Cyclic AMP regulation of Gs protein. Thyrotropin and forskolin increase the quantity of stimulatory guanine nucleotide-binding proteins in cultured thyroid follicles

This study was carried out to clarify the way in which thyrotropin (TSH) and forskolin regulate the adenylylcyclase complex in thyroid follicle cells. We examined the effects of chronic treatment of pig thyroid follicles with TSH or forskolin on the state of G proteins by (a) assaying adenylylcyclase activity, (b) analyzing the ADP-ribosylation of stimulatory G protein (Gs) by cholera toxin, and (c) quantifying the Gs subunits by Western blotting with antipeptide antibodies. Chronic exposure (18 h) of thyroid follicles to a low concentration of TSH (0.01-0.1 milliunit/ml) enhanced the subsequent response of adenylylcyclase to TSH. Higher concentration of TSH (1 milliunit/ml) induced a homologous desensitization of this response. In cells pretreated with forskolin, the TSH-stimulated adenylylcyclase activity was higher than in control cells. The forskolin-or guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p)-stimulated adenylylcyclase activity was always significantly increased after chronic treatment of cells with TSH or forskolin. Treatment of cultured thyroid follicle membranes with [32P]NAD and cholera toxin resulted in labeling of the Gs alpha (45-52-kDa) component. Culturing follicles with TSH (0.001-1 milliunit/ml) or forskolin (0.01-10 microM) greatly affected the cholera toxin-mediated ADP-ribosylation of the Gs alpha subunit. Gs alpha labeling increased progressively to level off at 1 milliunit/ml TSH or 1 microM forskolin (150-200%). Gs alpha immunoreactivity was increased in parallel (200-300%). The immunoreactivity of G beta subunits in cells cultured with TSH or forskolin was also increased compared with control cells. Cycloheximide abolished the effects of TSH and forskolin on the follicles, suggesting that new protein synthesis is required. These results indicate that Gs protein subunits are up-regulated by TSH and forskolin and suggest that their synthesis in thyroid cells is mediated, at least in part, by a cyclic AMP-dependent mechanism.     

Effects of 4-methyluracil and carnosine on healing of skin wounds in rats

In experiments in vivo and in vitro the authors studied antioxidative properties of 4-methyluracil and carnosine, their capacity to inhibit sex and accelerate healing of skin wounds. 4-methyluracil and carnosine discover almost the same capacity to decrease in the tissues of the wound and the blood serum in the formation of various intermediate products of free radical oxidation. Data are given on the study of the dynamics of wound healing after a 5-day treatment with equimolar quantities.     

Two novel mutations in the MHC class II transactivator CIITA in a second patient from MHC class II deficiency complementation group A

Congenital MHC class II deficiency or bare lymphocyte syndrome (BLS; McKusick 209920) is caused by defects in trans-acting regulatory factors that control MHC class II expression and is therefore a disease of gene regulation. There are at least four complementation groups and the genetic and molecular dissection of this rare disease has contributed considerably to our current understanding of the molecular mechanisms governing MHC class II expression. Identification of the gene that is defective in BLS complementation group A, CIITA (MHC class II transactivator), has led to the discovery that CIITA acts as a master control factor of MHC class II expression. We have identified the CIITA mutations in a second patient from BLS group A. Two novel mutations abolish CIITA function, as shown by transfection experiments. Molecular analysis of these two novel mutations, together with the one described earlier in the first patient, is informative in terms of CIITA structure-function relationships. Received: 19 October 1996 / Revised: 25 November 1996     

Study of the biosynthesis of fumonisins B1, B2 and B3 by different strains of Fusarium moniliforme

The relationship between fungal growth and the production of fumonisin on maize grain by 25 strains of Fusarium moniliforme of different origins has been investigated. Although sporulation was essentially the same for all the strains (about 10⁸ propagules g⁻¹ dry matter), ergosterol assays revealed marked variations in fungal biomass. All strains studied produced highly variable amounts of fumonisin B1, the highest levels being observed in strains of ergosterol content above 400 μg g⁻¹. However, no correlation could be established between the synthesized biomass and the quantity of fumonisins produced. We verified that ergosterol is an indicator of mycelial growth, and therefore of the potential toxicity of the analysed grain.     

Kinetics of beta-tubulin exchange following translation. Evidence for a slow conformational change in beta-tubulin necessary for incorporation into heterodimers

Cell-free translation of beta-tubulin mRNA generates full length beta-tubulin polypeptides distributed in three molecular forms: a high molecular weight lysate-associated form, the free beta-tubulin subunit, and the alpha beta-heterodimer (Yaffe, M.B., Farr, G. W., and Sternlicht, H. (1988) J. Biol. Chem. 263, 16023-16031). A quantitative assay system for these three forms was developed and used to measure the rates of incorporation/exchange of the newly synthesized free beta-subunit and the high molecular weight form into tubulin heterodimers following incubation of the 35S-translation products with unlabeled bovine tubulin dimer. This exchange process was found to be slow and strongly temperature-dependent. The half-lives for exchange ranged from 12.5 min at 37 degrees C to 17.5 h at 0 degree C with a measured activation energy of 22.5 kcal/mol. Microtubule-associated proteins appeared to play no role in the exchange process, since identical exchange rates were observed regardless of whether microtubule protein or phosphocellulose-purified tubulin was used as the source of tubulin dimer. Surprisingly, the exchange rates were found to be independent of dimer concentration. We interpret these results as evidence for a rate-limiting, slow conformational change that occurs within the newly synthesized beta-subunits prior to their association with alpha-tubulin to generate the alpha beta-hetero-dimer.     

Translocation of pro-OmpA across inner membrane vesicles of Escherichia coli occurs in two consecutive energetically distinct steps

The rate of energy-dependent transfer of pro-OmpA across Escherichia coli inner membrane vesicles in vitro was found to be a function of the ATP concentration. At concentrations above 0.1 mM ATP, the addition of a transmembrane electrochemical potential (proton motive force or pmf) increased the rate of pro-OmpA translocation. Additional experiments demonstrated that the overall reaction proceeded by at least two distinct energy-requiring steps. The first step required only ATP, was nearly unaffected by the pmf, and resulted in the insertion of the amino-terminal domain of pro-OmpA across the membrane. The insertion exposed the signal sequence cleavage site to the periplasmic side of the membrane, as measured by the appearance of a mature length translocation intermediate. However, this intermediate was partially exposed to the cytoplasmic side of the membrane. In a second energy-dependent step, either ATP or the pmf was sufficient to complete the translocation of mature length OmpA across the membrane.     

The bactericidal effect of isoascorbic acid combined with mild heat

The thermal inactivation of Salmonella thompson, Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Candida zeylanoides, Enterococcus faecium and E. faecalis was accelerated by the addition of sodium isoascorbate (1 mmol/l) to phosphate-buffer heating medium but not to complex food mixtures. The lethal effect of isoascorbate was nullified by heating under anaerobic conditions or by the addition of catalase. The scavengers of hydroxyl radicals, mannitol and formate were not protective whereas histidine was. Histidine may have protected by slowing the rate of isoascorbate autoxidation, a property common to other amino acids tested. Superoxide dismutase was not protective. Dehydroascorbic acid also enhanced heat killing and its action was also reversed by catalase. The bactericidal effects of mild heat plus isoascorbate or dehydroascorbic acid both apparently depend on oxidative processes but their relative effectiveness was not related to their respective rates of oxygen consumption or peroxide production. We speculate that site-specific redox reactions, involving amino-carbonyl intermediates are involved in the inactivation mechanism.