A convenient synthesis of dinorbile acids: oxidative hydrolysis of norbile acid nitriles

We report a convenient method for the synthesis of dinorbile acids (23,24-dinor-5beta-cholan-22-oic acids, pregnane-20-carboxylic acids) in fair to good yields from norbile acid nitriles in one step by oxidative hydrolysis with oxygen in the presence of potassium-t-butoxide. The method results in stepwise overall removal of two carbon atoms in bile acid side chains in two steps. Dinorbile acids corresponding to several common bile acids have been prepared and their structures confirmed by spectroscopic methods. This simple method for synthesis of dinorbile acids may facilitate their study metabolically.     

Metagenomic analyses of an uncultured viral community from human feces

Here we present the first metagenomic analyses of an uncultured viral community from human feces, using partial shotgun sequencing. Most of the sequences were unrelated to anything previously reported. The recognizable viruses were mostly siphophages, and the community contained an estimated 1,200 viral genotypes.     

Human herpesvirus 7 open reading frame U12 encodes a functional beta-chemokine receptor

Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily, infects mainly CD4+ T cells in vitro and infects children during infancy. After the primary infection, HHV-7 becomes latent. HHV-7 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 gene, we cloned the gene and expressed the U12 protein in cells. The U12 gene encoded a calcium-mobilizing receptor for the EBI1 ligand chemokine-macrophage inflammatory protein 3beta (ELC/MIP-3beta) but not for other chemokines, suggesting that the chemokine selectivity of the U12 gene product is distinct from that of the known mammalian chemokine receptors. These studies revealed that U12 activates distinct transmembrane signaling pathways that may mediate biological functions by binding with a beta-chemokine, ELC/MIP-3beta.     

Estimating predation pressure in ecological studies: controlling bias imposed by using sentinel plasticine prey

Sentinel plasticine prey has been increasingly used to estimate predation pressure. The use of plasticine prey may, however, bias the results, as this method was originally designed to account for predation by organisms that can visually recognize the shapes and colors of their prey. To evaluate the limitations of using sentinel plasticine prey, we compared predator attack rates between real prey – dead and live mealworms, Tenebrio molitor L. (Coleoptera: Tenebrionidae) – and plasticine models in a monsoonal tropical rainforest of southeastern China. The attack rates by invertebrates were highest on dead prey followed by live prey and plasticine models, whereas the attack rates by vertebrates were lowest on dead prey, and did not differ between live prey and plasticine models. These results confirm that bias imposed by using the plasticine models is affected by the type of predators. In addition, we tested the validity and generality of the premise that predators can distinguish the shapes of plasticine model prey and preferentially attack a caterpillar-like shape over other shapes. To test this hypothesis, we conducted three independent experiments in China, Papua New Guinea, and Finland. In the two latter localities, predation rates on plasticine caterpillars were higher than on models of other shapes, whereas in China, these differences were not significant. Taken together, our study suggests that plasticine models may underestimate the predation by invertebrates to a greater extent than predation by vertebrates, and the preference of model shape by predators may be locality-specific, presumably due to differences in the composition of the predator community. We propose that predation be estimated on both live and plasticine prey in future studies to measure the potential bias imposed by using plasticine models and its variation among various habitats and predator groups.     

Absolute configuration of pentahydroxy bile alcohols excreted by patients with cerebrotendinous xanthomatosis: a circular dichroism study

The absolute configurations of the C27 pentahydroxy bile alcohols present in bile and feces of two patients with cerebrotendinous xanthomatosis (CTX) were determined by circular dichroism (CD) spectroscopy. The CD spectra of 5beta-cholestane-3alpha,7alpha,12alpha,24alpha,25-pentol in the presence of Eu(fod)3 [tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyloctane-4,6-dionato) europium (III)] exhibited a negative Cotton effect and was assigned to 24R absolute configuration. Conversely, 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol showed a strong positive Cotton effect and was assigned the 24S configuration. These assignments were based upon comparison with a model compound, 5-cholestene-3beta,24(R),25-triol, whose single-crystal X-ray structure has been determined. The importance of these data is to establish a structural mechanism for the conversion of 5beta-cholestane-3alpha,7alpha,12alpha,24S,25-pentol rather than 5beta-cholestane-3alpha,7alpha,12alpha,24R,25-pentol into cholic acid in man as well as in animals.     

