Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact with TOM1 mutants defective in ribosome synthesis.

The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.

Steroidogenic factor-1 influences protein-deoxyribonucleic acid interactions within the cyclic adenosine 3,5-monophosphate-responsive regions of the murine steroidogenic acute regulatory protein gene.

De novo synthesis of the steroidogenic acute regulatory protein (StAR) in response to trophic hormonal stimulation of steroidogenic cells is required for the delivery of cholesterol from the mitochondrial outer membrane to the mitochondrial inner membrane and the cytochrome P450 side-chain cleavage enzyme. StAR expression is transcriptionally regulated by cAMP-mediated mechanisms, and we have identified a 45-bp region within the mouse promoter that is important for cAMP responsiveness of the gene. This region, located between -105 and -60 of the start site of transcription, contains a SF-1-binding site, a highly conserved C/EBPbeta-AP-1-nuclear receptor half-site sequences (CAN region), and a GATA-4-binding site. The SF-1 element and CAN region are required for full basal activity, whereas the GATA-4 element may account for 20% of the cAMP response in MA-10 mouse Leydig tumor cells. A cAMP-dependent protein-DNA complex was observed with the CAN region and mutation of a nonconsensus AP-1 site within this region greatly diminished promoter strength. Complex protein-DNA interactions within the cAMP response region (-105/-60) were shown to require the SF-1 element (-95), suggesting that SF-1 is required for protein-DNA interaction at the CAN (-79) region and maximal activity of the promoter.

Metabolism of dibasic esters by rat nasal mucosal carboxylesterase.

Inhalation exposure of rats to dibasic esters revealed lesions of the nasal olfactory epithelium similar to those observed with other ester solvents. Female rats are more sensitive to these effects than are male rats. It has been proposed that carboxylesterase conversion of inhaled esters within nasal tissues to organic acids may be a critical biochemical step in converting these chemicals to toxic substances. These experiments measured the kinetic parameters Vmax, KM, Ksi, and V/K for the hydrolysis of the dibasic esters in the target nasal tissue, olfactory mucosa, and nontarget tissue, respiratory mucosa. It was determined that under the conditions of these experiments, diacid metabolites are not formed. Esterase activity was inhibited by pretreatment with bis p-nitrophenyl phosphate. Vmax values for the three dibasic esters were 5- to 13-fold greater in olfactory mucosa than respiratory mucosa for male or female rats. V/K values were 4- to 11-fold greater in olfactory mucosa than respiratory mucosa for male or female rats. V/K was similar between male and female olfactory mucosa when dimethyl glutarate was used as the substrate. With dimethyl succinate or dimethyl adipate as the substrate, V/K for female olfactory tissue was 0.5- or 2-fold that of males, respectively. Differences in V/K were mainly due to decreases in KM associated with increasing carbon chain length. Substrate inhibition was observed at dibasic ester concentrations greater than approximately 25 mM, which are unlikely to be achieved in vivo. These results lend further support to the hypothesis that organic acid accumulation in the target tissue, olfactory mucosa, plays a significant role in the pathogenesis of dibasic ester-induced nasal lesions. This mechanism may be applicable to a wide range of inhaled esters.

Subchronic inhalation study with vinyl fluoride: effects on hepatic cell proliferation and urinary fluoride excretion.

Vinyl fluoride is used widely in the manufacture of fluoropolymers. Based in part on the structural similarity of vinyl fluoride to the hepatocarcinogens vinyl chloride and vinyl bromide, a TSCA Section 4 test rule mandated the testing of vinyl fluoride for oncogenicity. This report presents the results of a 90-day inhalation study in rats and mice with vinyl fluoride designed to set test concentrations for a subsequent oncogenicity study. Groups of 15 male and female rats and mice were exposed 6 hr per day, 5 days per week for approximately 90 days to target concentrations of 0, 200, 2000, or 20,000 ppm vinyl fluoride. Clinical chemical, hematological, and urine analyses were performed on rats after 45 and 90 days of exposure. A hematological evaluation was performed on mice following 45 and 90 days of exposure. A complete gross and microscopic evaluation was conducted at the end of the study. After 93 days on test, groups of five rats and five mice per sex were implanted with osmotic minipumps containing [3H]thymidine and were exposed for an additional 5 days to measure cell proliferation in liver, kidney, lung, and nasal cavity tissues. Results of the histopathological, clinical chemical, and hematological evaluations showed no significant effects of vinyl fluoride exposure at any concentration following either 45 or 90 days of exposure. A concentration-related increase in fluoride ion in urine was observed in rats at 45 and 90 days of exposure. A plateau in urinary fluoride excretion was observed at approximately 2000 ppm, suggesting saturation of vinyl fluoride metabolism. Vinyl fluoride-related cell proliferation effects were largely restricted to liver. Hepatic cell proliferation in male and female rats and mice was elevated at all concentrations. The response was similar at concentrations of either 2000 or 20,000 ppm and was consistent with concentration-response relationships for other haloethylenes. Taken together, the urinary fluoride excretion and hepatic cell proliferation data suggest a mechanistic link between the two effects. On the basis of these findings and experience with other haloethylenes, concentrations of vinyl fluoride to be tested for oncogenicity should be chosen such that the full linear range of the concentration-response curve is evaluated. The present study demonstrates through example the value of incorporating cell proliferation studies in standard testing protocols.