Human prostatic steroid 5α-reductase isoforms—A comparative study of selective inhibitors

The present study describes the independent expression of the type 1 and 2 isoforms of human 5α-reductase in the baculovirus-directed insect cell expression system and the selectivity of their inhibition. The catalytic properties and kinetic parameters of the recombinant isozymes were consistent with published data. The type 1 isoform displayed a neutral (range 6–8) pH optimum and the type 2 isoform an acidic (5–6) pH optimum. The type 2 isoform had higher affinity for testosterone than did the type 1 isoform (K_m = 0.5 and 2.9 μM, respectively). Finasteride and turosteride were selective inhibitors of the type 2 isoform (K_i (type 2) = 7.3 and 21.7 nM compared to K_i (type 1) = 108 and 330 nM, respectively). 4-MA and the lipido-sterol extract of Serenoa repens (LSESr) markedly inhibited both isozymes (K_i (type 1) = 8.4 nM and 7.2 μg/ml, respectively; K_i (type 2) = 7.4 nM and 4.9 μg/ml, respectively). The three azasteroids were competitive inhibitors vs substrate, whereas LSESr displayed non-competitive inhibition of the type 1 isozyme and uncompetitive inhibition of the type 2 isozyme. These observations suggest that the lipid component of LSESr might be responsible for its inhibitory effect by modulating the membrane environment of 5α-reductase. Partially purified recombinant 5α-reductase type 1 activity was preserved by the presence of lipids indicating that lipids can exert either stimulatory or inhibitory effects on human 5α-reductase.     

Decrease in glutathione levels of kidney and liver after injection of methionine sulfoximine into rats

After rats were injected with the convulsant methionine sulfoximine, there was a rapid decrease in the glutathione concentrations of the kidney and liver, but there was no measurable effect (within 5 hours) on brain glutathione. The maximum decreases in the glutathione concentrations of kidney and liver were observed 1 hr after injection and were about 60 and 40%, respectively, of the control levels. The findings suggest that there may be at least two pools of tissue glutathione. Studies in which other amino acids were injected, and earlier $\text{in vitro}$ studies, are consistent with the conclusion that methionine sulfoximine affects glutathione synthesis $\text{in vivo}$ by inhibiting γ-glutamylcysteine synthetase. Injection of glycylglycine also decreased glutathione levels, an effect probably mediated by γ-glutamyltranspeptidase.     

Application of tree rings [dendrochemistry] for detecting historical trends in air Hg concentrations across multiple scales

Tree rings are a useful long term monitor of climate change indicators and certain atmospheric constituents. Since we have no long-term historical record of air mercury (Hg) concentrations, and previous research demonstrates atmospheric uptake of Hg into vegetative biomass, we hypothesized that tree rings would be a useful archive for monitoring changes in local, regional, and global air Hg concentrations. We developed and tested a method for measurement of Hg in tree rings, and then investigated our hypothesis using species within the genus Pinus. Mercury concentrations declined along a coast-to-interior (California-to-Nevada, USA) gradient with highest mean concentrations (5.7 ng/g) at the coast, and lowest (<1.2 ng/g) at inland locations. Tree ring Hg concentrations recorded the impact of local sources and indicate increasing global concentrations over time. The data also indicate that concentrations are higher in areas impacted by marine air, and this suggests that the atmospheric chemistry is influencing assimilation. Future research should address species- and age-specific variation in the sensitivity of tree-ring samples as proxy data for atmospheric Hg concentrations. Tree rings show great potential as passive recorders of atmospheric Hg for long time periods (centuries to millennia) at time steps of ≤5 years.     

The codon specificity of eubacterial release factors is determined by the sequence and size of the recognition loop

The two codon-specific eubacterial release factors (RF1: UAA/UAG and RF2: UAA/UGA) have specific tripeptide motifs (PXT/SPF) within an exposed recognition loop shown in recent structures to interact with stop codons during protein synthesis termination. The motifs have been inferred to be critical for codon specificity, but this study shows that they are insufficient to determine specificity alone. Swapping the motifs or the entire loop between factors resulted in a loss of codon recognition rather than a switch of codon specificity. From a study of chimeric eubacterial RF1/RF2 recognition loops and an atypical shorter variant in Caenorhabditis elegans mitochondrial RF1 that lacks the classical tripeptide motif PXT, key determinants throughout the whole loop have been defined. It reveals that more than one configuration of the recognition loop based on specific sequence and size can achieve the same desired codon specificity. This study has provided unexpected insight into why a combination of the two factors is necessary in eubacteria to exclude recognition of UGG as stop.     

