Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation.

Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that activation of the Fas receptor present on a human breast epithelial cell line resulted in a conformational change in the N terminus of the pro-apoptotic protein Bax. This conformational change appeared to occur in the cytosol and precede Bax translocation to the mitochondria. Overexpression of the anti-apoptotic protein Bcl-2 inhibited both the conformational change of Bax as well as its relocalization to the mitochondria. Bcl-2 overexpression did not, however, inhibit Fas-induced cleavage of both procaspase-8 and the pro-apoptotic protein Bid, indicating that Bcl-2 functions downstream of these events. These results suggest that the mechanism by which Bcl-2 inhibits Bax mitochondrial translocation and subsequent amplification of the apoptotic cascade is not by providing a physical barrier to Bax, but rather by inhibiting an upstream event necessary for Bax conformational change.

Bax membrane insertion during Fas(CD95)-induced apoptosis precedes cytochrome c release and is inhibited by Bcl-2.

Ligation of the Fas cell surface receptor leads to activation of caspases and subsequent apoptosis. Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that the pro-apoptotic protein, Bax, translocates from the cytosol specifically to the mitochondria following Fas ligation in MCF10A1 breast epithelial cells. Bax translocation was dependent on caspase activation, and preceded the release of cytochrome c and loss of mitochondrial respiratory activity. Bax translocation occurred in concert with activation of downstream caspases as determined by cleavage of a synthetic substrate, proteolysis of poly(ADP-ribose) polymerase, and processing of procaspase-3 and -7. Overexpression of the anti-apoptotic protein, Bcl-2, prevented Bax insertion, cytochrome c release, complete processing of caspase-3 and -7, and full activation of DEVD-specific cleavage activity. These data establish a role for Bax mitochondrial insertion during Fas-mediated apoptosis, and support a model in which Bax insertion amplifies the Fas apoptotic cascade through cytochrome c release and complete processing of caspases-3 and -7. In addition, our findings indicate that prevention of Bax insertion into the mitochondria represents a novel mechanism by which Bcl-2 inhibits Fas-induced apoptosis.

Induction of cold shock proteins in Bacillus subtilis.

The cold shock response in the Gram-positive soil bacterium Bacillus subtilis is described. Cells were exposed to sudden decreases in temperature from their optimal growth temperature of 37 degrees C. The B. subtilis cells were cold shocked at 25 degrees C, 20 degrees C, 15 degrees C, and 10 degrees C. A total of 53 polypeptides were induced at the various cold shock temperatures and were revealed by two-dimensional gel electrophoresis. General stress proteins were identified by a comparative analysis with the heat shock response of B. subtilis. Some unique, prominent cold shock proteins such as the 115 kDa, 97 kDa, and 21 kDa polypeptides were microsequenced. Sequence comparison demonstrated that the 115-kDa protein had homology to the TCA cycle enzyme, aconitase.

Bacteriophage phi 105clz induces the GroEL-homologue protein in Bacillus subtilis.

Using two-dimensional polyacrylamide gel electrophoresis, the GroEL homologue of Bacillus subtilis was shown to be induced upon infection with phi 105clz, a clear plaque mutant of the temperate bacteriophage phi 105. Western blotting of one dimensional polyacrylamide gels also showed the induction of the GroEL homologue when cells were infected with phi 105clz.

Heat shock in Bacillus subtilis: genetic characterization of a mutant.

Bacillus subtilis strain BUL786, a transposon-generated mutant, lacks a 97 kD protein following heat shock and has unique protein secretion properties. In this study, transduction analysis demonstrated that the properties of high temperature protease secretion, heat shock protein distribution, and loss of the 97 kD heat shock protein were all transferred as a single trait to recipient bacteria. The high temperature-specific protease released by this mutant into the supernatant was also shown to be produced during heat shock by the parental cells, but is retained intracellularly.

Basic features of the staphylococcal heat shock response.

The major heat shock proteins of Staphylococcus aureus had apparent Mrs of 84,000, 76,000, and 60,000, and other prominent proteins of Mrs 66,000, 51,000, 43,000 and 24,000 were also induced. Staphylococcus epidermidis showed a similar response. These proteins were also induced by CdCl2, ethanol and apparently osmotic stress (1.71 M NaCl or 2.25 M sucrose). Most of the proteins sedimented with the membrane fraction, but the Mr 60,000 protein remained in the cytoplasm.

