Site-directed mutagenesis of La protease. A catalytically active serine residue.

Comparative sequence analysis of Escherichia coli ATP-dependent La protease led to the suggestion that Ser679 is the catalytically active enzyme residue. Site-directed mutagenesis Ser679----Ala, investigation of the cells containing the mutant plasmid, and study of the partially purified mutant protein produced results in favour of this suggestion.     

Interaction of menadione and duroquinone with Q-cycle during DT-diaphorase function]

The interaction of quinones (menadione and duroquinone) with DT-diaphorase and mitochondrial electron transport chain translocators at low (120 mosM) and high (400 mosM) values of the medium tonicity in the quinone concentration range of 6-90 microM was studied. It was shown that with a rise in menadione (K3) concentration the number of electron transport carriers interacting with it increase. At K3 concentration of 6 microM the latter is reduced by DT-diaphorase and fully oxidized via the Q-cycle. At K3 concentration of 15 microM the latter is also reduced by DT-diaphorase via the Q-cycle, but in this case the oxidation is incomplete (about 30% K3H2 is oxidized by the terminal part of the respiratory chain). At 90 microM K3 50% of quinone is reduced by DT-diaphorase and 50% by the respiratory chain NADH dehydrogenase complex enzymes; about 30% of K3H2 is oxidized via the Q-cycle, about 20%--by the terminal part of the respiratory chain and about 50%--by O2 without cytochrome oxidase. Unlike menadione, duroquinone (6-90 microM) is reduced only by DT-diaphorase and is oxidized in all cases by cytochrome oxidase. It was shown that the increase in the mitochondrial matrix volume in low tonicity media decreases the rate of the DT-diaphorase shunt operation.     

Features of peroxidase oxidation of amidopyrine and its analogs]

Peroxidatic oxidation of N-alkyl and sulfalkyl-substituted 4-aminopyrazolones (amidopyrine and metapyrine) is mediated by oxyperoxidase, whereas the oxidation of non-substituted 4-aminoantipyrine occurs via the classical peroxidase cycle, without oxyperoxidase accumulation. The free radicals formed at the first step of the oxidation cycle show a tendency for disproportionation and exchange. During catalysis in heavy water the oxidation of substituted aminopyrazolones is accelerated by plant peroxidase. This effect is due to the activation of the second oxidative state of the enzyme. Complete peroxidatic oxidation of amidopyrine results in the formation of several reaction products. The main product is not identified as dioxypyramidone formed via nonenzymatic peroxidatic oxidation. The oxidation of the indicator (azopyram) which represents an amidopyrine-aniline mixture results first in the formation of amidopyrine radicals. The reaction product (dye) is formed via the binding of these radicals to aniline radicals. The latter are predominantly formed via a nonenzymatic route during the reduction of the amidopyrine radicals by aniline. Similar to the formation of azopyram and the full oxidation of amidopyrine, this reaction is accompanied by the displacement of the substituents from the 4-amino group.     

A- and B-type lamins are differentially expressed in normal human tissues

 A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies. Accepted: 4 February 1997     

Effect of overproduction of heat shock chaperones GroESL and DnaK on human procollagenase production in Escherichia coli.

The effect of overexpression of the heat shock chaperone genes dnaK and groESL on heterologous protein production in Escherichia coli was examined, using a set of related human procollagenase proteins. A diverse range of effects on protein solubility, secretion, and accumulation was observed, and these effects were highly dependent on the particular chaperone/procollagenase pairing involved. Both chaperones caused a large increase in the apparent solubility of a fusion of the LamB signal peptide to procollagenase. GroESL had no effect on the accumulation of mature (secreted) procollagenase, while DnaK suppressed secretion considerably. In the absence of a signal peptide, overexpression of either chaperone resulted in a dramatic increase in both solubility and accumulation of procollagenase. The 10-fold increase in accumulation was associated with an increase in in vivo protein half-life.     

Respiratory activity generated by semi-isolated medullo-spinal preparation and recorded from the phrenic nerve of newborn rats

Inspiratory activity generated by superfusedin situ semi-isolated medullo-spinal preparations of newborn (one-day-old) and four- to five-day-old rats was recorded from then. phrenicus before and after transverse sectioning of the ventrolateral part of the medulla (VLPM) at different levels. Under similar experimental conditions, the frequency of inspiratory discharges (ID) and their integral intensity, reflecting the volume and temporal parameters of inspiration, are much lower in one-day-old rats, as compared with those in four- to five-day-old animals. Specific roles of different VLPM levels in respiration control in young rats are demonstrated. Transection of the VLPM below the most rostral VLPM portion, corresponding to theM chemosensitive zone, caused a significant increase in the ID frequency and a decrease in the ID integral intensity. Transection performed below the intermediate VLPM region, corresponding to theS chemosensitive zone, resulted in a significant decrease in both ID frequency and ID integral intensity, up to total ID blockade in 5 of 12 1-day-old preparations. This finding can be interpreted as an indication of morphofunctional immaturity of the respiratory network in the caudal VLPM regions in newborn animals. Comparative analysis of ID pattern showed that this activity in one-day-old rats is more or less gasping-like, while that in four- to five-day-old animals is eupnoe-like. The results allow us to conclude that the level of maturity of morphofunctional organization of medullary respiratory networks considerably differs in newborn and older animals. The mechanisms responsible for formation and control of respiratory activity in early postnatal period of rats are discussed.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 387–395, September–December, 1995.     

Genetic organization of transfer region of F-like plasmid pAP18-1

The results of complementation analysis of nitrosoguanidine-induced mutants of F-like drd-plasmid pAP18-1 (Tc, ColV) testified to the existence of at least 3 tra regions (tra1, tra2, tra3) and regulating locus fin V in the genome of this plasmid. By means of molecular cloning of tra2 region and locus fin V of plasmid pAP18-1drd were located in Sall-fragment f5 (3.9 MD).