Isolation of Clostridium botulinum type G from Swiss soil specimens by using sequential steps in an identification scheme.

After Clostridium botulinum type G organisms and toxin were identified in necropsy specimens in cases of unexplained death in adults and infants (O. Sonnabend, W. Sonnabend, R. Heinzle, T. Sigrist, R Dirnhofer, and U. Krech, J. Infect. Dis. 143:22-27, 1981), extensive research to detect C. botulinum type G in soil samples from Switzerland was done. A total of 41 specimens from virgin soil and from cultivated land were examined for the presence of C. botulinum type G and other toxin types. Because of the lack of the lipase marker in type G, the detection of C. botulinum type G was based on the demonstration of type G organisms in enrichment cultures by a type G-specific enzyme-linked immunosorbent assay to detect both the type G toxin and antigen; enrichment cultures in which type G toxin or antigen was identified by enzyme-linked immunosorbent assay were then tested by a type G-specific gel immunodiffusion agar procedure. This method not only isolated strains of type G but also strains of Clostridium subterminale, a nontoxigenic variant of C. botulinum type G. As a consequence of the observed cross-reactions caused by strains of C. subterminale within this test system, all isolates of type G had to be definitively confirmed by mouse bioassay. The sequential steps of these methods seem to be very useful for detecting C. botulinum type G organisms. C. botulinum type G strains were isolated in five soil samples from different locations in close association with cultivated land.(ABSTRACT TRUNCATED AT 250 WORDS)     

NIH 3T3 cells transfected with human tumor DNA lose the transformed phenotype when treated with swainsonine

Phenotypic expression of transformation was inhibited by swainsonine at concentrations which affect the late stages of glycoprotein processing but not growth of cells. In the presence of swainsonine, NIH 3T3 fibroblasts transfected with human tumor DNA (al-l) no longer grew in soft agar or expressed complex type oligosaccharides characteristic of transformed cells. Thus, it appears that glycoproteins with fully processed oligosaccharides are necessary for the maintenance of the transformed phenotype in these cells.     

Differential expression of multiple protein kinase C subspecies in rat central nervous tissue

Protein kinase C from a number of areas of rat central nervous tissue was resolved into three distinct fractions upon hydroxyapatite column chromatography. One of the enzyme fractions, designated type II, could be further distinguished into two subspecies with polyclonal antisera, which were raised against synthetic peptides specific for the predicted amino acid sequences of two alternative cDNA clones encoding this enzyme type. Using a combination of these biochemical and immunological techniques, the relative activity of the multiple subspecies of protein kinase C was assessed for each brain area. A distinct regional pattern of expression was found, which per se may be an important factor in determining the response of different neuronal cell types to extracellular stimuli.     

Vasopressin increases 45Ca2+ influx in rat aortic smooth muscle cells

[Arg8]Vasopressin (AVP)-induced 45Ca2+ influx was examined in vascular smooth muscle cells derived from rat aorta. AVP stimulated the 45Ca2+ influx in a concentration-dependent manner. The effect was abolished in the presence of La3+. The dihydropyridine calcium channel antagonist darodipine did not affect the AVP-induced influx of 45Ca2+. These data suggest that AVP stimulates in these cultured aortic smooth muscle cells a receptor-operated channel (ROC) that is permeable to Ca2+.     

Activity of long chain acyl-CoA synthetase in isolated alveolar type II cells

Fatty-acyl-CoA synthetase activity was determined in rat alveolar type II cells. Compared to whole-lung homogenate, the enzyme specific activity with palmitic acid was 3.6-fold higher in isolated type II alveolar cells. The enzyme in rat alveolar type II cells did not discriminate among various fatty acids, suggesting that supply of fatty acids rather than specificity might be an important factor for their activation in these cells.     

