracy. A classification scheme for errors in homology modeling is proposed. Proteins, Suppl. 1:29-37, 1997.© 1998 Wiley-Liss, Inc.

Notizen: We constructed five comparative models in a blind manner for the second meeting on the Critical Assessment of protein Structure Prediction methods (CASP2). The method used is based on a novel graph-theoretic clique-finding approach, and attempts to address the problem of interconnected structural changes in the comparative modeling of protein structures. We discuss briefly how the method is used for protein structure prediction, and detail how it performs in the blind tests. We find that compared to CASP1, significant improvements in building insertions and deletions and sidechain conformations have been achieved. Proteins, Suppl. 1:43-49, 1997. © 1998 Wiley-Liss, Inc.

Notizen: An assessment is presented for all submissions to the comparative modeling challenge in the 1996 Critical Assessment of Structure Prediction (CASP2). Of the original 12 target structures, 9 were solved prior to the meeting: 8 by X-ray crystallography and 1 by NMR spectroscopy. These targets varied over a large range of difficulty, as assessed by the percentage sequence identity with the principal parent structure, which ranged from 20% up to 85%. The overall quality of the models reflected the similarity of the principal parent. As expected, when the sequence alignment was correct, the core was accurately modeled, with the largest deviations occurring in the loops. Models were built which gave Cα root-mean-square deviations (RMSDs) compared with the observed structure of 〈1 Å for targets with high parental similarity; even at 26% sequence identity, the best model structures had Cα deviations of only 2.2 Å. Overall, these deviations are comparable with those observed between the parent structure and the target, but locally there are several examples where the model approaches closer to the target than does the parent. There were three targets below 25% sequence identity, and the models generated for these targets were, in general, significantly less accurate. This principally reflects errors in the alignment which, if systematically shifted, can generate Cα RMSDs 〈18 Å. Compared with CASP1, the geometry of the models was significantly improved with no D-amino acids. By far the major contribution to RMSD error was the alignment accuracy, which varied from 100% down to 7% over the range of targets. In the structurally variable regions, global shifts, caused by hinge bending, were the major source of error, giving significantly lower local RMSDs than global RMSDs. In over 50% of these noncore regions, the difference between global and local RMSDs was more than 3 Å, and was as high as 10 Å for one structurally variable region. For the side chains, the χ1 RMSDs are strongly correlated with the Cα RMSDs. For models with Cα deviations less than 1 Å, on average 78.5% of side chains are placed in the correct rotamer, although the χ1 RMSDs, though clearly better than random, were disappointing at around 46°. As the backbone deviations increased, the side chain placement became less accurate, with an average χ1 RMSD of 75° on a 1.5-2.5 Å Cα backbone (average 51.4% correct rotamer). Refinement by energy minimization or molecular dynamics made only minor adjustments to improve local geometry and generally made small, but not significant, improvements to the RMSD. In total, 19 groups submitted 62 models (89 coordinate sets) that could be assessed. Most modelers used manual adjustments to sequence alignments and, in general, good alignments were obtained down to 25% sequence identity. The modeling methods ranged from “classical” modeling, involving core building followed by loop and side chain addition, to more sophisticated approaches based on probability distributions, Monte Carlo sampling or distance geometry. For each target, several groups produced equally good models, given the expected errors in the structures (about 0.5 Å). No one method came out as clearly superior, although the approaches that inherit directly from the parents generally performed better than the more radical techniques. However, for each target there were some poor models, usually reflecting a poor sequence alignment, and the range of accuracy for each target is therefore large. Fully automated methods are able to perform very well for “easy” targets (85% sequence identity with parent), but when modeling using a distantly related parent, care and expertise, especially in performing the alignment, still appear to be important factors in generating accurate models. Proteins, Suppl. 1:14-28, 1997. © 1998 Wiley-Liss, Inc.

Notizen: Comparative modeling targets 1, 3, 9, and 17 were predicted by alignment of multiple sequences and structures, when available, followed by minimization using the program AMMP. The minimization used improved potentials, and distance restraints for regions of common structure. New prediction procedures were evaluated. Three tested solvent corrections did not significantly improve the predictions. Target 17 had 85.3% sequence identity with the parent and no insertions or deletions. The prediction had a root-mean-square deviation from target 17 of 0.56 Å on Cα atoms, and 0.59 Å for the ligand atoms, which verified the accuracy of the minimization. Targets 1, 3, and 9 had 36.4%, 46.7%, and 33.3% identity with the parent sequences, and predictions resulted in root-mean-square deviations for 79-85% of Cα atoms of 1.49, 1.11, and 1.24 Å, respectively. Conformational differences between parent and target crystal structures were difficult to predict. The use of distance restraints and multiple structures improved the positioning of gaps in sequence alignment. Distance restraints did not overcome errors in sequence alignment or ambiguities due to conformational variation in proteins. Predictions for targets 3 and 9 successfully reduced large deviations between parent and target structures. Proteins, Suppl. 1:68-73, 1997. © 1998 Wiley-Liss, Inc.

Notizen: No abstract.

Notizen: The stability of the α helix as an element of secondary structure is examined in the absence of solvation, in the gas phase. Mass-analyzed ion kinetic energy (MIKE) spectrometry was applied to measure intercharge repulsion and intercharge distance in multiply protonated melittin, a polypeptide known to possess a stable helical structure in a number of different environments. The experimental results, interpreted in combination with molecular mechanics calculations, suggest that triply charged melittin retains its secondary structure in the gas phase. The stability if the α-helical conformation of the polypeptide in the absence of solvent molecules reflects the fact that a network of intrinsic helical hydrogen bonds is energetically more favorable than unfolded conformations. © 1997 Wiley-Liss, Inc.

Notizen: The structure of a complex between a hexapeptide-based inhibitor, MVT-101, and the chemically synthesized (Aba 67,95,167,195; Aba: l-α-amino-n-butyric acid) protease from the human immunodeficiency virus (HIV-1), reported previously at 2.3 Å has now been refined to a crystallographic R factor of 15.4% at 2.0 Å resolution. Root mean square deviations from ideality are 0.18 Å for bond lengths and 2.4° for the angles. The inhibitor can be fitted to the difference electron density map in two alternative orientations. Drastic differences are observed for positions and interactions at P3/S3 and P3′/S3′ subsites of the two orientations due to different crystallographic environments. © 1997 Wiley-Liss, Inc.

Notizen: Six models of the catalytic site of HIV-1 protease complexed with a reduced peptide inhibitor, MVT-101, were investigated. These studies focused on the details of protonation of the active site, its total net charge and hydrogen bonding pattern, which was consistent with both the observed coplanar configuration of the acidic groups of the catalytic aspartates (Asp-25 and Asp-125) and the observed binding mode of the inhibitor. Molecular dynamic simulations using AMBER 4.0 indicated that the active site should be neutral. The planarity of the aspartate dyad may be due to the formation of two hydrogen bonds: one between the inner Oδ1oxygen atoms of the two catalytic aspartates and another between the Oδ2atom of Asp-125 and the nitrogen atom of the reduced peptide bond of the bound inhibitor. This would require two additional protonations, either of both aspartates, or of one Asp and the amido nitrogen atom of Nle-204. Our results favor the Asp-inhibitor protonation but the other one is not excluded. Implications of these findings for the mechanism of enzymatic catalysis are discussed. Dynamic properties of the hydrogen bond network in the active site and an analysis of the interaction energy between the inhibitor and the protease are presented. © 1997 Wiley-Liss, Inc.