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Biomedical subjects

J Chaloupka

Publications and source records attributed to J Chaloupka.

At least 19 recordsLinked to original sources
PubMed1990

Effect of actinomycin D on viability, sporulation and nucleotide pool of Bacillus megaterium.

A transient 7-fold rise of ppGpp concentration, 2-3-fold increase of pppGpp concentration and 50% drop of the concentration of GTP in Bacillus megaterium cells immediately after their transfer to the sporulation medium were observed. Actinomycin D, in concentrations inhibiting RNA synthesis by 95%, blocked the rise of the (p)ppGpp pool and caused an instant several-fold increase of the GTP level. When the cells were exposed to actinomycin D in the sporulation medium for a 1-h period (time 0-1 h, 1-2 h or 2.20-3.20-h), they were able to form colonies on nutrient agar after being kept, in addition for 1-2 h in the sporulation medium free of the antibiotic. The ability of sporulation was, however, markedly limited. The share of cells that could sporulate increased when the irreversible sporulation phase was reached.

Adenosine Triphosphate↗
PubMed1990

Characteristics of intracellular proteolytic activities of Bacillus megaterium.

Intracellular proteolytic activities of B. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called CI was stable, had a relative molecular mass of Mr = 105,000 (M = 105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated CII, was labile and had a relative molecular mass of Mr = 46,000 (M = 46 kg/mol). Because of its lability it could not be characterized in detail. In the "periplasm" only a single proteolytic enzyme P (Mr = 28,000; M = 28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12 that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the cytoplasmic proteinase CI, could be demonstrated.

Bacillus megaterium↗
PubMed1989

Regulation of extracellular proteins and alpha-amylase secretion by temperature in Bacillus subtilis.

alpha-Amylase was found to be the main protein secreted by Bacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45 degrees C, respectively. A change in temperature can affect the pattern of proteins secreted as detected by gel electrophoresis. 14C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30 degrees C than that at 40 or 45 degrees C. The effect of temperature on alpha-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.

Bacillus subtilis↗
PubMed1988

Netropsin increases formation of mRNA coding for a neutral metalloproteinase in Bacillus megaterium.

The anticancer drug netropsin increases the synthesis of an exocellular metalloproteinase during exponential growth as well as in the stationary phase of a sporulating strain of Bacillus megaterium. Its effect is due to a stimulation of the synthesis of the mRNA coding for the proteinase, determined as a residual synthesis of the enzyme in the presence of actinomycin D. The half-life of the proteinase mRNA (5-6 min at 35 degrees C) is not affected by netropsin. Netropsin relieves partially the repression of the proteinase mRNA caused by amino acids, whereas the repression brought about by an increased temperature is almost unaffected by the drug.

Amino Acids↗
PubMed1988

Effect of temperature on alpha-amylase formation and DNA replication in Bacillus subtilis.

The effect of temperature on extracellular alpha-amylase synthesis and chromosomal and plasmid DNA replication in Bacillus subtilis A18 carrying plasmid pMI10 was studied. The specific growth rate mu increased with elevated temperature up to 42.5 degrees C, while the activities of alpha-amylase per population dry mass decreased. No obvious quantitative changes of 14C-thymidine incorporation per dry mass increase and no basic differences in plasmid copy number in the range of temperatures from 25 to 40 degrees C were found.

Bacillus subtilis↗
PubMed1986

Turnover of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae: differences between in vivo and in vitro degradation.

Degradation of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae in vivo was compared with that in cell-free extracts. Protein degradation in vivo, when the cells were labelled with 14C-leucine during growth in the presence of ethionine, was affected by the concentration of the analogue used. Proteins synthesized in the presence of 0.2-1 mM ethionine were degraded most rapidly in both organisms. The proteolytic enzyme system of yeast degraded the analogue-containing proteins in vitro faster than the normal proteins. This holds also for proteins synthesized in the presence of 5 mM ethionine, whose degradation in vivo was impaired. The proteolytic system of B. megaterium, on the other hand, was unable in vitro to differentiate between normal and abnormal proteins. Denatured proteins underwent preferential degradation over normal and ethionine-containing proteins.

Bacillus megaterium↗
PubMed1986

Interference with sporulation can stimulate the rate of a proteinase synthesis in Bacillus megaterium.

Bacillus megaterium, in which sporulation was blocked either by mutation or with netropsin, synthesizes during the stationary phase more exocellular proteinase than the sporulating culture. The asporogenic mutant synthesizes the enzyme at a higher rate and for a longer time than does the sporulating population. The culture, whose sporulation was inhibited by netropsin, produces the proteinase at a higher rate, although for only a limited time interval.

Bacillus megaterium↗
PubMed1985

Temperature as a factor regulating the synthesis of microbial enzymes.

Temperature affects the formation of several enzymes permanently during long-term growth (e.g. penicillinases, proteases, respiratory enzymes) or transiently immediately after a heat or cold shock (RNA polymerase, aminoacyl-t-RNA synthetases, ribonucleases and others). The synthesis of developmentally regulated enzymes may be suppressed by temperatures permissive for growth but not for differentiation (e.g. sporulation). The control of enzyme formation by temperature may be operating at the level of template multiplication (plasmid DNA), transcription, translation or formation of low-molecular-weight effectors.

