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Biomedical subjects

D Schlessinger

Publications and source records attributed to D Schlessinger.

At least 199 records · Page 11Linked to original sources
PubMed1977

Escherichia coli DNA-directed beta-galactosidase synthesis in presence and absence of Ca2+.

DNA-dependent synthesis of beta-galactosidase was optimized in extracts made from cells lysed by a standard French pressure cell. Extracts made at 3200 psi synthesized up to 25-fold more beta-galactosidase than extracts made at 7500 psi. beta-Galactosidase synthesis was cyclic 3', 5' AMP dependent, as expected, and in optimal conditions transcription and translation proceeded at 8.6 nucleotides and 2.7 amino acids per s, respectively. The high pressure extracts were stimulated 3- to 5-fold by Ca2+, especially at low Mg2+ concentrations. In contrast, extracts prepared at low pressure were inhibited as much as 50-fold by Ca2+ ions. The inhibition by Ca2+ was analyzed further. Addition of kasugamycin, an antibiotic that acts on ribosomes, to reactions containing Ca2+ stimulated beta-galactosidase synthesis to nearly control levels. Extracts from a kasugamycin resistant mutant were neither inhibited by Ca2+ nor stimulated by the addition of kasugamycin to in vitro reactions containing Ca2+. The change in the mutant was ascribed to the ribosomes by testing combinations of soluble proteins, ribosome wash, and ribosomes from parental and mutant strains. These results suggest that Ca2+ ions inhibit translation by ribosomes, very likely at an initiation step; and that they enhance enzyme synthesis only in conditions where translation is inefficient (high-pressure extracts at low concentrations of Mg2+, for example). This latter effect is probably a consequence of increased RNA stability in the presence of Ca2+ (Cremer, K., and Schlessinger, D. (1974), J. Biol. Chem. 249,4730).

Aminoglycosides↗
PubMed1977

Amphotericin B and filipin effects on L and HeLa cells: dose response.

Amphotericin B (AmB) and filipin effects on L and HeLa cells were compared by monitoring drug-induced potassium leakage from cells, changes in radioactive uridine incorporation into cellular ribonucleic acid, protein leakage from cells, and cell viability. L cells were much more susceptible to both AmB and filipin than were HeLa cells, but the overall dose response was similar. For AmB, the various effects were easily separable. Potassium leakage occurred at the lowest concentrations of AmB and was reversible. Inhibition of uridine incorporation and loss of viability occurred at intermediate levels, and protein loss occurred at higher levels. In contrast, filipin was much more potent; its effects on potassium leakage were only minimally reversible, and the separation of the permeabilizing effects from complete cell lysis was possible only over a limited concentration range and for a short time.

Amphotericin B↗
PubMed1977

Inhibition of isolated yeast and mycelial phase RNA polymerases of Histoplasma capsulatum by rifamycin derivatives.

Various derivatives of rifamycin were shown to inhibit the RNA polymerases of the yeast and mycelial phases of Histoplasma capsulatum. The relative potency of each of the derivatives against the isolated polymerases was the same as the potency of each against the viable organism. RNA polymerase PC III from the yeast phase was more susceptible to the rifamycin derivatives than yeast phase polymerases PC I and PC II and the biggest differences in susceptibility were seen with the derivative AF/ABDP (2,6-dimethyl-4-benzyl-4-demethyl-rifamycin). The susceptibility pattern of the mycelial polymerase activity was identical to the yeast polymerase PC III.

Cell Differentiation↗
PubMed1976

Coupling of rates of transcription, translation, and messenger ribonucleic acid degradation in streptomycin-dependent mutants of Escherichia coli.

