Fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases. Application to subtilisins.

A fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases is described. It consists of: selective labelling of the corresponding subsites with a fluorescent group by using N alpha-dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ated peptide chloromethanes containing different numbers of amino acid residues, and probing the immediate environment of the subsites by quenching experiments using ionic and neutral quenchers. Intramolecular distances between the subsites and particular chromophores can be also determined. The technique is of general applicability to all serine proteinases. The above mentioned approach was applied to two proteinases: subtilisin Novo and mesentericopeptidase. It was concluded that the substrate-binding site of mesentericopeptidase is considerably more polar than that of subtilisin Novo. Intramolecular distances between the labelled subsites and tryptophan residues in the two proteinases were determined.     

Anti-My-26: a monoclonal antibody inhibiting human phagocyte chemiluminescence

Anti-My-26, a mouse monoclonal IgG1 antibody, was raised against human granulocytes and has been shown to inhibit luminol-enhanced, glucose-independent chemiluminescence (CL) of human granulocytes (or monocytes) responding to the soluble secretagogues A23187 or ionomycin (calcium ionophores) and phorbol myristate acetate (PMA). Anti-My-26 inhibition of CL was reversible and was dependent on both secretatogue and monoclonal antibody concentration. This inhibition appeared to be directed at the component of granulocyte CL that is independent of NAD(P)H-oxidase-catalyzed formation of superoxide anion, because neither opsonized zymosan-stimulated CL nor the PMA-induced decrease in NAD (P)H-associated autofluorescence was affected by anti-My-26. In addition, ionomycin, over a wide concentration range, failed to generate any decrease in granulocyte autofluorescence. The A23187-induced CL inhibited by anti-My-26 was correlated with its depression of oxygen consumption. Furthermore, anti-My-26 was not cytotoxic and did not itself induce oxidative metabolism when used as a stimulant. Binding of anti-My-26 to phagocytic cells was not decreased by pre-exposure of cells to either A23187 or PMA. Evidence is presented to suggest that the binding of anti-My-26 to the granulocyte surface inhibits the oxidative response to calcium ionophore and PMA by blocking a common pathway(s) stimulated by these different secretagogues.     

A specific decrease in collagen synthesis in acutely fasted, vitamin C-supplemented, guinea pigs

Weight loss often results from various experimental conditions including scurvy in guinea pigs, where we showed that decreased collagen synthesis was directly related to weight loss, rather than to defective proline hydroxylation (Chojkier, M., Spanheimer, R., and Peterkofsky, B. (1983) J. Clin. Invest. 72, 826-835). In the study described here, this effect was reproduced by acutely fasting normal guinea pigs receiving vitamin C, as determined by measuring collagen and non-collagen protein production after labeling tissues in vitro with [3H]proline. Collagen production (dpm/microgram of DNA) decreased soon after initiating fasting and by 96 h it had reached levels 8-12% of control values. Effects on non-collagen protein were much less severe, so that the percentage of collagen synthesis relative to total protein synthesis was 20-25% of control values after a 96-h fast. These effects were not due to changes in the specific radioactivity of free proline. Refeeding reversed the effects on non-collagen protein production within 24 h, but collagen production did not return to normal until 96 h. The effect of fasting on collagen production was independent of age, sex, ascorbate status, species of animal, and type of connective tissue and also was seen with in vivo labeling. Pulse-chase experiments and analysis of labeled and pre-existing proteins by gel electrophoresis showed no evidence of increased collagen degradation as a result of fasting. Procollagen mRNA was decreased in tissues of fasted animals as determined by cell-free translation and dot-blot hybridization with cDNA probes. In contrast, there was no decrease in translatable mRNAs for non-collagen proteins. These results suggest that loss of nutritional factors other than vitamin C lead to a rapid, specific decrease in collagen synthesis mainly through modulation of mRNA levels.     

Haematological correlates of burrowing in Ctenomys

Haematological parameters related to O2 transport and regulation of acid-base equilibrium were determined for two species of Ctenomys. The oxygen capacity values of Ctenomys blood are similar to those of other fossorial mammals. Ctenomys blood has almost half the number of red blood cells of Rattus blood but the amount of Hb in each blood cell is 2-2.5 times higher. Blood pH is within typical mammalian values. Concentration of inorganic phosphate is higher in Ctenomys than in rats while bicarbonate and protein values are within typical mammalian range.     

Phenotype frequencies of vitamin D binding protein (Gc) and of posttransferrin-2 (Ptf-2) in Belgian cattle breeds*

The vitamin D binding protein (Gc) and posttransferrin-2 (Ptf-2) phenotypes have been determined in a number of Belgian cattle breeds. A very slow migrating variant of the Gc protein — Gc C — has been found in White and Red East Flemish breed. This variant was absent from the other breeds studied. This slow variant was identified as a vitamin D binding protein by autoradiography. The Gc C protein was shown to be controlled by a codominant autosomal allele Gc C at the Gclocus. The Gc C protein is probably identical with a fraction previously described in buffalo and an Italian cattle breed. The allele frequencies for the Gc and Pft-2 systems are reported for several Belgian breeds of cattle.     

Comparison of estrogen priming effects with body weight restoration effects on the gonadotropin pattern of patients with anorexia nervosa

Plasma estradiol (E2), serum LH and FSH, and the gonadotropin response to two consecutive LHRH administrations (10 and 100 micrograms with an interval of 2 h) were determined in 19 patients with anorexia nervosa (AN) at the emaciation phase, before and after estradiol benzoate (E2B) injections (3 micrograms/kg/day for 7 days). The same investigations were repeated after weight restoration in 9 AN patients who remained amenorrheic. Both at the emaciation phase and after weight restoration, E2B enhanced the second LH response to LHRH and decreased serum FSH, suggesting that the functional capacities of the pituitary gonadotrophs are normal in AN. Unlike E2B injections, weight restoration increased all the hormone values, suggesting that the weight restoration effects on the abnormal gonadotropin secretory pattern of AN depend on another mechanism than the E2 lowering. That mechanism is probably a disorder of the hypothalamic LHRH secretion, the consequences of which could be reinforced by the low E2 levels.     

Tunicamycin-resistant mutants of Bacillus amyloliquefaciens are deficient in amylase, protease and penicillinase synthesis and have altered sensitivity to antibiotics and autolysis

Mutants of Bacillus amyloliquefaciens resistant to at least 10 micrograms/ml of tunicamycin were isolated and shown to be pleiotropic. The mutants were more resistant to streptomycin, chloramphenicol, kanamycin and neomycin than was the parent strain but less resistant to penicillin G and tetracycline. They were more autolytic, presumably due to an altered cell wall. The mutants produced reduced levels of amylase, penicillinase and both metal and serine protease besides having an enhanced sporulation frequency and being more motile.