Facile autoplast generation and transformation in Bacillus thuringiensis subsp. kurstaki.

We describe a method for maximizing the rate of conversion of Bacillus thuringiensis subsp. kurstaki vegetative cells to osmotically fragile forms in the absence of exogenously added enzymes. Optimal generation of autoplasts occurred in 50 mM sodium acetate buffer (pH 7.0) at 37 degrees C with 10% (wt/vol) polyethylene glycol as an osmotic stabilizer. The maximum autolytic rate resulted in a conversion of greater than 90% of bacilli to spherical autoplasts in 6 min. Autoplasts regained bacillary morphology upon plating on DM3-G regeneration medium, with reversion frequencies ranging from 1.2 x 10(-1) to 5.3 x 10(-3). The autoplasts could efficiently take up exogenously added plasmid DNA. The presence of plasmids was verified by Southern hybridization analysis.

Isolation and initial characterization of a Bacillus subtilis mutant with novel protease secretion capability.

A bank of pTV32 (Tn 917 lacZ) - generated Bacillus subtilis mutants were examined on milk agar for the ability to produce proteases at 48 degrees C. A single mutant, BUL786, was isolated, which could hydrolyze casein after overnight incubation at 48 degrees C. This mutant secreted protease 10 fold more at 48 degrees C when compared to 37 degrees C, and part of the activity appears to be 48 degrees C-specific. At high temperatures, other strains of B. subtilis, including hyperprotease secretors, were unable to secrete protease to any significant degree. The BUL786 strain is missing the 97K major heat shock protein. Since a number of other proteins also appear to be secreted at 48 degrees C, this mutant may be a hypersecretor of exported proteins at temperatures greater than 45 degrees C.

Heat shock proteins in bacilli.

Five strains of bacilli, including a nonsporulating strain, when heat shocked, accelerated the synthesis of a specific subset of proteins. The major heat shock protein in all bacilli had a molecular weight of 66,000. The response persisted for at least 40 min and could be eliminated upon a shift down to 37 degrees C.

Effect of aromatic carcinogens and noncarcinogenic analogs on the induction of murine alpha/beta interferon.

The effects of several aromatic carcinogens and their noncarcinogenic analogs on the production of alpha/beta interferon by mouse embryo fibroblasts were determined. The carcinogens 2-naphthylamine and 9,10-dimethylanthracene significantly depressed alpha/beta interferon production, while their poorly or noncarcinogenic analogs, 1-naphthylamine and anthracene, respectively, had no significant effect. Neither the carcinogen benzidine nor its poorly carcinogenic analog 3,3',5,5'-tetramethylbenzidine had any effect on alpha/beta interferon production. These data, taken together with previous findings, suggest the possibility of drawing a high correlation between the carcinogenic potential of a chemical and its effect on interferon production.

Insertion and fate of the cell wall in Bacillus subtilis.

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.

Dosage limitations of the effects of difficult-to-detect carcinogens on in vitro interferon induction.

Several carcinogens have been shown to depress in vitro interferon (IFN) induction, while closely matched weakly or noncarcinogenic analogues had no effect on IFN induction. In this study, specific carcinogens that were difficult to detect by the Ames Salmonella assay were tested for their effects of IFN-alpha/beta induction. The carcinogens were applied to mouse embryo fibroblast cultures, and were then removed. IFN-alpha/beta induction was then carried out using polyriboinosinic-polyribocytidylic acid. Application of urethane, which was undetectable by the Salmonella assay except under special conditions, significantly depressed IFN-alpha/beta induction. Treatment with cyclophosphamide or dimethylcarbomyl chloride, both of which required extremely large dosages to be detected by the Salmonella assay, had no effect on IFN-alpha/beta induction at the dosages used. Higher dosages of these carcinogens were toxic to the cells used in this system.

Differential effects of amorphous and crystalline nickel sulfide on murine alpha/beta interferon production.