Incorporation of highly purified melittin into phosphatidylcholine bilayer vesicles

Melittin free of phospholipase A2 was prepared. In the absence of salt this highly pure protein starts to aggregate in solution at a protein concentration of Cp greater than 10(-3) M. In high salt solution (2 M) aggregation starts at Cp greater than 10(-6) M. This was determined from the blue shift of the intrinsic fluorescence of the protein. Reinvestigation of the quenching behaviour clearly shows that self-aggregation cannot be deduced from quenching experiments using nitrate or 2,2,6,6-tetramethylpiperidine-1-oxyl as quencher. The incorporation of melittin into phosphatidylcholine bilayer vesicles was studied by fluorescence quenching and by energy-transfer experiments using 2- and 6-anthroyloxypalmitic acid as acceptor and peptide tryptophan as donor. Incorporation of melittin into small unilamellar vesicles was found to be reduced below the lipid phase transition temperature, Tt, whereas it incorporates and distributes more randomly above Tt. Cooling the temperature below Tt after incubation at T greater than Tt leads to a deeper incorporation of the peptide into the lipid bilayer due to electrostatic interaction between the lipid phosphate groups and the positively charged amino acids. This stabilizing effect is lost above Tt and melittin is extruded to the polar phase. Quenching experiments support this finding. EPR measurements clearly demonstrate that even in the presence of high amounts of melittin up to 10 mol% with respect to the lipid broadening of the phase transition curves was only observed with fatty acid spin labels, where the doxyl group is localized near the bilayer surface. The order degree of the inner part of the bilayer remains almost unchanged even in the presence of high melittin content.     

Glutamic acid-dihydrogen phosphate hydrogen-bonded networks: their proton polarizability as a function of cations present. Infrared investigations.

Glutamic acid [(L-glu)n] + dihydrogen phosphate systems are studied by infrared (IR) spectroscopy dried and hydrated at 75% relative humidity, as a function of both the phosphate-glutamic acid residue (Pi/glu) ratio and the type of cations present. It is shown that the glutamic acid groups form hydrogen-bonded chains with the phosphates. In these chains the positive charge fluctuates, and they show very large proton polarizability which increases in the series Li+,Na+,K+ systems. These chains are cross-linked via phosphate-phosphate hydrogen bonds, in which the proton is almost localized at one Pi. The comparison of the (L-glu)n + dihydrogen phosphate systems with the results obtained earlier in the case of (L-glu)n + hydrogen phosphate systems shows that the behavior of (L-glu)n + Pi systems strongly depends on the pH. Only with decreasing pH the conducting chains are formed. Finally, a hypothesis is discussed with regard to the charge conduction in the F0 subunit of the H+-ATPase in mitochondria.     

Combined submerged and solid substrate fermentation for the bioconversion of lignocellulose

A novel two-stage bioreactor has been designed for a combined submerged (SF) and solid substrate fermentation (SSF) of wheat straw. The straw was pretreated with steam, and cellulases from the culture fluid of Trichoderma reesei were adsorbed on it for increased bioconvertibility. SSF was conducted in the top part of the bioreactor by inoculating the straw with a 36-h mycelial culture of T. reesei, or Coriolus versicolor. In the bottom part of the fermenter, Endomycopsis fibuliger was grown in SF. The SF liquor was recirculated through the SSF stage at 24 h intervals to remove glucose and other metabolites that may inhibit growth, and to maintain optimum moisture level and temperature. The removed glucose and other metabolites provided nutrients for the yeast in the SF stage. The combined fermentation resulted in overall higher biomass yield, increased bioconversion, increased cellulase production, and increased digestibility compared with single SSF or SF.     

Red blood cell T-activation and hemolysis in surgical intensive care patients with severe infections

The exposure of Thomsen-Friedenreich (T) antigens on RBCs, serum neuraminidase, and serum hemoglobin levels were investigated in 53 adult surgical intensive care unit (ICU) patients with septicemia. Unmasked T-antigens were assayed by a hemagglutination test using peanut agglutinin (PNA) (direct anti-T test), and by an indirect anti-T test employing rabbit anti-PNA globulin. RBC T-activation was demonstrated in 17/53 patients (32%); in 2/53 patients (4%) the direct anti-T test was positive, indicating strong T-exposure. No polyagglutination phenomena were observed. Serum neuraminidase was elevated in 12/17 (71%) patients with T-activation and in 7/36 (19%) patients without T-activation. Free serum hemoglobin was elevated in 12/17 (71%) patients with T-activation and in 5/36 (14%) patients without T-activation. Correlations between T-activation and serum neuraminidase and between T-activation and serum hemoglobin were significant (p less than 0.001). Potentially neuraminidase-releasing bacteria were demonstrated in 13/17 (76%) patients with RBC T-exposure. We conclude that neuraminidase-induced RBC T-activation and subsequent hemolysis may be involved in the pathomechanism of hemolytic anemia in patients with severe infections.