Two human liver cDNAs encode UDP-glucuronosyltransferases with 2 log differences in activity toward parallel substrates including hyodeoxycholic acid and certain estrogen derivatives.

Two human liver UDP-glucuronosyltransferase cDNA clones, HLUG25 [Jackson, M. R., et al. (1987) Biochem. J. 242, 581-588] and UDPGTh-2 [Ritter, J. K., et al. (1990) J. Biol. Chem. 266, 7900-7906] have previously been shown to encode isozymes active in the glucuronidation of hyodeoxycholic acid (HDCA) and certain estrogen derivatives (estriols and 3,4-catechol estrogens), respectively. Here we report that the UDPGTh-2-encoded isoform (udpgth-2) and the HLUG25-encoded isoform (udpgth-1) have parallel aglycon specificities. Following expression in COS-1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol and 17-epiestriol, and HDCA, but the udpgth-2 isozyme is 100-fold more efficient than udpgth-1. udpgth-1 and udpgth-2 are 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which confers isoform substrate specificity. The data indicate that a high level of conservation in the amino terminus is not required for the preservation of substrate selectivity. Analysis of glucuronidation activity encoded by UDPGTh-1/UDPGTh-2 chimeric cDNAs constructed at their common restriction sites, SacI (codon 297), NcoI (codon 385), and HhaI (codon 469), showed that nine amino acids between residues 385 and 469 are important for catalytic efficiency, suggesting that this region represents a domain which is critical for catalysis but distinct from that responsible for aglycon selection.(ABSTRACT TRUNCATED AT 250 WORDS)     

WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.     

Maize C4 photosynthesis involves differential regulation of phosphoenolpyruvate carboxylase genes

Maize as a C4 plant partitions CO2 fixation in two consecutive, spatially separated steps, thus eliminating photorespiration. The crucial enzyme for primary CO2 fixation is a C4-specific phosphoenolpyruvate carboxylase (PEPC). The differential expression of the unique C4-specific gene pepcZm1 and two non-C4-specific genes, pepcZm2A and pepcZm3B, in leaf, root, and stem is reported here. It is shown, in a transient homologous system, that this tissue-specific regulation is mainly controlled by their distinct promoters. The light induction of the C4-specific pepcZm1 in illuminated etiolated (greening) leaves probably relies on light-dependent developmental changes instead of an immediate responsiveness found for other maize genes. Analyses of deleted, mutated, and hybrid promoters revealed the redundant nature of a 14mer which is repeated four times and a decisive function of the TATA box-like motif, TATTT, and the sequences directly preceding it. No consensus sequences to other photosynthetic gene promoters were uncovered. Although light induces the expression of C4 PEPC and other photosynthetic genes in maize, this co-ordination is apparently mediated through different signal transduction pathways and distinct regulatory elements. This study indicates that the acquisition of a new promoter is at least partially responsible for the C4-specific expression of pepcZm1 essential for C4 photosynthesis.     

Secondary rhinoplasty.

Secondary rhinoplasty is a highly complex, judgmental type of surgery. It is only through analysis of the successful treatment of various problems in secondary rhinoplasty that meaningful information can be obtained.     

Frequency-dependent viability in mutant strains of Drosophila melanogaster

We investigated the effects of genotypic frequencies on egg-to-adult viabilities in pairwise combinations of four strains of Drosophila melanogaster. The experiments involved mixture of a total of 42,000 eggs in varying proportions under controlled densities and observation of surviving adults. Viabilities were found to depend on frequencies in several genotypic combinations. In the most extreme case, the absolute viability of cn;bw females increased monotonically from 54% when common to 70% when rare. The results illustrate several statistical and methodological problems that might explain why some experiments have failed to detect frequency-dependent viabilities. These problems include heterogeneity between replications, sex differences in susceptibility to competition, and strong dependence of the experimental outcome on the choice of competitor genotypes.     

Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes.

I describe here the organization of maize C4 chloroplast and non-C4 cytosolic pyruvate, orthophosphate dikinase (PPDK) genes and the molecular mechanisms underlying their differential expression. The maize C4 chloroplast PPDK gene (C4ppdkZm1) appears to have been created by the addition of an exon encoding the chloroplast transit peptide at a site upstream of a cytosolic PPDK gene (cyppdkZm1). A splice acceptor sequence located in the first exon of cyppdkZm1 allows the fusion of the transit peptide to the cyppdkZm1 sequences. A second cyPPDK gene (cyppdkZm2) shares extensive homology with cyppdkZm1 in the coding region and in the 5' flanking region up to the TATA box. By a novel protoplast transient expression method, I show that the light-inducible expression of C4ppdkZm1 is controlled by two expression programs mediated through separate upstream regulatory elements that are active in leaf, but inactive in root and stem. Light-mediated C4ppdkZm1 expression in maize is apparently uncoupled from leaf development and partially associated with chloroplast development. For cyppdkZm1 expression, distinct upstream elements and a specific TATA promoter element, located in the first intron of C4ppdkZm1, are required. The low expression of cyppdkZm2 can be attributed to an absence of upstream positive elements and weak activity of the TATA promoter element.