A role of mitogen and stress-activated protein kinase 1/2 in survival of lipopolysaccharide-stimulated RAW 264.7 macrophages

The effect of inhibition of mitogen and stress-activated protein kinases 1/2 (MSK1/2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was investigated. Pretreatment with Ro 31-8220, an inhibitor of MSK1/2, induced cell death in LPS-stimulated RAW 264.7 cells. In contrast, calphostin C, another inhibitor of protein kinase C, did not cause cell death. Cell death was not mediated by the release of pro-inflammatory mediators from LPS-stimulated RAW 264.7 cells. Cell death was accompanied by DNA fragmentation and annexin V binding, suggesting apoptotic cell death. Further, several caspase inhibitors did not prevent LPS-induced cell death of Ro 31-8220-pretreated RAW 264.7 cells. Nuclear translocation of apoptosis-inducing factor (AIF) was detected in Ro 31-8220-pretreated cells after LPS stimulation. Cell death was due to mitochondrial damage. Ro 31-8220 exclusively inhibited the phosphorylation of cAMP-responsive element binding protein (CREB), a substrate of MSK1/2. RAW 264.7 cells transfected with the dominant-negative MSK1 clones underwent cell death in response to LPS. Hence, it was suggested that MSK1/2 might play a critical role in the survival of LPS-stimulated RAW 264.7 cells.     

PHACCS,an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information

Background  

Phages, viruses that infect prokaryotes, are the most abundant microbes in the world. A major limitation to studying these viruses is the difficulty of cultivating the appropriate prokaryotic hosts. One way around this limitation is to directly clone and sequence shotgun libraries of uncultured viral communities (i.e., metagenomic analyses). PHACCS, Phage Communities from Contig Spectrum, is an online bioinformatic tool to assess the biodiversity of uncultured viral communities. PHACCS uses the contig spectrum from shotgun DNA sequence assemblies to mathematically model the structure of viral communities and make predictions about diversity.     

Calcium Movements in CGRP-treated Cultured Skeletal Muscle Cells: Is There a Role for CGRP in Tension Headaches?

It was previously determined that the site of action of calcitonin gene-related peptide (CGRP) in cardiomyocytes was predominantly at the sarcolemmal calcium release channel, and studies have shown that CGRP has major effects on intracellular cardiomyocyte calcium concentrations. We postulated that CGRP would have similar effects on striated skeletal muscle and determined the effects of CGRP on calcium levels in cultured chick myotubes by fluorescence imaging. Myoblasts were cultured until they were continuous myotubes. Deconvolution fluorescence imaging was employed to visualize subcellular organelles and construct 3D renditions. Myotubes were treated with a high (1 μM) and a low (1 nM) concentration of CGRP for 1 h or 24 h time periods, and real-time fluorescence spectrophotometry with a calcium specific fluoroprobe permitted the acquisition of images and calcium transients. Experiments also used CGRP 8–37 to ensure specificity of action of the full-length neuropeptide. CGRP localizations by image stacking were made using fluorescence deconvolution microscopy and distributions on the myotubes were shown. Myotube contractions and intracellular calcium levels were dose dependent, a high CGRP concentration producing calcium overload. CGRP 8–37 had no effect on contractions or calcium levels. Reconstructed images revealed the neuropeptide to be localized to juxta-nuclear areas, supporting the likelihood of site specific actions. CGRP has dramatic effects on intracellular calcium in striated muscle, high concentrations producing sustained contractions and calcium overload. The results give support to a mechanistic role for CGRP in muscle tension headaches, and underscore the importance in the development of CGRP analogues or receptor antagonists for treatment.