Bacillopeptidase F of Bacillus subtilis: purification of the protein and cloning of the gene.

We have purified a minor extracellular serine protease from Bacillus subtilis. Characterization of this enzyme indicated that it was most likely the previously reported enzyme bacillopeptidase F. The amino-terminal sequence of the purified protein was determined, and a "guess-mer" oligonucleotide hybridization probe was constructed on the basis of that sequence. This probe was used to identify and clone the structural gene (bpr) for bacillopeptidase F. The deduced amino acid sequence for the mature protein (496 amino acids) was preceded by a putative signal sequence of 30 residues and a putative propeptide region of 164 amino acids. The bpr gene mapped near pyrD on the chromosome and was not required for growth or sporulation.     

Occurrence,properties and biological implications of the active uptake of water vapour from the atmosphere in Psocoptera

Water vapour absorption is shown to occur in 22 species of Psocoptera inhabiting diverse environments and representing all major groups of this insect order. Evidently the faculty is a common feature of the whole order and it seems not to be related to specific environmental conditions. For the first time water vapour uptake could be demonstrated in fully winged and flying insects. The critical equilibrium humidities vary considerably among different species ranging from 58 to 85% r.h. Marked interspecific differences are also observed in water loss and uptake rates but no clear correlation with habitat or systematic group is recognizable. The uptake rates of Psocoptera are among the highest of all arthropods investigated so far. From weight recordings with a sensitive microbalance it could be seen that continuous operation of the uptake mechanism is restricted to limited periods of time of less than 1 hr regardless of the water status of the animals. Initiation and termination of the uptake process are abrupt and continuous uptake proceeds at a constant rate at a given relative humidity. Uptake rates are humidity-dependent decreasing with falling relative humidity whereas the adjustment of the equilibrium level of body water is independent of ambient humidity. Equilibrium is maintained by intermittent operation of the uptake mechanism within ca. 3% of body water mass. The uptake mechanism exhibits marked sensitivity to starvation in most members of the Psocomorpha. Some features of the uptake process of Psocoptera are in close agreement with those of Mallophaga reflecting the close relationship between the two groups.     

Restriction of PGK-B to spermatogenic cells: Evidence from experimentally cryptorchid mice

The hypothesis that PGK-B, like LDH-C₄, is restricted to spermatogenic cells was explored by examining isozyme patterns in testes from mice depleted of germinal cells by surgical cryptorchism. In experimentally cryptorchized C57BL/10Sn males, decline in PGK-B activity paralleled decline in LDH-C₄ activity and was correlated with degeneration of spermatocytes, spermatids, and spermatozoa. Trace amounts of these sperm isozymes found in cryptorchid testes after the depletion of maturing germ cells probably came from degenerated spermatids and spermatocytes and not from somatic testicular cells.     

CYTODYNAMICS IN THE THYMUS OF YOUNG ADULT MICE:

Cell proliferation and cell loss in the thymic blast cell population were studied in young adult mice by (1) stathmokinetic methods combined with an analysis of the PLMe-curve after a pulse ³H-TdR, and (2) nigrosin-dye exclusion combined with ³H-TdR-autoradiography. It was calculated that about 17% of the blast cells do not progress into mitosis within the period of an average cell cycle. The dye exclusion studies indicated a rate of blast cell death of about 2–5 %/hr. The two methods of assessing blast cell loss (death) support each other very well. In spite of these findings scintillation countings on thymuses removed from 1 to 17 hr after ³H-TdR injection showed fairly constant levels of thymic radioactivity. This suggests a very extensive reutilization of ³H-labelled break-down products from dying blast cells. The very sparse labelling of pyknotic thymocytes strongly suggests that thymic blast cells do not become pyknotic. The rate of small thymocyte production and disappearance was studied by pulse and repeated ³H-TdR labelling techniques combined with dye exclusion studies and pyknotic counts. The data from the repeated labelling experiment were analysed by use of a model based on the assumption of first order kinetics of small viable, dead, and pyknotic thymocytes. The rate of cell production was estimated to 1–6 %/hr whereas the rates of cell loss due to disintegration, i.e. supravital stainability and nuclear pyknosis, were calculated to 0–02 %/hr and 0–0006 %/hr respectively. Cell loss due to disintegration was less than 2 % of the total loss of small thymocytes. It was concluded that pyknotic counts are a useless method of assessing the cell death in the population of thymic blast cells and small thymocytes. On the basis of a model for thymocyte proliferation, production and loss it is suggested that about 45 % of the small viable thymocytes re-enter the generative cell pool, whereas about 55% disappear by emigration.     

Uptake and Elimination of Poliovirus by West Coast Oysters

Accumulation of poliovirus Lsc-2ab by West Coast oysters was determined by using a stationary seawater system, and depuration was determined by using both stationary and free-flow systems. Results indicate that these shellfish have the same pattern of accumulation and localization of viruses as do East Coast species. However, uptake appeared to occur more rapidly than described for East Coast shellfish. There appeared to be a gradual diffusion of virus from the digestive area into the body. Depuration was found to occur more rapidly and completely under free-flow conditions than in a stationary system.     

Isoproterenol inhibition of horse serum cholinesterase is connected with subunit dissociation

Tetrameric cholinesterase from horse serum undergoes concentration-dependent dissociation. The dimer is highly stable so that even on SDS polyacrylamide gels subunit dissociation to the 80-kDa polypeptide chains is incomplete. Glutaraldehyde cross-linking confirms this finding, giving rise to a tetramer: dimer ratio of approximately 1:1. The beta-adrenergic agent isoproterenol acts as an inhibitor of the enzyme with respect to butyrylthiocholine hydrolysis; inhibition kinetics point to a dissociative effect of the ligand as the underlying mechanism (S?ylemez, Z. & Ozer, I. (1985) Comp. Biochem. Physiol. 81c, 433-437). Evidence from sedimentation analysis confirms this hypothetical mechanism: the sedimentation coefficient in the presence of saturating concentrations of both the substrate butyrylthiocholine and the inhibitor isoproterenol shows a 35 +/- 5% decrease; in high speed sedimentation equilibria the weight average molecular mass is shifted from the tetramer (Mr = 312 +/- 12 kDa) to the dimer (Mr = 160 +/- 10 kDa). The transition is complete at isoproterenol concentrations below saturation. Applying glutaraldehyde cross-linking to monitor the particle distribution at varying isoproterenol concentrations confirms the change in quaternary structure in a qualitative way. Enzyme concentrations applied in the present experiments are in the range of the concentration of cholinesterase in horse serum. Therefore the dissociative mechanism of isoproterenol on the enzyme may be of biological significance.     

Interaction of cibacron blue 3G-A and related dyes with nucleotide-requiring enzymes

Cibacron Blue 3G-A (I), the chromophore in Blue Dextran, its structural isomer Cibacron Brilliant Blue BR-P (II), and two other structural analogs (III, IV) were used to probe the nucleotide binding sites of selected kinases and dehydrogenases. Inhibition studies indicate that the portion of the dye molecule necessary for effective inhibition of nucleotide binding is a structure similar to 1-amino-4(4′-aminophenylamino)-anthraquinone-2,3′-disulfonic acid (ASSO; III). The strong inhibition exhibited by these dyes is likely to be due to interaction with specific nucleotide binding sites, irrespective of the presence of a “dinucleotide fold” in the proteins' supersecondary structure.