Mouse embryo fibroblast cultures were exposed to various concentrations of amorphous or crystalline NiS (nickel sulfide). After removal of the NiS from the cultures, polyriboinosinic-polyribocytidylic acid was added to induce alpha/beta interferon. Pretreatment of the cultures with crystalline NiS significantly inhibited the production of alpha/beta interferon at relatively noncytotoxic concentrations (i.e., 1 microgram/ml) while amorphous NiS had no significant effect on alpha/beta interferon production at 10 micrograms/ml. Treatment of cells with reduced glutathione did not block the inhibitory effects of crystalline NiS on alpha/beta interferon production, suggesting that these effects are not mediated by a free-radical mechanism.

Effect of environmental carcinogens and other chemicals on murine alpha/beta interferon production.

Mouse embryo fibroblast cultures were pretreated with a variety of chemicals found in the environment. After chemical treatment, polyriboinosinic-polyribocytidylic acid was added to the cultures to induce alpha/beta interferon. Pretreatment of the cell cultures with the chemical carcinogens chloroform and beta-propiolactone severely inhibited the production of alpha/beta interferon, while pretreatment of the cell cultures with their poorly or noncarcinogenic analogs 1,1,1-trichloroethane and gamma-butyrolactone had no effect on interferon induction. Pretreatment of the cell cultures with the possible carcinogen diethylstilbestrol had no effect on alpha/beta interferon induction. Pretreatment of the cells with the poor or noncarcinogens pyrene and ascorbic acid did not effect interferon induction; in fact, treatment with ascorbic acid may have enhanced interferon production. These results augment previous findings that most potent carcinogens can inhibit the induction of alpha/beta interferon.

Extracellular proteases increase tolerance of Bacillus subtilis to nafcillin.

Mutants of Bacillus subtilis capable of secreting high amounts of protease were highly tolerant to the lethal and lytic effects of nafcillin. Protease-deficient mutants were more susceptible. However, when subtilisin was added to exogenously to a protease-deficient strain, the organism assumed the characteristics of nafcillin tolerance. Similarly, when phenylmethylsulfonyl fluoride, a serine protease inhibitor, was added to the tolerant strains, they became susceptible to nafcillin-induced lysis. The effects of nafcillin on B. subtilis were studied with both viability determinations and assay of cellular lysis. The minimum inhibitory concentrations of nafcillin tended to be higher for the protease hyperproducing strains, but these values could be reduced by the protease inhibitor. No loss of antibiotic activity was observed when nafcillin was incubated with either subtilisin or trypsin. Furthermore, protease and autolysin from B. subtilis were not modified by nafcillin. The results showed that extracellular proteases could render B. subtilis relatively tolerant to the killing and lytic effects of a cell wall antibiotic. The proteases were probably acting on the autolysins of the organism, thereby increasing tolerance to nafcillin.

Effect of 7,12-dimethylbenz[a]anthracene on production and action of gamma interferon.

Spleens were removed from Swiss Webster mice, and cultures of spleen cells were prepared. Gamma (ty pe II immune) interferon (IFN) production was induced in these cells by addition of the purified protein form of phytohemagglutinin (PHA-P) to the cultures. When 7,12-dimethylbenz[a]anthracene (DMBA) was added to the cultures either before or after addition of PHA-P, the production of gamma IFN was inhibited. The degree of inhibition was greater when the cells were treated with DMBA before addition of PHA-P. In experiments to determine the effects of DMBA on the antivirus activity of previously prepared gamma IFN preparations, DMBA was either added to target cells before the addition of exogenous gamma IFN or mixed together with exogenous gamma IFN. In both cases. the antivirus activity of this exogenous gamma IFN was not affected. These data suggest that DMBA treatment can inhibit the production but not the antivirus activity of murine gamma IFN.

Transport and incorporation of N-acetyl-D-glucosamine in Bacillus subtilis.