Bacteria↗
PubMed1984

Mutants of Bacillus megaterium with altered synthesis of an exocellular neutral proteinase.

Germinated spores of Bacillus megaterium were mutagenized with ethyl methanesulphonate and spread on test agar with caseinate. Colonies with altered proteolytic zones or morphology were isolated and tested in liquid media. The mutants can be divided into four groups: A) those producing more proteinase in both growth and sporulation media, B) those producing the same amount of the enzyme in growth medium but higher amount in sporulation medium, C) those producing less proteinase in the growth medium and more in the sporulation one, D) those producing less or no enzyme. Clones of the first three groups were phenotypically asporogenic. All mutants producing more enzyme during growth retained their sensitivity to repression by amino acids. Isolation of mutants of types B) and C) supports the idea of differences in the control of proteinase synthesis during growth and during sporulation.

Bacillus megaterium↗
PubMed1984

Repression of the synthesis of exocellular and intracellular proteinases in Bacillus megaterium.

The synthesis of exocellular proteinase decreases with increasing concentration of amino acids in the growth medium. After removal of amino acids the enzyme synthesis is gradually restored to normal values. The presence of inhibitors of transcription (actinomycin D) or translation (chloramphenicol) blocks the restoration of enzyme synthesis. No active or inactive precursors of the exocellular enzyme could be detected in the cell. It is likely that the enzyme synthesis is regulated by amino acids at the level of specific mRNA synthesis rather than at the translation level or at the level of secretion. The activity of the enzyme that has already been secreted to the external medium is partially inhibited by amino acids. The periplasmic proteinase is repressed by amino acids to the same extent as the exocellular enzyme. The content of the enzyme(s) inside the protoplast is also decreased during growth in the presence of amino acids.

Amino Acids↗
PubMed1983

Suppression of an exocellular proteinase synthesis in Bacillus megaterium by increased temperature.

During cultivation of Bacillus megaterium at 42 degrees C the amount of the exocellular protease produced by growing cells sharply decreases as compared with temperatures of 28 and 35 degrees C. Within the above range the growth rate and incorporation of amino acids increase with increasing temperature. The culture adapted to 42 degrees C does not produce more proteinase at this temperature than the non-adapted culture. The high temperature does not induce accumulation of the enzyme in the cells. Total protein excretion was slightly lower at 42 degrees C than at 28 and 35 degrees C.

Bacillus megaterium↗
PubMed1983

Combined effect of temperature and nutrients on protein turnover in Bacillus megaterium.

Protein turnover was followed in populations of Bacillus megaterium growing in temperature range of 17-48 degrees C in different media. Higher temperature stimulated the protein turnover (expressed as the amount of protein degraded during 3.3 h) in all the media tested up to the optimal growth temperature (40-42 degrees C). Protein turnover in a medium containing amino acids continued to be stimulated by temperature even above this point; no further significant increase of turnover was found in the other media.

Amino Acids↗
PubMed1981

Synthesis of exocellular proteins during the exponential and stationary phase of growth of Bacillus megaterium.

Synthesis of exocellular metalloprotease and cellular and exocellular proteins in the sporogenic strain Bacillus megaterium J-27 and asporogenic strain KM 1 was investigated. Both organisms excrete the enzyme into the medium during growth and during the stationary phase. In the asporogenic strain the excretion decreases at the end of the exponential phase. In the sporogenic strain it continues during the transition to the stationary phase at the original rate and proteolytic activity in the medium increases two to three times during 2 h after the end of the exponential phase. Both organisms synthesize relatively more exocellular proteins during the exponential phase than during the stationary phase. The proportion of exocellular protein synthesized during the exponential phase does not exceed 3 % of total proteins, during the stationary phase this proportion usually decreases to less than 1 %.

Bacillus megaterium↗
PubMed1981

Protein turnover in growing cultures of Bacillus megaterium.

A growing population of Bacillus megaterium, prelabelled with 14C-amino acids displays protein turnover, the rate of which is influenced by the growth rate. The size of the pool of short-lived proteins, degraded with a half-life of less than 1 h, is directly proportional to the mass doubling time during subsequent growth of the population. However, their degradation constant is almost the same under all conditions. The degradation constant of residual long-lived proteins is influenced by the growth rate. The labile fraction can be replenished by a combination of shift-up and shift-down treatments. Tetracycline decreases the size of the labile (short-lived) fraction and almost stops the degradation of long-lived proteins. Correlation of protein turnover and intracellular proteolytic activity indicates that other factors in addition to the proteinase are necessary for the degradation of short-lived, as well as long-lived proteins.

Bacillus megaterium↗
PubMed1980

Turnover of proteins in asporogenic Bacillus megaterium. Evidence for a gradual decrease of the turnover rate.

The rate of protein turnover in asporogenic Bacillus megaterium decreases continuously during incubation in a sporulation medium. The capability of equilibration of external amino acids with amino acids in the metabolic pool of non-growing cells was retained for at least 5 h. Leucine, while repressing the synthesis of the exocellular protease, does not significantly influence the course of protein degradation in vivo. Transfer of non-growing cells after 4 h to a fresh sporulation medium does not influence the rate of protein degradation. The gradual decrease of the rate of protein turnover in non-growing cells of the asporogenic variant is thus not an artifact caused by a decreased uptake of amino acids by cells or by conditions under which the protein turnover is determined.

Amino Acids↗