Biotransformation of 17-alkylsteroids in the equine: gas chromatographic-mass spectral identification of ten intermediate metabolites of methyltestosterone

The metabolism of the orally active anabolic steroid methyltestosterone in the equine was investigated by administration of the drug along with a tritiated traces. In this study some of the metabolites were identified and a radio immunoassay screen and immunoaffinity chromatography gel for methyltestosterone were also evaluated. Pathway intermediates, in particular the 17-methylandrostanediols, were studied to gain an insight into the most likely stereochemistry of the major metabolites. The predominant phase I biotransformations involve reduction of the A ring 3-oxo and 4-ene groups to yield predominantly 3β-hydroxy-5-androstane products and hydroxylation of the steroid nucleus at several positions. Epimerisation of the 17-methyl group also occurred. Ten steroids could be positively identified by comparison with authentic references materials and many other triol, tetrol and pentols were also observed. Phase II metabolites and sulphate conjugates in particular, were common.     

Monitoring the health of selected eastern arc forests in Tanzania

The eastern arc mountains (EAMs) are a chain of isolated mountains (534,000 ha) in Kenya and Tanzania surrounded by arid woodlands and influenced by the Indian Ocean. In 1900 there was three times the amount of forest cover there is today. Much of the original forests have been converted into agricultural crops. These mountains are recognized as a globally important ‘hot spot’ by World Wide Fund for Nature (WWF) and World Conservation of Union (IUCN) for forest biodiversity and are major national, regional and local sources of hydropower, water and wide array of forest‐based benefits and agricultural production. In February 2000 we began a 3‐year evaluation of forest health, land‐use change and information sharing in the EAMs. Permanent sample plots were used to evaluate forest health. This paper summarizes the baseline data from eighteen permanent sample plots in the east Usambara's and nine in Ulugurus. The eastern arc web site ( http://www.easternarc.org ) was established, continues to expand and contains some detailed information on the project. Information gathered will assist land managers, planners, policy makers, interested communities (villages) and individuals in decision‐making. These data can also provide an early warning system of stresses on forest catchments.     

Differential modification of seawater carbonate chemistry by major coral reef benthic communities

Ocean acidification (OA) resulting from uptake of anthropogenic CO₂ may negatively affect coral reefs by causing decreased rates of biogenic calcification and increased rates of CaCO₃ dissolution and bioerosion. However, in addition to the gradual decrease in seawater pH and Ω _a resulting from anthropogenic activities, seawater carbonate chemistry in these coastal ecosystems is also strongly influenced by the benthic metabolism which can either exacerbate or alleviate OA through net community calcification (NCC = calcification – CaCO₃ dissolution) and net community organic carbon production (NCP = primary production ? respiration). Therefore, to project OA on coral reefs, it is necessary to understand how different benthic communities modify the reef seawater carbonate chemistry. In this study, we used flow-through mesocosms to investigate the modification of seawater carbonate chemistry by benthic metabolism of five distinct reef communities [carbonate sand, crustose coralline algae (CCA), corals, fleshy algae, and a mixed community] under ambient and acidified conditions during summer and winter. The results showed that different communities had distinct influences on carbonate chemistry related to the relative importance of NCC and NCP. Sand, CCA, and corals exerted relatively small influences on seawater pH and Ω _a over diel cycles due to closely balanced NCC and NCP rates, whereas fleshy algae and mixed communities strongly elevated daytime pH and Ω _a due to high NCP rates. Interestingly, the influence on seawater pH at night was relatively small and quite similar across communities. NCC and NCP rates were not significantly affected by short-term acidification, but larger diel variability in pH was observed due to decreased seawater buffering capacity. Except for corals, increased net dissolution was observed at night for all communities under OA, partially buffering against nighttime acidification. Thus, algal-dominated areas of coral reefs and increased net CaCO₃ dissolution may partially counteract reductions in seawater pH associated with anthropogenic OA at the local scale.     

Relative sensitivity of five Hawaiian coral species to high temperature under high-pCO2 conditions

Coral reef ecosystems are presently undergoing decline due to anthropogenic climate change. The chief detrimental factors are increased temperature and increased pCO₂. The purpose of this study was to evaluate the effect of these two stressors operating independently and in unison on the biological response of common Hawaiian reef corals. Manipulative experiments were performed using five species (Porites compressa, Pocillopora damicornis, Fungia scutaria, Montipora capitata, and Leptastrea purpurea) in a continuous-flow mesocosm system under natural sunlight conditions. Corals were grown together as a community under treatments of high temperature (2 °C above normal maximum summer temperature), high pCO₂ (twice present-day conditions), and with both factors acting in unison. Control corals were grown under present-day pCO₂ and at normal summer temperatures. Leptastrea purpurea proved to be an extremely hardy coral. No change in calcification or mortality occurred under treatments of high temperature, high pCO₂, or combined high temperature–high pCO₂. The remaining four species showed reduced calcification in the high-temperature treatment. Two species (L. purpurea and M. capitata) showed no response to increased pCO₂. Also, high pCO₂ ameliorated the negative effect of high temperature on the calcification rates of P. damicornis. Mortality was driven primarily by high temperature, with a negative synergistic effect in P. compressa only in the high-pCO₂–high-temperature treatment. Results support the observation that biological response to temperature and pCO₂ elevation is highly species-specific, so generalizations based on response of a single species might not apply to a diverse and complex coral reef community.

     

Conformational dynamics of stem II of the U2 snRNA

The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Förster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg²⁺ and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg²⁺ and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg²⁺ may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg²⁺ or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg²⁺ toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg²⁺-dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.     

Occurrence of Nontuberculous Mycobacteria in Environmental Samples

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).     

Solution structure of the DNA binding domain of AraC protein

We report the solution structure of the DNA binding domain of the Escherichia coli regulatory protein AraC determined in the absence of DNA. The 20 lowest energy structures, determined on the basis of 1507 unambiguous nuclear Overhauser restraints and 180 angle restraints, are well resolved with a pair wise backbone root mean square deviation of 0.7 Å. The protein, free of DNA, is well folded in solution and contains seven helices arranged in two semi‐independent sub domains, each containing one helix‐turn‐helix DNA binding motif, joined by a 19 residue central helix. This solution structure is discussed in the context of extensive biochemical and physiological data on AraC and with respect to the DNA‐bound structures of the MarA and Rob homologs. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Opposite allosteric mechanisms in TetR and CAP

Regulation of the DNA binding affinity of an oligomeric protein can be considered to consist of an intrinsic component, in which the affinity of an individual DNA‐binding domain is modulated in response to effector binding, and an extrinsic component, in which the relative position of the protein's two DNA‐binding domains are altered so that they can or cannot contact both half‐site operators simultaneously. We demonstrated directly that the TetR repressor utilizes an extrinsic mechanism and CAP, the catabolite activator protein, utilizes an intrinsic mechanism.