Renaturation of dextranase activity from culture supernatant fluids of Streptococcus sobrinus after sodium dodecylsulfate polyacrylamide gel electrophoresis

A rapid and reproducible method for the assay of individual fractions of the multicomponent dextranase activity of Streptococcus sobrinus after reduction/denaturation and electrophoresis in acrylamide gels is described. Multiple forms of dextranase, possible virulence factors in the formation of dental caries by oral Streptococci (S. mutans, S. sobrinus, S. sanguis, S. cricetus, and S. rattus), have been separated in sodium dodecylsulfate-polyacrylamide gels into which an indicator substrate, blue dextran, has been incorporated, and identified after renaturation to remove the reducing/denaturing agents of the Laemmli buffer system.     

Protein kinase C activity in renal microvillus membranes

Protein kinase C activity was found in rabbit renal microvillus membrane vesicles. C-kinase activity was assayed by examining H1 histone phosphorylation using microvillus membrane vesicles dispersed with Triton X. Calcium-activated protein kinase activity was only demonstrable in the presence of phosphatidylserine (PS). With PS (15 micrograms/ml) the Ka for activation by calcium was 1.04 microM. This was reduced to 0.38 microM by addition of diolein (3.75 micrograms/ml). These activations were dose-dependent and their combined synergistic activation could be reproduced by the combination of PS (15 micrograms/ml) and the phorbol ester, TPA (1.17 ng/ml). During microvillus membrane purification, protein kinase C activity enriched 5-fold relative to its activity in the homogenates. The activity was not due to trapped cytosol or adventitious association with microvillus membranes during homogenization. During further purification on sucrose gradients, the C-kinase activity coenriched with brush border and not with basolateral enzyme markers. We conclude that protein kinase C is a normal component of the renal microvillus membrane.     

Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Human liver cathepsin L.

Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.     

Genetic and physical analyses of Caulobacter crescentus trp genes.

Caulobacter crescentus trp mutants were identified from a collection of auxotrophs. Precursor feeding experiments, accumulation studies, and complementation experiments resulted in the identification of six genes corresponding to trpA, trpB, trpC, trpD, trpE, and trpF. Genetic mapping experiments demonstrated that the trp genes were in two clusters, trpCDE and trpFBA, and a 5.4-kilobase restriction fragment from the C. crescentus chromosome was isolated that contained the trpFBA gene cluster. Complementation experiments with clones containing the 5.4-kilobase fragment indicated that trpF was expressed in Escherichia coli and that all three genes were expressed in Pseudomonas putida. This expression was lost in both organisms when the pBR322 tet gene promoter was inactivated, indicating that all three genes were transcribed in the same orientation from the tet promoter. Thus, the C. crescentus promoters do not seem to be expressed in E. coli or P. putida. Complementation of the C. crescentus trp mutants indicated that the tet promoter was not necessary for expression in C. crescentus and suggested that at least two native promoters were present for expression of the trpF, trpB, and trpA genes. Taken together, these results indicate that C. crescentus promoters may have structures that are significantly different from the promoters of other gram-negative species.     

Differential recovery of circulating T cell subsets after nodal irradiation for Hodgkin's disease

To better understand the immunologic effects of lymphoid irradiation (LI), blood levels of T cell subsets were sequentially monitored in 15 patients before, during, and after irradiation treatment for Hodgkin's disease. Blood levels of all lymphocytes, T cells, and T cell subsets (defined by OKT4 and OKT8) fell dramatically and in similar proportions during early therapy, reaching levels less than 20 to 25% of control by the completion of mantle irradiation, and continuing at very depressed levels through the completion of therapy. Blood levels of OKT8-reactive (OKT8+) cells returned to pretreatment levels (402 +/- 38/mm3 vs 360 +/- 32/mm3 pretreatment) between 6 to 8 mo after LI, whereas blood levels of OKT4-reactive (OKT4+) cells returned to only 42% of previous values (242 +/- 22/mm3 vs 584 +/- 34/mm3 pretreatment) over the same period. The pre-LI ratio of OKT4+ to OKT8+ cells was 1.85 +/- 0.24 and fell to 0.65 +/- 0.05 between 6 to 8 mo after LI. During the recovery period, discrepancies of 208 +/- 32 cells/mm3 (3 to 5 months post LI) and 198 +/- 32 cells/mm3 (6 to 8 mo post LI) developed between the blood levels of OKT3+ cells and the sum of OKT4+ and OKT8+ cells. This suggests the emergence of OKT4+/OKT3-, OKT8+/OKT3-, and/or OKT4+/OKT8+ cells. In five patients, the sum of OKT4+ and OKT8+ cells was compared with the number of cells simultaneously co-stained by OKT4 and OKT8. It appeared that a significant proportion of the cells were OKT4+/OKT3- and OKT8+/OKT3- lymphocytes. We concluded that LI is similarly cytotoxic to peripheral blood T cell subpopulations. The reversed ratio after LI is a result of a slower repopulation of the peripheral blood by OKT4+ cells relative to OKT8+ cells. T cells after LI show a high degree of antigenic immaturity. It is postulated that the bone marrow that lies outside the fields of treatment and contains predominantly immature OKT8+/OKT3- cells is a major source of T cells repopulating the peripheral blood after LI.     

