Stable ester conjugate between the Saccharomyces cerevisiae RAD6 protein and ubiquitin has no biological activity.

The RAD6 gene of Saccharomyces cerevisiae, which encodes a ubiquitin-conjugating enzyme, is required for DNA repair, DNA damage-induced mutagenesis and sporulation. To evaluate the biological relevance of the thioester adduct between RAD6 protein and ubiquitin, formed as an obligatory, transient intermediate during ubiquitin conjugation to substrates, we altered cysteine 88 in RAD6 to serine. Esterification with ubiquitin occurs at serine 88 in the mutant protein, but conjugation of ubiquitin to the test substrate histone H2A is inactivated. Phenotypically, strains harboring the rad6 Ser88 allele are indistinguishable from rad6 deletion (rad6 delta) mutant cells. These findings argue against ligation of ubiquitin at cysteine 88 acting as a functional switch of a cryptic biochemical activity in RAD6.     

Role of kallikrein-kininogen system in insulin-stimulated glucose transport after muscle contractions.

Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum +/- kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum +/- bradykinin receptor-2 inhibitors; or 4) serum-free buffer +/- bradykinin. 3-O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum +/- kallikrein inhibitors tended (P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.     

Immersion staining of coarse fish in the Thames

Thirty-one vital dyes were screened for marking bleak, roach and gudgeon by total body immersion in aqueous solutions of the dye. Only six dyes stained without killing, and of these Nile blue, neutral red and Bismarck brown were used successfully in the field. This approach to estimation of population size was abandoned because (1) the toxicity of the dyes varied at different times of year, (2) the numbers needed for release were impractically high and (3) fingerlings showed extremely high mortality on handling.     

Chromatin degradation in white blood cells of irradiated rats

A study was made of the dose-time relationship during chromatin degradation in white blood cells of non-irradiated and irradiated rats. There was a linear increase in the release of PDN from leukocytes 1,2 and 3 days after irradiation (1-3 Gy) followed by the deceleration of the chromatin degradation at doses exceeding 3 Gy.     

How to become comfortable talking about sex to your patients.

Dealing with patients'' sex-related complaints is uncomfortable for many physicians. It can become more comfortable if the physician learns to apply his or her clinical knowledge of sexuality in combination with specific interpersonal interviewing skills. The level of comfort can be increased if the physician accepts his or her professional limitations in providing therapy and knows what other sources of care are available.     

Determination of the biological activity of Clostridium difficile toxins in in vivo and in vitro experiments

The biological activity of the filtrates of 29 C. difficile strains was studied in vivo (suckling white mice) and in vitro (cell cultures of different species and origin). The action of the filtrates on the experimental models in vivo was evaluated from the cytotoxic effect index, while in vitro the intensity of the cytotoxic effect was evaluated from the percentage of dead cells in the monolayer. The results of the comparative determination of toxicity characteristics in vivo and in vitro demonstrated that cell cultures were more sensitive experimental models than suckling white mice. The use of cell cultures permitted the quantitative evaluation of the cytotoxic activity of the filtrates under study, as well as the detection of their cell-directed action at minimal concentrations.     

Effect of alpha-fetoprotein on isolated mouse oocytes

Data are presented which indicate a possible action of alpha-fetoprotein (AFP) on female germinal cells. The in vitro maturation of mature mice oocytes was significantly inhibited when mouse AFP replaced albumin in the culture medium. In addition, the degenerative aspect of oocytes cultured with AFP seemed to indicate that this me?otic inhibition was caused by a premature degeneration of oocytes rather than by a blockage at a specific stage of maturation. Thus AFP, perhaps through its ligands, may play a role in the reduction of germinal cells during fetal and immediate post-natal life rather than in the arrest of me?osis at the diplotene stage.     

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.