Analysis in vivo of translational mutants of the rIIB cistron of bacteriophage T4

We have mapped the mutants isolated by Nelson et al. (1981) that reduce the amount of rIIB protein synthesized during bacteriophage T4 infection of Escherichia coli B and characterized their rIIB expression in vivo. These mutants fall into four distinct groups in terms of mapping and phenotype. We have located the probable site of each mutation on the DNA sequence. We have also analyzed a number of other mutations near the initiating AUG of rIIB with respect to their rIIB expression. In some of these mutants, ribosomal recognition of the wild-type initiating AUG is precluded and so initiation occurs at a different AUG, which, in some instances, we have identified.     

Crystal structure studies and inhibition kinetics of tripeptide chloromethyl ketone inhibitors with Streptomyces griseus protease B

The bacterial serine protease, SGPB, was inhibited by two specific tripeptide chloromethyl ketones, N-t-butyloxycarbonyl-l-alanylglycyl-l-phenylalanine chloromethyl ketone (BocAGFCK) and N-t-butyloxycarbonyl-glycyl-l-leucyl-l-phenylalanine chloromethyl ketone (BocGLFCK). Crystals of the inhibited complexes were grown and examined by X-ray crystallographic methods. The peptide backbone of each inhibitor is bound by three hydrogen bonds to the main chain of residues Ser214 to Gly216. There are two well-characterized hydrophobic pockets, S₁ and S₂, on the surface of SGPB which accommodate the P₁ and P₂ side-chains of the BocGLFCK inhibitor. A conformational change of Tyr171 is induced by the binding of this inhibitor. Both inhibitors make two covalent bonds to the SGPB enzyme. The imidazole ring of His57 is alkylated at the N^?2 atom and O^γ of Ser195 forms a hemiketal bond with the carbonyl-carbon atom of the inhibitor. Comparison of the binding modes of the two tripeptides in conjunction with the differences in their inhibition constants (K_I) allows one to estimate the binding energy of the leucyl side-chain as ?2.6 kcal mol^?1. The importance of an electrophilic component in the serine protease mechanism, which involves the polarization of the susceptible carbonyl bond of a substrate or inhibitor by the peptide NH groups of Gly193 and Ser195 is discussed.     

Bacillus sphaericus for the control of mosquitoes

From 1972 to 1977 a large laboratory effort was devoted to determining data on efficacy, safety, environmental impact (on nontarget organisms), and some preliminary field work using several isolates of Bacillus sphaericus. The B. sphaericus strains were found to be specific in their mosquito larvicidal activity, not causing mammalian toxicity nor apparent perturbation of the environment. During this period several fermentation and industrialization problems were investigated so that by 1978, using new strains and cultures, it was possible to have prepared kilogram amounts of an active dry stable powder, of strain 1593, for field evaluation. These field evolution. These field evaluations are presently still in progress. Control has been seen particularly against Culex, Anopheles, and Psorophora species, with some what less control aganst Aedes species. Unlike the agriculturally oriented Bacillus thuringiensis candidates, B. sphaericus bacterial cell, which is digested in the larval midgut (within a peritrophic membrane), releasing a toxin as early as 15 min after ingestion. Subsequent death of the larva ensues. Recent evidence suggests that applied B. sphaericus powder will survive in aquatic situations (ditches, ponds, and tree holes) for at least nine month. Comparisons of the B. sphaeicus strains with recently isolated strains of B. thuringiensis (var. israelensis), the latter being particularly active against Aedes species, indicates that they may be useful complements of each other in overall mosquito control strategies. The recent isolation of several new strains of B. thuringiensis, from WHO-CCBC accessions from Roumania, indicate that although the B. thuringiensis isolate is a rare event when compared to the occurrence of B. sphaericus isolates (they usually occur together in accessions from which B. thuringiensis is isolated), several new useful strains of B. thuringiensis should be anticipated. The longevity of the B. thuringiensis strains in the wild has not yet been investigated.     

Effects of tetracyclines on aldosterone- and insulin-mediated Na+ transport in the toad urinary bladder

The effect of oxytetracycline and demethylchlortetracycline on aldosterone- and insulin-mediated Na⁺ transport (short-circuit current) were examined in toad urinary bladders mounted in modified Ussing chambers. Oxytetracycline had little or no effect on either basal or aldosterone-mediated Na⁺ transport. In contrast, demethylchlortetracycline markedly inhibited both basal and aldosterone-mediated Na⁺ transport. Furthermore, demethylchlortetracycline inhibited the aldosterone response significantly out of proportion to its effects on basal Na⁺ transport. Neither of the drugs had an effect on insulin-mediated Na⁺ transport. Consequently, the natriuresis observed in certain patients treated with demethylchlortetracyline may be related to drug-induced renal resistance to the effects of aldosterone.     

The participation of alpha-actinin in the capping of cell membrane components.

By means of double fluorescence staining experiments, intracellular alpha-actinin was found to accumulate under caps and patches induced in several cells by a variety of ligands. This phenomenon was demonstrated in lymphocytes and lymphoma cells treated with anti-H-2 sera; spleen lymphocytes treated with concanavalin A or anti-immunoglobulin antibodies, and VSV-infected mouse fibroblast line MC57 treated with antiserum against viral antigens. It occurred during both rapid and slow capping processes, and could be obtained by either direct or indirect ligand-induced redistribution. These observations were carried out on whole cells. For other cytoskeletal proteins such as filamin, tropomyosin and myosin, a similar accumulation under caps was not readily apparent using whole cell mounts, although earlier experiments with frozen-sectioned cells had shown such an enrichment of myosin (as well as actin). The enrichment of alpha-actinin under the clustered surface molecules was already apparent in early stages (patching) of the capping process, with or without 10 mM sodium azide present. Prolonged incubation of the cells with the different ligands resulted in endocytosis of the ligand-receptor complex. alpha-Actinin was not associated with the inernalized complex, however, suggesting that it may dissociate from the patched or capped surface structures at some stage during endocytosis.     

Laryngotracheobronchitis: 2 years' experience with racemic epinephrine.

A relatively new form of treatment of laryngotracheobronchitis, administration of racemic epinephrine by intermittent positive-pressure breathing, was begun in 1973 in the pediatric unit of a large community hospital. A review of 2 years'' experience with this treatment, compared with the experience of the 3 years prior to its introduction, has shown that it has reduced significantly the necessity for tracheostomy, to nearly zero, and the duration of hospital stay. A total of 119 children (33.15% of those admitted) received this treatment, the average number of treatments required being 1.8. There were no important complications of treatment and no deaths.     

Steroids and hematopoiesis. I. The effect of steroids on in vitro erythroid colony growth: structure/activity relationships.

When substituted steroids of several classes are added to cultures of rat bone marrow cells in the presence of erythropoietin a consistent enhancement of the number of colonies of hemoglobin synthesizing cells is obtained. Maximum steroid effectiveness was found to be between 10(-6) and 10(-7) M. Representative compounds of several classes of steroids were examined for their ability to enhance colony growth, including delta 4-estrenes, delta 4-androstenes, 5alpha-H androstanes and estranes, 5beta-H estranes, pregnanes and androstanes. While testosterone and its 5alpha-H derivatives had little or no activity, many synthetic derivatives of testosterone were highly active in increasing erythroid colony growth. All 5beta-H androstanes, estranes, and all but one 5beta-H pregnane were active. Cortisol consistently inhibited colony growth and estradiol and progesterone had no significant effect.     

Plasma membrane localization of alkaline phosphatase in HeLa cells.

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.