Bacillus subtilis 168 has been found to possess a high-affinity transport system for N-acetyl-D-glucosamine (GlcNAC). The Km for uptake was approximately 3.7 microM GlcNAc, regardless of the nutritional background of the cells. Apparent increases in Vmax were noted when the bacteria were grown in the presence of GlcNAc. The uptake of GlcNAc by B. subtilis was highly stereoselective; D-glucose, D-glucosamine, N-acetyl-D-galactosamine, D-galactose, D-mannose, and N-acetylmuramic acid did not inhibit GlcNAc uptake. In contrast, glycerol was an effective inhibitor of [3H]GlcNAc transport and incorporation. Partial inhibition of GlcNAc uptake was observed with azide, fluoride, and cyanide anions, carbonyl cyanide-m-chlorophenyl hydrazone, methyltriphenylphosphonium bromide, N,N'-dicyclohexylcarbodiimide, gramicidin, valinomycin, monensin, and nigericin. Two anions, arsenite and iodoacetate, were potent inhibitors of the uptake of GlcNAc in B. subtilis. Results from paper chromatography showed that there was no intracellular pool of free GlcNAc and that the acetylamino sugar was probably phosphorylated during transport. A modification of the Park-Hancock cell fractionation scheme indicated that cells grown on glycerol or D-glucose incorporated [3H]GlcNAc primarily into the cell wall fraction. When GlcNAc was used as the sole carbon source, label could be demonstrated in fractions susceptible to protease and nuclease, as well as lysozyme, showing that the N-acetylamino sugar was utilized in macromolecular synthesis and energy metabolism.

The energized membrane and cellular autolysis in Bacillus subtilis.

Lysis of exponential cultures of B. subtilis follows the addition of reagents that dissipate either the electrical or pH gradients of cellular membranes. Stationary-phase cells or cultures that have been inhibited in division by macromolecular-synthesis inhibitors also lyse when uncoupling agents or ionophores are added to the growth medium. Autolysis occurs after brief starvation for a carbon source. Protoplasts are unaffected by azide or other lysis-inducing agents. Electron-donating agents, such as phenazine methosulfate and ascorbate, are effective in retarding autolysis. The addition of an oxidizable carbon source to starved and lysing cultures prevents their autolysis. These results suggest that cellular lysis in B. subtilis and energized membrane are tightly coupled. The fluorescence intensity and the wavelength of maximal fluorescence of 8-anilino-1-naphthalene sulfonic acid, when added to bacterial suspensions, appear to be qualitatively related to the rate of cell lysis. Analyses show that ATP limitations are probably not involved in the elicitation of lysis by ionophores, uncoupling agents or starvation. Measurements of protonmotive forces in the lysis-prone cells suggest that a threshold force of more than 85 mV may be required to maintain cellular integrity. Lipoteichoic acids, polyelectrolytes such as dextran sulfate or phospholipids do not modify the rate of cellular lysis when added to suspensions containing azide or other reagents that eliminate transmembrane protonmotive forces. We interpret the results to suggest that the in vivo control of autolysin activity in B. subtilis is related to the energized membrane

Transformation of a Bacillus subtilis L-form with bacteriophage deoxyribonucleic acid.

A stable L-form, sal-1, of Bacillus subtilis was transformed with deoxyribonucleic acid (DNA) from bacteriophages phi 25 and phi 29 to determine whether exogenous DNA can be introduced into this organism. The viral transformation (transfection) was successful with the use of polyethylene glycol. In the presence of the fusogen, bacteriophage phi 25 DNA initiated a single cycle of infection. When compared with transfection of competent cells of Bacillus subtilis, the appearance of viral particles was delayed and their production occurred over a longer time period. L-form cells were best able to support intracellular replication of phi 25 viral particles when in balanced growth in a rich medium. The addition of polyethylene glycol also induced infection of sal-1 with whole bacteriophage phi 25 particles which could not otherwise infect the L-form and enhanced infection by intact phi 29 particles. Primary recombination was shown to be required for polyethylene glycol-mediated phi 25 transfection, but not phi 29 transfection or for whole bacteriophage phi 25 infection mediated by polyethylene glycol. Successful transfection of sal-1 suggests that the L-form may be amenable to genetic modification with exogenous DNA.

Effect of carcinogens and analogs on interferon induction.

Pretreatment of mouse embryo fibroblasts with several chemicals, including 7,12-dimethylbenz-(a)-anthracene, 2-aminofluorene, aflatoxin B1, benzo-(a)-pyrene, styrene oxide, and the No. 4 fraction of tobacco smoke condensate, resulted in severely reduced production of interferon when the cells were challenged with Newcastle disease virus. All of the above chemicals are proven or strongly suggested carcinogens. When the analogs methyl methanesulfonate, a potent carcinogen, and ethyl methanesulfonate, a weak carcinogen, were applied to cells, interferon induction was only inhibited by the methyl methanesulfonate. Therefore, carcinogens may directly inhibit the induction of interferon by Newcastle disease virus.