A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with M _r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.     

Intragranular co-storage of neuropeptide Y and arginine vasopressin in the paraventricular magnocellular neurons of the rat hypothalamus

Summary Certain populations of arginine vasopressin (AVP) neurons in the magnocellular paraventricular nucleus became immunoreactive for neuropeptide Y (NPY) when rats were treated with colchicine or monosodium glutamate (MSG). The co-storage of these peptides was examined by empooying a post-embedding electron-microscopic immunohistochemistry technique using goldlabeled antibodies to the two peptides. In colchicinetreated rats, the neuronal perikarya contained numerous secretory granules showing co-storage of the two peptides. The cells of the MSG-treated rats were characterized by having well-developed Golgi bodies with the granular structures also co-storing the two peptides, although the secretory granules in the perikarya were rather fewer than in the colchicine-treated rats. It is concluded that the destruction of the arcuate nucleus by MSG-treatment may potentiate the synthesis of NPY in AVP neurons, the synthesis of which is latent in intact animals.     

Oxidation of glycerol,lactate, and propionate by Propionibacterium freudenreichii in a poised-potential amperometric culture system

Growth of Propionibacterium freudenreichii was studied with glycerol, lactate, and propionate as energy sources and a three-electrode poised-potential amperometric electrode system with hexacyanoferrate (III) as mediator. In batch culture experiments with glycerol and lactate as substrates, hexacyanoferrate (III) was completely reduced. Growth yields increased and the fermentation patterns were shifted towards higher acetate formation with increasing hexacyanoferrate (III) concentrations (0.25–8.0 mM). In experiments with regulated electrodes, glycerol, lactate, and propionate were oxidized to acetate and CO₂, and the electrons were quantitatively transferred to the working electrode. Growth yields of 29.0, 13.4 and 14.2 g cell material per mol were calculated, respectively. The high cell yield obtained during propionate oxidation cannot be explained solely by substrate level phosphorylation indicating that additional energy was conserved via electron transport phosphorylation. Furthermore, this result indicated complete reversibility of the methyl-malonyl-CoA pathway in propionic acid bacteria.     

Cellular localization of cytochrome c 553 in the N2-fixing cyanobacterium Anabaena variabilis

The in situ location of the electron carrier protein cytochrome C ₅₅₃ (cyt c ₅₅₃) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c ₅₅₃ specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c ₅₅₃ during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c ₅₅₃ is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c ₅₅₃ cytochrome c ₅₅₃ - PBS phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4) - PMSF phenylmethylsulfonyl fluoride Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz.     

Evidence that the capsule around mycobacteria grown in axenic media contains mycobacterial antigens: implications at the level of cell envelope architecture

The intracellular growth of pathogenic mycobacteria has been linked to the presence of an electron transparent zone (ETZ or capsule), which surrounds the phagocytized bacteria and prevents the diffusion of lysosomal enzymes in infected macrophages. Recently, it was suggested that this capsule may be a bacterial structures, even being present in test tube-grown pathogenic mycobacteria (FEMS Microbiol. Lett. 1988, 56, 225-230). In the present paper, we show that under special fixation and embedding conditions, this capsule was clearly observed among 7 strains of mycobacteria grown in axenic media and also in M. leprae extracted and purified from experimentally infected armadillo or nude mice. In the case of bacteria treated likewise but subject to a prior dehydration step, this capsular structure disappeared suggesting its lipidic nature. Ultrathin sections of M. intracellular after immunolabelling showed for the first time that this capsule obtained mycobacterial antigens confirming its mycobacterial origin. It is suggested that the mycobacterial capsule may be formed of inert lipids, in which surface antigens are embedded.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.     

The efferent innervation of the genital chamber by an identified serotonergic neuron in the female cricket Acheta domestica

Summary The serotonergic innervation of the genital chamber of the female cricket, Acheta domestica, has been investigated applying anti-serotonin (5-HT) immunocyto-chemistry at both light- and electron-microscopic levels as well as using conventional electron microscopy. Whole mount and pre-embedding chopper techniques of immuno-cytochemistry reveal a dense 5-HT-immunoreactive network of varicose fibers in the musculature of the genital chamber. All of these immunoreactive fibers originate from the efferent serotonergic neuron projecting through the nerve 8v to the genital chamber (Hustert and Topel 1986; Elekes et al. 1987). At the electron-microscopic level, 5-HT-immunoreactive nerve terminals, which contain small (50–60 nm) and large ( 100 nm) agranular vesicles as well as granular vesicles (100nm), contact the muscle fibers or the sarcoplasmic processes without establishing specialized neuromuscular connections. In addition to the 5-HT-immunoreactive axons, two types of immunonegative axons can also be found in the musculature. By use of conventional electron microscopy, three ultrastructurally distinct types of axon processes can be observed, one of which resembles 5-HT-immunoreactive axons. While the majority of the varicosities do not synapse on the muscle fibers, terminals containing small (50–60 nm) agranular vesicles occasionally form specialized neuromuscular contacts. It is suggested that the 5-HTergic innervation plays a non-synaptic modulatory role in the regulation circular musculature in the genital chamber of the cricket, while the musculature as a whole may be influenced by both synaptic and modulatory mechanisms.Fellow of the Alexander von Humboldt-Stiftung     

Light- and electron-microscopic immunocytochemistry of peptidergic neurons innervating thoracico-abdominal neurohaemal areas in the blowfly

Summary Ventral thoracic neurosecretory cells (VTNCs) of the blowflies, Calliphora erythrocephala and C. vomitoria, innervating thoracic neuropil and the dorsal neural sheath of the thoracico-abdominal ganglion have been shown to be immunoreactive to a variety of mammalian peptide antisera. In the neural sheath the VTNC terminals form an extensive neurohaemal network that is especially dense over the abdominal ganglia. The same areas are invaded by separate, ut overlapping serotonin-immunoreactive (5-HT-IR) projections derived from neuronal cell bodies in the suboesophageal ganglion. Immunocytochemical studies with different antisera, applied to adjacent sections at the lightmicroscopic level, combined with extensive cross-absorption tests, suggest that the perikarya of the VTNCs contain co-localized peptides related to gastrin/cholecystokinin (CCK), bovine pancreatic polypeptide (PP), Met- and Leuenkephalin and Met-enk-Arg⁶-Phe⁷ (Met-enk-RF). Electron-microscopic immunogold-labeling shows that some of the terminals in the dorsal sheath react with several of the individual peptide antisera, whilst others with similar cytology are non-immunoreactive. In the same region, separate terminals with different cytological characteristics contain 5-HT-IR. Both 5-HT-IR and peptidergic terminals are localized outside the cellular perineurium beneath the acellular permeable sheath adjacent to the haemocoel. Hence, we propose that various bioactive substances may be released from thoracic neurosecretory neurons into the circulating haemolymph to act on peripheral targets. The same neurons may also interact by synaptic or modulatory action in the CNS in different neuropil regions of the thoracic ganglion.