Macrocyclic trichothecene toxins produced by Stachybotrys atra strains isolated in Middle Europe.

A total of 17 strains of Stachybotrys atra isolated in Hungary and Czechoslovakia were cultured on Sabouraud agar, and the toxins produced by them were chemically analyzed by gas-liquid chromatography, high-pressure liquid chromatography, and mass spectroscopy. Furthermore, brine shrimp (Artemia salina) bioassay was used for the determination of toxicity of the compounds examined. Macrocyclic trichothecenes (satratoxins H and G, roridin E, and verrucarin J as well as two other unidentified macrocyclic trichothecenes) were found in all of the cultures tested. The identities of satratoxins H and G, roridin E, and verrucarin J were qualitatively determined by high-pressure liquid chromatography and gas-liquid chromatography. The ratio of satratoxins H and G and roridin E was found to be similar in each of the strains tested, but the amount of verrucarin J found was different in each of them. One of the unidentified macrocyclic trichothecenes was equivalent to the compound isolated by Harrach et al. (Harrach et al., Appl. Environ. Microbiol. 41:1428-1433, 1981). The other one proved to be a newly isolated macrocyclic trichothecene toxin. Stachybotryotoxicosis, one of the oldest mycotoxicoses known, and a serious problem in Middle Europe (Gy. Danko, Magy. Allatorv. Lapja 31:226-232, 1976), is believed to be caused by macrocyclic trichothecene toxins produced by Stachybotrys atra (R. M. Eppley, in Rodricks et al., ed., Mycotoxins in Human and Animal Health, p. 285-293, 1977). Forty years ago, the death of animals in the Soviet Union was associated with this fungus (C. U. Ruhliada, in Proceedings of the All-Union Sci. and Tech. Conf., p. 47-51, 1980).(ABSTRACT TRUNCATED AT 250 WORDS)     

Impairment of the calcium pump of human erythrocytes by divicine

Divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, produces a remarkable and consistent inactivation of the Ca2+-ATPase activity of the erythrocyte calcium pump. The patterns of inactivation are similar in normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes. Inactivation of Ca2+-ATPase is apparently unrelated to the cellular GSH system, to the proteolytic machinery of mature erythrocytes, and to calmodulin, and also occurs in hemoglobin-free, unsealed erythrocytes membranes at 50-100 microM concentrations of divicine. Analysis of erythrocytes that have escaped destruction during the acute hemolytic crisis of a number of favic patients revealed a dramatic elevation of erythrocyte calcium and a significant decrease of Ca2+-ATPase activity. These results support the view that divicine plays a toxic role in the pathogenesis of favism and suggest that acute electrolyte imbalances, mostly affecting calcium homeostasis, are involved in the mechanisms of erythrocyte damage and destruction in this hemolytic disease.     

Effects of glutathione depletion on gluconeogenesis in isolated hepatocytes

Glutathione-depleted hepatocytes, by incubation with diethylmaleate (DEM) or phorone (2,6-dimethyl-2,5-heptadiene-4-one), i.e., substrates of the GSH S-transferases (EC 2.5.1.18), showed rates of gluconeogenesis from various precursors significantly lower than controls; however the rate of glucose synthesis from fructose was similar to that of controls. Isolated hepatocytes from rats pretreated with those substrates 1 h before isolation to deplete hepatic glutathione (GSH) also showed a decrease of the rate of gluconeogenesis from lactate plus pyruvate. Incubation of hepatocytes with L-buthionine sulfoximine, a specific inhibitor of gamma-glutamyl-cysteine synthetase (EC 6.3.2.2), resulted in a decreased rate of gluconeogenesis from lactate plus pyruvate only when GSH values were lower than 1 mumol/g cells. Freeze-clamped livers from GSH-depleted rats showed a higher concentration of malate and glycerol 3-phosphate, indicating that GSH depletion probably affects phosphoenolpyruvate carboxykinase and glycerol-3-phosphate dehydrogenase activities. Several indicators of cell viability, such as lactate dehydrogenase leakage, malondialdehyde accumulation, ATP concentration, or urea synthesis from different precursors, were not affected by GSH depletion under the experimental conditions used here. Besides, the GSH/GSSG ratio remained unchanged in all cases.     

Control of sugar transport in human fibroblasts independent of glucose metabolism or carrier-substrate interaction

Transport regulation by different metabolizable and nonmetabolizable sugars was studied in human fibroblasts. Sugars were classed as glucose-like (D-mannose, 3-0-methyl-D-glucose, thio-D-glucose, and D-allose) and starvation-like (D-galactose, D-fructose, L-glucose, D-xylose, 6-deoxy-D-glucose and 2-deoxy-D-glucose) based on their competence in curbing glucose starvation enhanced transport. No significant correlation existed between the ability of a sugar to curb hexose transport and the KI of that sugar in inhibiting hexose transport. Independence of the transport curb from glucose metabolism was observed since nonmetabolizable analogs of D-glucose when substituted for D-glucose in the culture medium effected glucose [i.e. 3-0-methyl-D-glucose (3-OMG)] and starvation-like (i.e. 6- and 2-deoxy-D-glucose) effects. The KI of inhibition pf 2-deoxy-D-glucose transport for 3-OMG was 8.5 mM, similar to those obtained for 6-deoxyglucose and 2-deoxyglucose on 2-deoxyglycose transport (7.5 and 3.5 mM, respectively) and on 3-0-methylglucose transport (3.5 and 2.5 mM, respectively). An equimolar mixture of D-glucose and 3-OMG (5.55 mM each) was more effective than 11.1 mM D-glucose or 3-OMG alone in curbing hexose transport or reversing hexose starvation induced increases in transport. The effect of 3-OMG may be independent of glucose metabolism but it is possible that 3-OMG structurally mimics a metabolite of glucose that may interact with intracellular regulators of carrier degradation and or expression.     

