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Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Deltatsc1 and Deltatsc2, were up-regulated in Deltabhd, and levels of specific intracellular amino acids known to be low in Deltatsc1 and Deltatsc2 were elevated in Deltabhd. This "opposite" profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1(+) dramatically increased permease expression levels in Deltabhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Deltabhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes.

Cystathionine gamma-synthase (CGS) catalyzes the first committed step of Met biosynthesis in plants. We have previously shown that expression of the gene for CGS is feedback-regulated at the level of mRNA stability, and that the amino acid sequence encoded by the first exon of the CGS gene itself is responsible for the regulation (Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M., and Naito, S. (1999) Science 286, 1371-1374). To identify the functional region within CGS exon 1, deletion analysis was performed. The results showed that the 41-amino acid region of exon 1 highly conserved among plants is necessary and sufficient for the regulation. Analyses of in vivo and in vitro generated mutations that abolish the regulation identified the functionally important amino acids as 11-13 residues within this conserved region. The importance of these residues was confirmed by deletion analysis within the conserved region. These studies identified the functional region of CGS exon 1 required for the posttranscriptional autoregulation of the CGS gene as (A)RRNCSNIGVAQ(I), with uncertainty of the first and last residues. This sequence is almost perfectly conserved among CGS sequences of higher plants but cannot be found elsewhere in the public databases.

The O2 dependence of cytochrome oxidase, cellular ATP/ADP ratio, and drug sulfation and glucuronidation were studied in isolated hepatocytes to examine secondary biochemical changes of hypoxia related to decreased cytochrome oxidase function. A decrease in cytochrome oxidase function as well as in cellular ATP/ADP ratio occurred at low O2 concentrations and their O2 dependencies gave p50 values of 3.2 and 2.8 microM O2, respectively. Changes in the ATP/ADP ratio corresponded directly with changes in the oxidation-reduction state of cytochrome oxidase. The p50 value for sulfation of acetaminophen (2.5 microM O2) corresponded with the values for ATP/ADP ratio and cytochrome oxidase. Addition of compounds that lowered cellular ATP/ADP ratio caused a corresponding inhibition of sulfation. Selective use of ethionine, an adenosyl-trapping agent, demonstrated that the O2 dependence of sulfation related directly to a decrease in the absolute ATP concentration. The p50 value for glucuronidation (5.2 microM O2) was higher than that for sulfation, but in spite of this difference, glucuronidation also correlated well with cellular ATP/ADP ratio when this ratio was decreased by either O2 limitation or metabolic inhibitors. These results demonstrate that the predominant limitation of drug sulfation and glucuronidation during hypoxia is due to decreased cytochrome oxidase function.

A substantial inhibition (50-70%) of GSH efflux by methionine was demonstrated in hepatocytes isolated from fed rats. Concurrent measurements of intracellular GSH revealed maintenance of a higher concentration in methionine-supplemented cells over the 1-h incubation. Analysis of total GSH suggested that maintenance of higher intracellular GSH by methionine could be quantitatively accounted for by inhibition of GSH efflux rather than by net GSH synthesis. This conclusion was supported by studies with propargylglycine, a potent inhibitor of cysteine synthesis from methionine. Identical results were obtained in incubations containing either propargylglycine and methionine or methionine alone, thereby suggesting that net synthesis of GSH from methionine was minimal under the assay conditions. Similar decreases (40-60%) in the rate of extracellular accumulation of GSH were observed with ethionine and buthionine, two higher homologs of methionine, but not with a wide range of other naturally occurring and synthetic amino acids. The inhibition of GSH efflux by methionine was not dependent on the presence of sodium in the medium and did not correlate with metabolic consumption of ATP.

Ribosome phosphorylation was studied by monitoring the phosphorylation state of small subunit protein S6 as visualized on two-dimensional electrophoretograms of ribosomal proteins isolated from rat liver. No phosphorylation of S6 was observed under conditions of ethionine-induced inhibition of protein synthesis. Moderate phosphorylation, detected as the appearance of S6 and four or five phosphorylated derivatives, was observed in saline-treated animals. Reversal of ethionine-induced inhibition of protein synthesis by treatment with adenine led to extensive phosphorylation of S6. A model for protein synthesis which includes requisite phosphorylation of ribosomes during initiation is proposed. Cyclic adenosine 3':5'-monophosphate concentration was significantly elevated in liver of both ethionine- and ethionine plus adenine-treated rats, relative to that of saline-treated animals.