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Biomedical subjects

R Marais

Publications and source records attributed to R Marais.

45 records · Page 3Linked to original sources
PubMed1989

Monoclonal antibodies to protein kinase C gamma. Functional relationship between epitopes and cofactor binding sites.

Four monoclonal antibodies (mAbs), derived from high-responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype gamma 1, kappa and 15G4 is of isotype gamma 2b, kappa. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific for the bovine gamma isoform of protein kinase C and cross-react with protein kinase C gamma from a variety of animal species. Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase C gamma cofactor-dependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor-independent catalytic activity of protein kinase C gamma. Competition analysis between these mAbs and phosphatidylserine, 12-O-tetradecanoylphorbol 13-acetate and Ca2+ showed that 36G9 and 5A2 block cofactor binding to protein kinase C gamma. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase C gamma.

Animals↗
PubMed1989

Dephosphorylation of the human T lymphocyte CD3 antigen.

Previous studies demonstrated that activation of T lymphocytes by phorbol ester or mitogenic lectin leads to phosphorylation of Ser 126 of the CD3 antigen gamma chain, whereas treatment with ionomycin results in phosphorylation of both Ser 123 and 126 [Davies, A. A. et al. (1987) J. Biol. Chem. 262, 10918-10921]. In the present study, the dephosphorylation of Ser 123 and Ser 126 of the gamma chain was investigated. Phorbol-ester-induced phosphorylation of the gamma-chain Ser 126 in vivo was reversed following removal of phorbol ester. Dephosphorylation of both Ser 123 and 126 was also observed in vitro using the microsome fraction of T lymphocytes. In order to identify the phosphatases acting at these two sites, the immunoprecipitated gamma chain was used as substrate either following treatment with protein kinase C in vitro, in which case phosphorylation occurs mainly at Ser 123, or following in vivo phosphorylation of Ser 126. Purified oligomeric forms of the polycation-stimulated phosphatases were more effective in dephosphorylating both phosphorylated forms of the gamma chain compared with equivalent amounts of ATP,Mg2+-dependent phosphatases or calcineurin. By using phosphopeptide analogues of the CD3 gamma chain containing Ser 123 or Ser 126 as substrates (A3 and A6), it was shown that polycation-stimulated phosphatases selectively dephosphorylated Ser 123 compared to Ser 126. In order to determine which phosphatases dephosphorylate the gamma chain in microsomes, A3 and A6 were used as substrates for characterising phosphatases in microsomes from human T leukaemia Jurkat 6 cells. Three phosphopeptide phosphatases (250-400 kDa) co-eluted through five purification steps with three forms of polycation-stimulated phosphorylase phosphatase. The partially purified A3/A6 phosphopeptide phosphatases were insensitive to Ca2+, calmodulin and inhibitor-1, and dephosphorylated A3 preferentially compared with A6. A latent form of microsomal ATP,Mg2+-dependent phosphorylase phosphatase was stimulated 10-fold by trypsinisation, but did not dephosphorylate phosphopeptides A3 and A6. The results show that high-Mr forms of polycation-stimulated phosphatases are the only enzymes in human T leukaemia cell microsomes which dephosphorylate gamma chain phosphopeptides. The data point to an important role for polycation-stimulated phosphatases in regulating the phosphorylation state, and so function(s), of the CD3 antigen.

Amino Acid Sequence↗
PubMed1987

The in-vitro activity of doxycycline and minocycline against anaerobic bacteria.

The likelihood of bacterial resistance now prevents the use of oxytetracycline in the empirical therapy of anaerobic infections. This study investigates the in-vitro activity of two semi-synthetic derivatives, doxycycline and minocycline, against a range of anaerobic bacteria. MICs for each antibiotic were determined by an agar incorporation technique. Doxycycline and minocycline were four to eight times more active against the majority of strains than oxytetracycline. With the exception of Bacteroides bivius, almost 90% of strains were inhibited by 4 mg/l of doxycycline or minocycline, but resistance to the same concentration of oxytetracycline was present in 60% of the B. fragilis group, 30% of Peptostreptococcus spp. and 24% of Clostridium perfringens. Doxycycline and minocycline represent an alternative therapy for anaerobic infections where bacterial sensitivities are known.

Bacteria, Anaerobic↗
PubMed1987

In vitro activity of vancomycin and teicoplanin against anaerobic bacteria.

The in vitro activity of vancomycin and teicoplanin, a new glycopeptide antimicrobial, was determined against a total of 286 anaerobic bacteria including Bacteroides fragilis group (100), B. melaninogenicus (21), B. bivius (16), Fusobacterium spp. (15), Peptococcus spp. (20), Peptostreptococcus spp. (21), Clostridium perfringens (23), C. difficile (41) and Propionibacterium acnes (29). Minimum inhibitory concentrations (MIC) were determined using an antimicrobial incorporation technique in Wilkins-Chalgren agar approximately 10(4) colony forming units (cfu) contained in 10 microliters Wilkins-Chalgren broth, which was applied to the surface of the agar plates using a multipoint inoculator. Following inoculation, plates were incubated for 48 h at 37 degrees C in an anaerobic atmosphere. Both vancomycin and teicoplanin were highly active against all the Gram-positive anaerobic bacteria examined, 90% of all isolates being inhibited by 0.5 micrograms/ml of either antimicrobial. Isolates of B. fragilis group and Fusobacterium spp. were resistant to vancomycin (MIC90 64 micrograms/ml) and teicoplanin (MIC90 128 micrograms/ml). Unexpectedly, isolates of B. melaninogenicus and B. bivius which were resistant to vancomycin (MIC90 64 and 128 micrograms/ml respectively) were sensitive to teicoplanin (MIC90 2 and 2 micrograms/ml respectively).

Bacteria, Anaerobic↗
PubMed1987

The effect of carbon dioxide on the in vitro activity of erythromycin and RU-28965 against anaerobic bacteria.

The in vitro activity of erythromycin and RU-28965 (a novel macrolide antimicrobial with improved pharmacokinetics) was determined against a variety of anaerobic bacteria in anaerobic atmospheres with and without added carbon dioxide. Minimum inhibitory concentrations (MIC) were determined using an antimicrobial incorporation technique in Wilkins-Chalgren agar (Oxoid, UK) containing saponinlysed horse blood to a final concentration of 10%. The inoculum used was approximately 10(4) colony forming units (cfu) contained in 10 microliters Wilkins-Chalgren broth, which was applied to the surface of the agar plates using a multipoint inoculator. Following inoculation, plates were incubated for 48 h at 37 degrees C in an anaerobic atmosphere containing 10% carbon dioxide or in hydrogen alone. The MIC of each antimicrobial for each organism examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum. The minimum concentrations required to inhibit the growth of 50% (MIC50) and 90% (MIC90) of the bacteria examined were also determined. The MICs of erythromycin and RU-28965 for isolates of the Bacteroides fragilis group, B. bivius and Fusobacterium spp. were generally 10-100 times greater when determined in the presence of carbon dioxide than when determined in hydrogen alone. The MICs of erythromycin and RU-28965 for B. melaninogenicus, Peptococcus spp., Peptostreptococcus spp., Clostridium perfringens, Cl. difficile and Propionibacterium acnes were less affected by the presence of carbon dioxide.

Anaerobiosis↗