The use of monoclonal antibodies in the study of the interaction between adrenal medullary cell membranes and chromaffin granules

Simultaneous incubation of bovine adrenal medullary plasma membranes (PM) with chromaffin granules (CG) resulted in the release of the soluble granular content. The molecular mechanism of this process was studied with several monoclonal antibodies (mAb) raised against different plasma membrane components. Specific inhibition of the catecholamine secretion was obtained upon incubation with the monoclonal antibody UIA/NEU/VI B17. The corresponding antigen had an apparent molecular weight of 54000 Dalton. These results suggest a specific recognition between proteins located on the plasma membrane and chromaffin granule membrane, the interaction of which mediates exocytosis.     

Possible mechanism of the allosteric activation of cAMP receptor protein

Secondary structure of cAMP receptor protein of E. coli was predicted and compared to its crystal structure in the complex with cAMP solved by McKay and Steitz. The two conformations coincide in the DNA binding domain but strikingly differ in the other domain which binds cAMP and causes protein dimerization. The comparison indicates that cAMP destabilizes a very long helix instead of which sheets are formed creating a hydrophobic pocket where cAMP binds. Consequently, the helix-sheets isomerization and a resulting change in the relative monomer disposition in the dimer appears to be the origin of cAMP-induced allosteric activation of the protein. Extremely long helices were also predicted in the regions of the regulatory subunit of cAMP-dependent protein kinase from bovine cardiac muscle where cAMP binds. It is thus likely that the proposed mechanism of the effect of cAMP on protein structure has wider implications.     

Requirements and functions of vesicular stomatitis virus L and NS proteins in the transcription process in vitro

The L and NS proteins of vesicular stomatitis virus were purified from transcribing ribonucleoprotein complex and were used to study their requirements and functions during reconstitution of RNA synthesis in vitro. The requirements for L and NS proteins for optimal RNA synthesis were found to be catalytic and stoichiometric, respectively. Addition of increasing amounts of NS protein to N-RNA template and saturating L protein, the ratio of N-mRNA to leader RNA synthesis increased linearly. In contrast, when the concentration of L protein was increased the corresponding ratio remained constant. These results, coupled with the observation that the L protein is involved in the initiation of RNA synthesis, suggest that the NS protein is involved in the RNA chain elongation step. The NS protein possibly interacts with both the L protein and the template N-RNA and unwinds the latter to facilitate the movement of L protein on the template RNA.     

The isolation and genetic analysis of aCaenorhabditis elegants translocation (szT1) strain bearing an X-chromosome balancer

A single X-chromosome balancer-bearingCelegans ♂+, as a founder of a strain (AF1), was isolated directly from Fl progeny of irradiated+dpy-8unc-3/lon-2++ hermaphrodites on the basis of the absence of recombinant F2 categories. The balancer chromosome (Bal-X-1) suppresses recombination over a two-thirds section of the X chromosome (between genesdpy-8 andlet-2) and is associated with a reciprocal translocation between linkage groups (LG) X and I. Animals homozygous for the translocation (szT1(X:1)) are nonviable. Hermaphrodites heterozygous for the translocation segregate male selfprogeny at a frequency of 0.08-0.12.Bal-X-l carries the marker mutationlon-2(e678) and can be detected cytologically. This balancer chromosome proved useful for rnaimaininga number of X-linked lethal mutations and deficiencies inC. elegans.     

Additional differentiating characters of the two subspecies of Staphylococcus hyicus

Forty-five strains of Staphylococcus hyicus subsp. hyicus and 36 strains of S. hyicus subsp. chromogenes were examined for bacteriolytic activity with the same assay system previously used in taxonomic studies on staphylococci. The two subspecies differed from each other chiefly in that for optimal lytic activity S. hyicus subsp. hyicus strains required a higher salt concentration in the test medium than S. hyicus subsp. chromogenes strains. The lack of lytic activity on B15TP1 medium was a major difference between S. hyicus and S. aureus, and the lack of activity on TP2P medium was a major difference between S. hyicus and S. intermedius. Penicillin-binding proteins (PBPs) were studied in 40 S. hyicus strains. The S. hyicus subsp. hyicus strains had only one PBP (mol. wt 79 000) while the S. hyicus subsp. chromogenes strains had three distinct PBPs (mol. wts 84 000, 82 000 and 79 000).     

The ftsA gene product: a possible connection between DNA replication and septation in Escherichia coli

The study of Escherichia coli strain D-2, which harbours the ftsA2(ts) allele, has shown that temperature-induced filaments of this strain can divide, at 30 degrees C, in the absence of DNA replication and translation. Strain D-2 is thermosensitive during a period coincident with that in which the termination protein should be synthesized and exert its action. The ftsA gene product, which participates in the structure of the septum, needs for its synthesis a short period of DNA replication. The FtsA protein could be involved in a mechanism that coordinates chromosome replication and cell division by a pathway different from and independent of the SOS-induced response.