Combinational effects of farnesoid X receptor antagonist and statin on plasma lipid levels and low-density lipoprotein clearance in guinea pigs

Aims

We previously reported anti-dyslipidemic effects of a farnesoid X receptor antagonist in monkeys. In this study, we compared the cholesterol-lowering effects of single and combined administration of a farnesoid X receptor antagonist, compound-T8, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor atorvastatin in a guinea pig model.

Main methods

Plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, were measured after a single administration of compound-T8. The effects of compound-T8 or atorvastatin on plasma cholesterol levels and low-density lipoprotein (LDL) clearance were investigated after 14 or 16 days of repeated dosing, respectively. Fractional catabolic rate of plasma LDL was estimated by intravenous injection of DiI-labeled human LDL. The cholesterol-lowering effects of combination therapy were investigated after 7 days of repeated treatment.

Key findings

Compound-T8 (10 and 30 mg/kg) increased plasma 7α-hydroxy-4-cholesten-3-one levels in a dose-dependent manner. Single administration of compound-T8 (30 mg/kg) and atorvastatin (30 mg/kg) reduced plasma non-high-density lipoprotein (non-HDL) cholesterol levels by 48% and 46%, respectively, and increased clearance of plasma DiI-labeled LDL by 29% and 35%, respectively. Compound-T8 (10 mg/kg) or atorvastatin (10 mg/kg) reduced non-HDL cholesterol levels by 19% and 25%, respectively, and combination therapy showed an additive effect and lowered cholesterol levels by 48%.

Significance

Similar to atorvastatin, compound-T8 reduced plasma non-HDL cholesterol levels accompanied with accelerated LDL clearance in guinea pigs. Combination therapy additively decreased plasma non-HDL cholesterol levels. Therefore, monotherapy with a farnesoid X receptor antagonist and combination therapy of a farnesoid X receptor antagonist with atorvastatin would be attractive dyslipidemia treatment options.     

Posttranslational N-myristoylation is required for the anti-apoptotic activity of human tGelsolin, the C-terminal caspase cleavage product of human gelsolin

Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal nine residues of the C-terminal caspase cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation. In this study, to analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase cleavage products expressed in COS-1 cells with [3H]myristic acid was performed. It was found that the C-terminal caspase cleavage product of human gelsolin (tGelsolin) was efficiently N-myristoylated. When COS-1 cells transiently transfected with gelsolin cDNA were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was generated, as demonstrated by in vivo metabolic labeling. The generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressed in HeLa cells. Immunofluorescence staining and subcellular fractionation experiment revealed that exogenously expressed tGelsolin did not localize to mitochondria but rather was diffusely distributed in the cytoplasm. To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using this plasmid significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis. These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin.     

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.     

Oxygen-Dependent Inhibition of Neutrophil Respiratory Burst by Nitric Oxide

Effect of nitric oxide (NO) on the respiratory burst of neutrophils was examined under different oxygen tensions. Phorbol myristate acetate (PMA) stimulated oxygen consumption and superoxide (O₂^-) generation in neutrophils by a mechanism which was inhibited reversibly by NO. The inhibitory effect of NO increased significantly with a decrease in oxygen tension in the medium. The inhibitory effect of NO was suppressed in medium containing oxyhemoglobin (HbO₂), a NO scavenging agent. In contrast, 3-morpholinosydnonimine (SIN-1), a compound that rapidly generates peroxynitrite (ONOO^-) from the released NO and O₂^-, slightly stimulated the PMA-induced respiratory burst. These results suggested that NO, but not ONOO, might reversibly inhibit superoxide generation by neutrophils especially at physiologically low oxygen tensions thereby decreasing oxygen toxicity particularly in and around hypoxic tissues.     

Molecular characterization of the essential response regulator protein YycF in Bacillus subtilis

The response regulator YycF is essential for cell growth in gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. To study the function of YycF in the essential process, we characterized a YycF (H215P) mutation that caused temperature-sensitive growth in B. subtilis. The response regulators YycF and YycF (H215P) were analyzed using circular dichroism spectroscopy, whose T(m) values were 56.0 and 45.9 degrees C, respectively, suggesting that YycF (H215P) significantly affects the protein structure with an increase in temperature. Furthermore, using the gel mobility shift assay and DNase I footprinting, we investigated the effect of YycF (H215P) on binding to the YycF box of ftsAZ operon of B. subtilis. The replacement of the histidine 215 with proline resulted in a decrease of the DNA-binding ability of YycF in vitro. In vivo, using Escherichia coli two-hybrid and homodimerization assays, we clarified that His 215 of YycF plays a crucial role in the homodimerization of the protein. Thus the essential genes involved in growth of B. subtilis appear to be regulated by the homodimer of YycF. These results suggest that the YycF dimerization is an excellent target for the discovery of novel antibiotics.     

Does genomic variation in a foundation species predict arthropod community structure in a riparian forest?

Understanding how genetic variation within a foundation species determines the structure of associated communities and ecosystem processes has been an emerging frontier in ecology. Previous studies in common gardens identified close links between intraspecific variation and multispecies community structure, and these findings are now being evaluated directly in the complex natural ecosystem. In this study, we examined to what extent genomic variation in a foundation tree species explains the structure of associated arthropod communities in the field, comparing with spatial, temporal and environmental factors. In a continuous mixed forest, arthropods were surveyed on 85 mature alders (Alnus hirsuta) in 2 years. Moreover, we estimated Nei's genetic distance among the alders based on 1,077 single nucleotide polymorphisms obtained from restricted‐site‐associated DNA sequencing of the alders’ genome. In both years, we detected significant correlations between genetic distance and dissimilarity of arthropod communities. A generalized dissimilarity modelling indicated that the genetic distance of alder populations was the most important predictor to explain the variance of arthropod communities. Among arthropod functional groups, carnivores were consistently correlated with genetic distance of the foundation species in both years. Furthermore, the extent of year‐to‐year changes in arthropod communities was more similar between more genetically closed alder populations. This study demonstrates that the genetic similarity rule would be primarily prominent in community assembly of plant‐associated arthropods under temporally and spatially variable environments in the field.