Purification of cathepsin B by a new form of affinity chromatography.

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2'-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.     

Temporal patterns of protein phosphorylation after angiotensin II, A23187 and/or 12-O-tetradecanoylphorbol 13-acetate in adrenal glomerulosa cells.

The temporal patterns of protein phosphorylation in the adrenal glomerulosa cell were analysed by two-dimensional electrophoresis after stimulation with 10 nM-angiotensin II or various agents [10 nM-12-O-tetradecanoylphorbol 13-acetate (TPA), 50 nM-A23187, 1 microM-nitrendipine], administered singly or in combination. These patterns were compared with the temporal patterns of aldosterone secretion induced by the same agonists and antagonists. After 1 and 30 min of stimulation with angiotensin II, different patterns of protein phosphorylation were observed. A comparison of these patterns reveals that: the phosphorylation of only one protein was persistently enhanced during the continuous incubation with angiotensin II; the phosphorylation of five proteins was transiently enhanced (at 1 min but not 30 min); and the phosphorylation of three proteins did not occur at 1 min but was seen at 30 min. Addition of the phorbol ester TPA alone, which at 30 min is without effect in enhancing aldosterone production, has no effect on protein phosphorylation. The combined addition of TPA and the Ca2+ ionophore, A23187, which, like angiotensin II, evokes a sustained increase in aldosterone production, reproduced the temporal patterns of protein phosphorylation seen after angiotensin II action. Manipulations (A23187 alone, angiotensin II plus nitrendipine) which evoke only a transient rise in aldosterone production rate induce a transient rise in cellular protein phosphorylation. The 1 min patterns of phosphorylation seen after A23187 or combined angiotensin II and nitrendipine (a Ca2+ channel antagonist) are similar to those observed after 1 min of angiotensin II stimulation. These results suggest that, when angiotensin II acts, the initial cellular response is mediated by a different mechanism than that responsible for the sustained response.     

Roles for menaquinone and the two trimethylamine oxide (TMAO) reductases in TMAO respiration in Salmonella typhimurium: Mu d(Apr lac) insertion mutations in men and tor.

Three groups of mutants defective in trimethylamine oxide (TMAO) reduction were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). Mutants were identified by their acidic reaction on a modified MacConkey-TMAO medium. Group I consisted of pleiotropic chlorate-resistant mutants which were devoid of TMAO reductase activity. None expressed the lac operon. Group II mutants were partially defective in TMAO reductase. Electrophoretic studies revealed that they lacked the inducible TMAO reductase, but retained the constitutive activity. The genotypic designation tor was suggested for these mutants. The tor mutation in one was located between 80 and 83 U on the S. typhimurium chromosome. Expression of the lac operon in these mutants was not affected by air, TMAO, or nitrate. Group III mutants reduced little or no TMAO in vivo, but their extracts retained full capacity to reduce it with methyl viologen. These mutants also failed to produce hydrogen sulfide from thiosulfate and could not grow anaerobically on glycerol-fumarate. Two subgroups were distinguished. Vitamin K5 restored wild-type phenotype in subgroup IIIa only; vitamin K1 restored wild-type phenotype in both IIIa and IIIb isolates. The genotypic designation men (menaquinone) was suggested for group III isolates. The mutation in IIIa mutants was cotransducible with glpT, which corresponds to the menBCD site in Escherichia coli. That in IIIb mutants was cotransducible with glpK, which corresponds to the menA site in E. coli. Expression of the lac operon in IIIa, but not IIIb, mutants was repressed by air. An additional mutant group isolated on the same medium consisted of strains defective in formate hydrogenlyase.