The proline biosynthesis gene proA from the thermophilic bacterium Thermus ruber: its cloning, sequencing and heterologous expression]

The proA proline biosynthesis gene of thermophilic bacterium Thermus ruber was cloned and sequenced, and several properties of the encoded enzyme, gamma-glutamylphosphate reductase (GPR) were studied. The proA open reading frame (ORF) was of 1286 bp. Nucleotide sequence analysis revealed the ATG initiation codon in position 36 and the TTA termination codon in position 1304. A deduced protein product of the gene was shown to be of 44,919 Da in molecular weight. The GC content was 66%, as is characteristic of various bacteria of the genus Thermus. An amino acid sequence encoded by the cloned gene showed the highest homology (up to 64%) with GPR of T. thermophilus. The maximum activity of GPR (8.2 x 10(-2) units/ml) was observed at 55 degrees C. A weak enzymatic activity was also detected at 70 degrees C. The enzyme can be used in biotechnological studies.     

Isotopic effect in enzymatic oxidation of methane]

The isotopic effect during oxidation of methane and deuteromethane by a suspension of Methylomonas rubrum cells, for which methane is the only source of carbon, was observed. The rate of CH4 oxidation is 12.5 times higher than that of CH4 oxidation. It is demonstrated that CD4 is a competitive inhibitor of CH4 oxidation. The results obtained suggest that the disruption of the C-H bond is the limiting step of enzymatic oxidation of methane.     

Development of a cellular immune response in a highly cancer-susceptible C3H/He strain of mice and its nonfactor substrain C3H/f after the transplantation of a syngeneic mammary gland tumor]

Syngeneic mammary gland tumor (MMT-1) grew slower in non-factor C3H/f (MTV-L) mice than in wirus-positive C3H/He (MTV-S) mice. The immunization of mice with tumor (MMT-1), inoculated and later excised, revealed a higher immune reactivity in the mice of the non-factor substrain. In studying the dynamics of the cytotoxic activity of lymphocytes in regional lymph nodes cytotoxically active lymphocytes were found to appear in C3H/f mice on day 6 after the inoculation of tumor (MMT-1), while the areactive state of lymphocytes was observed in C3H/He mice on days 3 and 24 and in C3H/f mice on day 24 after the inoculation of tumor (MMT-1). Spleen lymphocytes in C3H/f mice had no cytotoxic effect on tumor cells.     

Enzymatic and conformational stability of polymeric complexes of alpha-amylase

The enzymatic and conformational stability of Bacillus subtilis alpha-amylase and its polymeric complexes in acid media and subsequent renaturation in weakly alkaline media were investigated. The following parameters of alpha-amylase secondary structure were determined from circular dichroism spectra: helical units -25%, beta-structures -9%; beta-turns -13%; disordered conformations -53%. After complexation with polymethacrylic acid (PMAA) the alpha-amylase secondary structure did not change, and the tertiary structure underwent only small local changes. Complexation of alpha-amylase with linear and cross-linked PMAA led to an increase in both enzymatic and conformational stabilities in acid media. Purification of alpha-amylase using a biosorbent resulted in higher acid resistance of the free enzyme and of that in the complex with PMAA. Moreover, the degree of reversibility of the acid inactivation also increased.     

The role of intracellular processes in regulating neuronal sensitivity to acetylcholine

Intracellular regulatory mechanisms of neuronal response to acetylcholine were studied on intracellularly perfused isolated neurones of Lymnaea stagnalis using voltage-clamp technique. It was found that at the change of concentration of free intracellular Ca2+ from 0.06 to 0.7 microM the inhibitory effect of intracellularly added serotonin depends on Ca2+, and the modulation of acetylcholine responses by intracellular serotonin is unchanged. The blockers of calmoduline trifluoperazine and W-7 inhibit inward acetylcholine current at both intra- and extracellular introduction. Possible mechanisms mediating the effect of intracellularly added serotonin on the membrane cholinoreceptors of neurones are discussed.