Ribosomal proteins of Thermus thermophilus fused to beta-galactosidase are imported into the nucleus of eukaryotic cells

Archaea, Bacteria, and Eukarya have 34 homologous ribosomal protein (RP) families in common. Comparisons of published amino acid sequences prompted us to question whether RPs of the prokaryote Thermus thermophilus contain nuclear localization signals (NLSs), which are recognized by the nuclear import machinery of eukaryotic cells and are thereby translocated into the nucleoplasm ultimately accumulating in the nucleolus. Several RPs of T. thermophilus - specifically S12, S17, and L2 - were selected for this study since their three-dimensional structures as well as rRNA interaction patterns are precisely known at the molecular level. Fusion proteins of these RPs were constructed and subsequently expressed in COS cells. N-terminally tagged fusions with dimeric EGFP and C-terminally tagged hybrids with beta-galactosidase of prokaryotic RP S17 (S17p) were targeted to the nucleoplasm where they were visualized by direct fluorescence and by indirect immune staining, respectively. A region containing the classical monopartite NLS KRKR, which is known to physically interact with karyopherin alpha2, was delineated by tagging specific S17p fragments with beta-galactosidase. Unexpectedly, S12p and L2p hybrids accumulated in the nucleolus. Due to their size, RPs tagged with beta-galactosidase can only be imported into the nucleus when NLS-recognition is mediated by karyopherins since they are otherwise excluded from entry into the nucleoplasm of eukaryotic cells. Our results indicate that after the formation of the nuclear compartment during evolution, the newly established eukaryotic cell relied on the pre-existing basic amino acid clusters of the prokaryotic RPs for use as NLSs.     

Selective feeding by larval dytiscids (Coleoptera:Dytiscidae) and effects of fish predation on upper littoral zone macroinvertebrate communities of arctic lakes

Carbon and nitrogen stable isotopic data from the primary producers in mangrove ecosystems are needed to investigate trophic links and biogeochemical cycling. Compared with other mangrove species (e.g. Rhizophora mangle) very few measurements have been conducted on the white mangrove, Laguncularia racemosa. The carbon and nitrogen stable isotopic and elemental compositions of L. racemosa were analyzed and compared from Florida and Belize. ¹³C values of L. racemosa from Florida (mean = –26.4) were slightly higher than those from Twin Cays, Belize (mean = -27.4), which may be due to higher salinity in some parts of the Florida site. There was no difference between the ¹⁵N values from L. racemosa from these two sites (Florida mean = 0.6; Belize mean = 0.3), which are indicative of nitrogen derived from nitrogen fixation in a planktonic marine system. However, higher ¹⁵N values from L. racemosa at Man of War Cay in Belize (11.4 and 12.3), which is fertilized by roosting marine birds (14.0), illustrate that L. racemosa can sensitively reflect alternative nitrogen sources. Although the isotopic data could not distinguish between Avicennia germinans, R. mangle and L. racemosa in Belize the L. racemosa had considerably higher C/N ratios (46.5 – 116.1) compared with the Florida samples (42.2 – 76.0) or the other mangrove species. Unlike some previous findings from R. mangle, substrate characteristics (e.g. salinity, NH₄ ⁺, and H₂S) were not related to the isotopic or elemental composition of L. racemosa. ¹³C, ¹⁵N and C/N were analyzed for ecosystem components from L. racemosa habitats at Twin Cays, including other plants (e.g. R. mangle, A. germinans and seagrass), detritus, microbial mats and sediments. Results from mass-balance calculations show that mangrove detritus composes very little of the sediment, which is principally composed of microbial biomass (80 – 90%). Detritus at some sites is also influenced by sources other than that from L. racemosa, including seagrass leaves.     

Soil Carbon and Nitrogen Storage in Upper Montane Riparian Meadows

Though typically limited in aerial extent, soils of high-elevation riparian wetlands have among the highest density of soil carbon (C) and nitrogen (N) of terrestrial ecosystems and therefore contribute disproportionally to ecosystem services such as water retention, forage production, wildlife habitat, and reactive N removal. Because much soil C and N is stored in labile forms in anaerobic conditions, management activities or environmental changes that lead to drying cause mineralization of labile soil organic matter, and loss of C and N. Meadows are focal points of human activities in mountain regions, often with incised stream channels from historically heavy grazing exacerbated by extreme runoff events. To quantify soil C and N stores in montane riparian meadows across hydrologic conditions, 17 meadows between 1950- and 2675-m elevation were selected in the central Sierra Nevada Range, California, that were classified using the proper functioning condition (PFC) system. Results indicate that C and N density in whole-solum soil cores were equivalent at forest edge positions of properly functioning, functioning at-risk, and nonfunctioning condition. Soils under more moist meadow positions in properly functioning meadows have at least twice the C, N, dissolved organic C, and dissolved organic N (DON) than those under nonfunctioning meadows. Densities of total N and DON, but not C, of functioning at-risk meadows are significantly lower (P < 0.05) than those of properly functioning meadows at mid-slope and stream-bank positions, suggesting accelerated loss of N early in degradation processes. Though variable, the soil attributes measured correspond well to the PFC riparian wetland classification system.