25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes

A constitutive cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 was purified from rat liver microsomes. The enzyme fraction contained 16 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum molecular weight of 51,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cytochrome P-450 catalyzed 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and 1 alpha-hydroxyvitamin D3 up to 50 times more efficiently, and 25-hydroxylation of vitamin D3 about 150 times more efficiently than the microsomes. The cytochrome P-450 showed no detectable 25-hydroxylase activity towards vitamin D2 and was inactive in cholesterol 7 alpha-hydroxylation as well as in 12 alpha- and 26-hydroxylations of C27-steroids. It catalyzed hydroxylations of testosterone and demethylation of ethylmorphine at the same rates as, or lower rates than, microsomes. The 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and vitamin D3 with the purified cytochrome P-450 was not stimulated by addition of phospholipid or cytochrome b5 to the reconstituted system. Emulgen inhibited 25-hydroxylase activity towards both substrates. The possibility that 25-hydroxylation of C27-steroids and vitamin D3 is catalyzed by the same species of cytochrome P-450 is discussed.     

Ram ribosomes are defective proofreaders

We have studied the kinetics of poly(U) translation by three ribosomal ambiguity (Ram) mutants in an in vitro system with performance characteristics similar to those expressed in vivo. The leucine missense frequency supported by Ram ribosomes with tRNALeu2 increases between six and twelve-fold over that of wild-type ribosomes, while the corresponding increase with tRNALeu4 was between four and eight-fold, depending on the rpsD allele. We have used a steady-state assay for proofreading to identify the kinetic lesion responsible for the Ram phenotype. We were unable to detect any difference between Ram and wild-type ribosomes with respect to the initial kinetics of amino-acyl tRNA selection. All of the increased error rates could be associated with a decreased capacity of these Ram ribosomes to discard non-cognate aminoacyl-tRNA by proof reading.     

Isolation and characterization of a 23 kDa protein essential for photosynthetic oxygen evolution

A 23 kDa protein has recently been demonstrated to participate in photosynthetic oxygen evolution by reconstitution experiments on inside-out thylakoid vesicles (Åkerlund H-E, Jansson C and Andersson B (1982) Biochim Biophys Acta 681:1–10). Here we describe the isolation of the 23 kDa protein from a spinach chloroplast extract using ion-exchange chromatography. The protein was obtained in a yield of 25% and with less than 1% of contaminating proteins. The ability of the protein to stimulate oxygen evolution in inside-out thylakoids was preserved throughout the various fractionation steps. The isolated protein was highly water soluble and appeared as a monomer. Its isoelectric point was at pH=7.3. The amino acid composition showed a high content of polar amino acids, resulting in a polarity index of 49%. The isolated protein lacked metals and other prosthetic groups. Its function as a catalytic or regulating subunit in the oxygen evolving complex is discussed.     

Potentiation of antibody responses by specific IgM: specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.     

Outbreak of Giardiasis: Effect of a New Antiflagellate Drug,Tinidazole

We describe here an intensive outbreak of mostly symptomatic (90%) Giardia lamblia infestation in a Swedish student group visiting the U.S.S.R. A new antiflagellate drug, ethyl (2-(2-methyl-5-nitro-1-imidazolyl)ethyl) sulphone, tinidazole, Pfizer, was given in a dosage of 150 mg twice daily for seven days to 10 healthy volunteers and to 24 students infested with G. lamblia. The drug was found to be effective in curing giardiasis and in eradicating G. lamblia from the intestinal tract. All the students with symptomatic giardia infestation became free from gastrointestinal disturbance, usually soon after treatment was started. None of the 24 students had G. lamblia in their stools after tinidazole treatment was discontinued or at follow-up. No side effects of the drug were seen and all of the subjects tolerated it very well.     

Purification of two muscle enzymes by chromatography on immobilized ferric ions

Two enzymes, glycogen phosphorylase and lactate dehydrogenase, were purified simultaneously in a single step. Ferric ions immobilized on a chelating gel were used as the adsorbent. Adsorption and desorption steps were accomplished by changes in buffer composition. The recoveries were better than 80% and the capacities were about 5 mg of protein per milliliter of adsorbent. The procedure worked well both on a small and on a preparative scale. The homogeneity of the purified enzymes was checked by FPLC.     

Functional maturation of human T lymphocytes is accompanied by changes in the G-protein pattern

The putative guanine nucleotide binding (G)-protein involved in transduction of signals from the TCR/CD3 complex has not been identified. We have used a UV-photoaffinity labeling technique to covalently attach [alpha-32P]GTP to human lymphocyte and thymocyte membrane proteins. Ten bands specifically labeled with [32P]GTP were detected by SDS-PAGE and autoradiography in T lymphocyte membranes. Among these, a 40-kDa protein was identified by immunoblotting as the alpha-subunit of the adenylate cyclase-inhibiting G-protein, Gi, and two proteins of 44 and 46 kDa were identified as the alpha-subunits of adenylate cyclase stimulating G-protein (Gs). These proteins also served as substrates for ADP-ribosylation by pertussis toxin and cholera toxin, respectively. Comparison of GTP-labeled membrane proteins from immature and more mature thymocytes and blood T lymphocytes, revealed that bands of 26, 30, 34, 40, 44 and 46 kDa were absent or weakly labeled in immature thymocytes, intermediate in mature thymocytes, and strongest in blood T cells. Similar increases were seen in ADP ribosylation of the substrates for pertussis, cholera, and botulinum C3 toxin. However, corresponding quantitative changes in Gi and Gs were not detected by immunoblotting, which suggests that the increased labeling is caused by enhanced affinity of the proteins for GTP rather than by increased amount of protein during thymic maturation. A concomitant maturation of GTP-induced cAMP production was seen in the cell populations, but no such change occurred in direct activation of adenylate cyclase by forskolin. The changes in some (but not all) GTP-binding proteins during acquisition of immunocompetence indicates their importance in T lymphocyte physiology.     

Production of TNF-alpha and TNF-beta by staphylococcal enterotoxin A activated human T cells

Staphylococcal enterotoxin at concentrations of less than 1 pg/ml induces significant TNF activity in human peripheral blood T cells and monocytes. Maximal TNF activity is routinely detected after 48 to 72 h of culture. IL-2 and IL-4 were both growth promoting for human T cells but only IL-2 could efficiently induce TNF production. The production of TNF-alpha and TNF-beta differed greatly in kinetics. An early intracytoplasmatic production of TNF-alpha after 6 h was detected in both monocytes and T cells whereas a late production of TNF-beta (lymphotoxin) after 48 h, occurred in the T cell population. Induction of TNF-alpha and TNF-beta production by Staphylococcal enterotoxin requires the presence of both monocytes and T cells. The CD4+45R- but not CD4+45R+ and CD8+ cells supported TNF-alpha production in monocytes. The main lytic component from Staphylococcal enterotoxin-activated mononuclear cells is TNF-beta. CD4+ and CD8+ T cells produced about equal amounts of biologically active TNF into the culture supernatants but a fourfold higher frequency of TNF-beta producing cells was demonstrated among CD4+ vs CD8+ cells. The CD4+45R- T cell subset was an efficient producer of TNF-beta and IFN-gamma whereas the CD4+45R+ T cell subset produced significant amounts of TNF-beta but only marginal amounts of IFN-gamma.