Toxin Production in Clostridium botulinum as Demonstrated by Electron Microscopy

Spheroplasts of Clostridium botulinum 62A were prepared with the use of lysozyme. These spheroplasts were then exposed to ferritin-labeled type A antitoxin. Ultrathin sections of these specimens revealed the ferritin-labeled antibody symmetrically arranged around the outer spore coats but not within the spore cortex. The ferritin-labeled antibody was also observed in the bacterial cytoplasm. Here it was arranged in aggregates and strands, although it was not associated with any identifiable cell structure. Controls included sections of C. botulinum spheroplasts treated with a 1.5% solution of ferritin as well as spheroplasts of C. roseum and Bacillus subtilis treated with conjugated type A antitoxin or a 1.5% solution of ferritin. No intracellular or extracellular ferritin was demonstrable in these specimens.     

Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes

Crude striatum synaptosomes (P₂ fraction) from Fisher 344 female rats were incubated in the presence of ADP-chelated Fe³⁺ (0.5–50 M) and ascorbate (250 M). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by¹⁴CO₂ evolution froml-[1-¹⁴C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe³⁺/ascorbate was found with 50% inhibition occurring at 2.5 M Fe³⁺ concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe³⁺ in the absence of exogenous ascorbate was effective only above 25 M. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P₂ fractions using sucrose gradients. Membrane microviscosity of the purifled synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe³⁺/ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulhydryl content. It is concluded that ironinduced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

Oscillations in the electrical resistance of Nitellopsis obtusa nodes

Oscillatory changes of the electrical resistance across the nodal complex of Nitellopsis obtusa (Desv. in Lois) J. Gr. were observed in experiments performed for 40–150 min with the use of external electrodes and microelectrodes. Three main patterns of node resistance oscillations were similar to those found for membrane potential and resistance. The presented findings indicate an oscillatory behaviour of the plasmodesmata system at the node, which may be connected with e.g. pulsatile variations in the number of open plasmodesmata.     

Partial characterization of allatinhibin,a neurohormone of Manduca sexta

During the last larval stage, corpora allata (CA) of Manduca sexta are inactivated by a factor from the brain. Apparently the same factor (allatinhibin, Al) is secreted by day 4 Vth instar brains kept overnight in Grace's medium. Al is rapidly inactivated by heat or acid but withstands exposure to alkali and can be recovered after freezing and lyophilization. Exposure to pronase, chymotrypsin, carboxypeptidases-A and-Y, as well as leucine aminopeptidase eliminated Al activity completely, whereas after exposure to trypsin and protease XVII-S, some residual activity remained. Inactivation by pyroglutamate aminopeptidase is interprefed as being due to prolinase activity of this enzyme. Incubation of CA with gentamicin, an aminoglycoside antibiotic, affects neither their ability to produce JH in vitro nor their viability in implantation assays. However, Al did not inactivate CA in the presence of low concentrations of gentamicin. This effect was used to guard against false positive assay results possibly produced by allatotoxic contamination. Al was purified by chromatography on Sephadex G-25. All activity recovered emerged from the columns in intermediate fractions with an apparent Mr of 1,000–2,000. © 1993 Wiley-Liss, Inc.     

Acoustic observations of krill (Euphausia superba) at the ice edge (between elephant I. and south Orkney I., Dec. 1988/Jan. 1989)

The effect of the ice edge on Antarctic krill abundance, swarm parameters, distribution and migration, were investigated using acoustics. Two parameters, overall abundance and inter-swarm distance were found to increase with distance from the ice edge, while the number of swarms per unit distance decreased. Swarm dimensions, length and thickness do not seem to depend on proximity of ice. Krill near the ice-edge undergo diurnal vertical migration with a periodicity of 12 hours and an amplitude of about 6 m. Juvenile krill of 31 mm were dominant in the area investigated.     

Insoluble glycogen, a metabolizable internal adsorbent, decreases the lethality of endotoxin shock in rats

Insoluble glycogen is an enzymatically modified form of naturally occurring soluble glycogen with a great adsorbing capacity. It can be metabolized by phagocytes to glucose. In this study we used insoluble glycogen intravenously in the experimental endotoxin shock of rats. Wistar male rats were sensitized to endotoxin by Pb acetate. The survival of rats were compared in groups of animals endotoxin shock treated and non-treated with insoluble glycogen. Furthermore, we have determined in vitro the binding capacity of insoluble glycogen for endotoxin, tumour necrosis factor alpha, interleukin-1 and secretable phospholipase A2. Use of 10 mg/kg dose of insoluble glycogen could completely prevent the lethality of shock induced by LD50 quantity of endotoxin in rats. All animals treated survived. Insoluble glycogen is a form of 'metabolizable internal adsorbents'. It can potentially be used for treatment of septic shock.     

Replication variants of the human inactive X chromosome

Replication variants of the inactive X chromosome were investigated in lymphocytes from six donors by means of terminal BrdU or thymidine incorporation. There were interindividual differences in the incidence of particular variants. In endoreduplicated and tetraploid cells both allocyclic X chromosomes showed the same replication sequence. The Xp22 band of the allocyclic X chromosome seemed to replicate later than the homologous material in some cells. Initiation time of DNA synthesis within the inactive X chromosome was found to be stable; termination time, however, varied greatly relative to the other chromosomes. Early completion of replication within the heterochromatic X chromosome could be demonstrated preferentially for the Xq25–27 terminal sequence, but other variants expressed the phenomenon also. A variable replication rate of the inactive X chromosome is believed to be responsible for its asynchronous, independent replication. The biological significance of the phenomenon is discussed with respect to cell differentiation.     

Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH₂-10-CH₃PteGlu₄). When the time-dependent synthesis of MTX polyglutamates (4-NH₂-10-CH₃PteGlu₂ and higher) at extracellular concentrations of 10 and 100 μm methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μm is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μm intracellular methotrexate, respectively, after 24 h in the presence of 100 μm extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μm with both cell types which corresponded to 10 to 12 μm intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu_4–5) are more avidly retained by the cells than the shorter ones (Glu_2–3).