The growth rates of streptomycin-dependent mutants varied in proportion to the level of streptomycin supplied; growth also varied characteristically from one dependent strain to another at a given streptomycin concentration. When cells growing at different rates (over a threefold range) were treated with rifampin, direct proportionality was observed for three parameters: (i) the rates of shutoff of transcription of total ribonucleic acid (RNA) and ribosomal RNA, as measured by pulse labeling at later times; (ii) the translation time for molecules of beta-galactosidase; and (iii) the rate of chemical degradation of messenger RNA. In contrast, the rate of functional inactivation of both total and beta-galactosidase messenger RNA was about the same at all growth rates. None of the variations of growth or other parameters were observed in an otherwise isogenic streptomycin-resistant strain treated with streptomycin. Since the mutational change in strd mutants and the site of action of streptomycin are in the 30S ribosomal subunits, it is suggested that the rate of ribosome function is set by the dependent lesion (and the level of streptomycin). One possibility is that the other correlated effects are mechanistically "coupled" to ribosome function, but the apparent coupling could also be an indirect result of differential effects of streptomycin on variables such as ribosomal miscoding and nucleotide pool size. However, since the rate of functional inactivation of messenger RNA is constant even when the RNA is broken down two- to fourfold more slowly, translation yield tends to be proportional to the growth rate of the dependent strains.

Escherichia coli↗
PubMed1975

30 S pre-ribosomal RNA of Escherichia coli:primary and secondary processing.

The metabolism of newly-formed labeled 30 S pre-ribosomal RNA was studied in the Escherichia coli mutant strain AB301-105. Detailed kinetic analysis in rifampicin-treated cells showed that precursors of 23 S and 16 S rRNA are formed from the 30 S RNA species, even in phosphate-limited cultures. To establish the order fo segments along 30 S pre-rRNA, it was allowed to accumulate in replicate chloramphenicol-treated cultures, and pulse-labeled after the addition of rifampicin in segments that progressively contained more 3'-distal label with time. The purified RNAs from the various cultures were then cleaved to a limited extent by RNAase III; the specific activity of the fragments yielded an order from the 5'-end of 17.5S-X-25S, where X refers to some of the sequences released as additional small fragments. More extensive treatment the 25-S and 17.5-S pre-rRNA chains with RNAase III yielded additional fragments and produced RNA species with the mobility of the 23-S and 16-S RNA precursors made in normal cells treated with chloramphenicol. A processing scheme is suggested that distinguishes between early, site-specific cleavage of recognition sites in the RNA itself ('primary processing'), and later, "secondary processing' reactions. The secondary processing reactions are blocked chloramphenicol-treated wild-type cells.

Chloramphenicol↗
PubMed1975

Escherichia coli mutants deficient in RNA accumulation at high temperature.

A selection technique is described which has permitted isolation of 10 mutants that continue to form protein, but are deficient in the accumulation of rRNA at 42 degrees C. DNA-RNA hybridization experiments have demonstrated that most of these mutants are specifically defective in their ability to synthesize rRNA at the restrictive temperature. The bulk of the pulse-labeled RNA formed in such mutants at 42 degrees C appears to be mRNA, with normal instability as measured in the presence of rifampicin. The remaining mutants appear to synthesize normal levels of rRNA, but that rRNA does not accumulate in a stable form. Since all of these mutants continue to form protein, and most do not accumulate significant levels of ppGpp at 42 degrees C, it appears likely that the shut-off of rRNA synthesis at 42 degrees C does not act through a lesion in the rel locus. Thus, these mutants may reveal another element(s) required to promote ribosomal RNA formation.

Bacterial Proteins↗
PubMed1975

Stability of amphotericin B in fungal culture media.

We have found that amphotericin B is unstable in two commonly employed fungal culture media. This instability leads to inaccuracies in determining the actual level of susceptibility of slow-growing strains that require prolonged incubation for growth. To help compensate for this problem, we have described two rapid methods of susceptibility testing.

Amphotericin B↗
PubMed1975

Response of yeast and mycelial phases of Histoplasma capsulatum to amphotericin B and actinomycin D.

The dimorphic fungus Histoplasma capsulatum exists in two phases: a unicellular yeast form at 37 C and a mycelium at 25 C. We have found that these two phases have selective drug susceptibilities. The mycelial form of H. capsulatum was much more susceptible to the polyene antibiotic amphotericin B than the yeast form; in contrast, the yeast form was more susceptible to the antibiotic actinomycin D. The changes in susceptibility occurred early in the transition between the two phases and permitted the transitions to be blocked by sublethal concentrations of the appropriate drugs.

Amphotericin B↗