A rapid extraction method for detecting aflatoxin producing isolates

A rapid extraction method for screening aflatoxin producing potential ofAspergillus flavus group isolates is described. The method is performed using a moist wheat medium with ca. five infected grains extracted with 2 mL of chloroform, and using thin layer chromatography. This method was proved with 95A. flavus isolates from animal feeds.     

pH-dependent structural transition in rabbit skeletal troponin C

Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.     

Evidence for the regulation of protein synthesis by a wheat germ phosphoprotein factor

A 48-kilodalton phosphoprotein, termed T-protein or pT, isolated from wheat germ and purified to homogeneity is found to inhibit the translation of tobacco mosaic virus (TMV) RNA in both wheat germ and reticulocyte lysates. The translation of TMV RNA in both systems was inhibited over 80% by 8 microM pT. There was no evidence to indicate that the reticulocyte lysate also contained a pT-like protein. pT was rapidly phosphorylated in the wheat germ and reticulocyte lysates. Although the relationship between pT phosphorylation and inhibition of protein synthesis is not known, there is evidence to indicate that complete phosphorylation of pT is not required for inhibition. Furthermore, no significant differences in the kinetics of inhibition of protein synthesis between prephosphorylated and unmodified pT were observed. Investigation of the mechanism of inhibition indicated that neither the aminoacylation of tRNA nor the elongation of nascent polypeptide chains was affected by pT. On the other hand, pT was found to prevent the formation of the 80S initiation complex. This action of pT was not due to the binding of pT to the ribosomes. However, the effect of pT was found to vary with the concentrations and types of mRNA used in the translational system. These results suggest that pT may interact with specific region(s) of the mRNA and prevent its translation. Alternatively, pT could block the translation of mRNA by binding to one or more of the initiation factors that interact with mRNA to facilitate mRNA binding to the 43S preinitiation complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of viral and plasmid DNA with the nuclear matrix during productive infection

The association of simian virus 40 (SV40) DNA or plasmid DNA in subcellular fractions from either infected or transfected cells was examined. In lytically infected cells, approx. 25% of viral specific DNA during the infection cycle was retained in nuclei after washing with low ionic strength buffer and 1% Triton X-100. Viral replicating DNA found in the nuclear matrix was capable of performing limited DNA synthesis by the endogenous DNA polymerase in vitro. Viral DNA synthesized in vitro hybridized preferentially to SV40 Hind-III B and C fragments which are in proximity to the origin of replication. In plasmid-transfected COS-7 cells (SV40-transformed cells), the amount of plasmid DNA found in the nuclear matrix was related to its replication efficiency in cells. More than 80% of the plasmid DNA was tightly associated with subnuclear structures. Little or no plasmid DNA was found in the cytoplasmic fraction. The results suggest that, in extrachromosomal model systems, the association of DNA with nuclear matrix is important for the regulation of DNA replication.     

Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins

Summary Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, cysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.     

Production of enterotoxin A by supposedly nonenterotoxigenic Staphylococcus aureus strains

The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity.     

Structure of DNA hydration shells studied by Raman spectroscopy

We have used Raman scattering to study the water O-H stretching modes at approximately 3450 and approximately 3220 cm-1 in DNA films as a function of relative humidity (r.h.). The intensity of the 3220-cm-1 band vanishes as the r.h. is decreased from 98% to around 80%, which indicates that the hydrogen-bond network of water is disrupted in the primary hydration shell (which therefore cannot have an "ice-like" structure). The number of water molecules in the primary hydration shell was determined from the intensity of the approximately 3200-cm-1 band as about 30 water molecules per nucleotide pair. The approximately 3400-cm-1 O-H stretch band was used for determining the total water content, and this band persists at 0% r.h., implying that 5-6 tightly bound water molecules per nucleotide pair remain. The frequency of the approximately 3400-cm-1 O-H stretch mode is lower by 30 to 45 cm-1 in the primary hydration shell compared to free water. The water content as a function of r.h. obtained from these experiments agrees with gravimetric measurements. The disappearance of the approximately 3200-cm-1 band and the shift of the approximately 3400-cm-1 O-H stretch band provide a reliable way of measuring the hydration number of DNA.     

Sliding controller design for a nonlinear fermentation system

The design of a sliding controller for a continuous fermentation process is presented. The results obtained by simulation have proved the control scheme to be very robust. Regulation of substrate concentration at its optimal value has been achieved even though process parameters change their nominal values. Chattering effects are decreased by introducing a minor modification of the control variable around the sliding surface.     

Oligosaccharide synthesis by enzymatic transglycosylation

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