The conversion of cholest-7-en-3beta-ol into cholesterol. General comments on the mechanism of the introduction of double bonds in enzymic reactions

Convenient syntheses of 6β-tritiated Δ⁷-cholestenol and 3α-tritiated Δ⁷-cholestene-3β,5α-diol are described. It was shown that the conversion of 6β-tritiated Δ⁷-cholestenol into cholesterol is accompanied by the complete retention of label. It was unambiguously established that the overall reaction leading to the introduction of the double bond in the 5,6-position in cholesterol occurs via a cis-elimination involving the 5α- and 6α-hydrogen atoms and that during this process the 6β-hydrogen atom remains completely undisturbed. Metabolic studies with 3α-tritiated Δ⁷-cholestene-3β,5α-diol revealed that under anaerobic conditions the compound is not converted into cholesterol. This observation, coupled with the previous work of Slaytor & Bloch (1965), is interpreted to exclude a hydroxylation–dehydration mechanism for the origin of the 5,6-double bond in cholesterol. It was also shown that under aerobic conditions 3α-tritiated Δ⁷-cholestene-3β,5α-diol is efficiently converted into cholesterol and that this conversion occurs through the intermediacy of 7-dehydrocholesterol. Cumulative experimental evidence presented in this paper and elsewhere is used to suggest that the 5,6-double bond in cholesterol originates through an oxygen-dependent dehydrogenation process and a hypothetical mechanism for this and related reactions is outlined.     

Anti-anabolic effects of adenosine 3′:5′-cyclic monophosphate. Inhibition of protein synthesis

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.     

Changes in Gut Microbiota in Rats Fed a High Fat Diet Correlate with Obesity-Associated Metabolic Parameters

The gut microbiota is emerging as a new factor in the development of obesity. Many studies have described changes in microbiota composition in response to obesity and high fat diet (HFD) at the phylum level. In this study we used 16s RNA high throughput sequencing on faecal samples from rats chronically fed HFD or control chow (n = 10 per group, 16 weeks) to investigate changes in gut microbiota composition at the species level. 53.17% dissimilarity between groups was observed at the species level. Lactobacillus intestinalis dominated the microbiota in rats under the chow diet. However this species was considerably less abundant in rats fed HFD (P<0.0001), this being compensated by an increase in abundance of propionate/acetate producing species. To further understand the influence of these species on the development of the obese phenotype, we correlated their abundance with metabolic parameters associated with obesity. Of the taxa contributing the most to dissimilarity between groups, 10 presented significant correlations with at least one of the tested parameters, three of them correlated positively with all metabolic parameters: Phascolarctobacterium, Proteus mirabilis and Veillonellaceae, all propionate/acetate producers. Lactobacillus intestinalis was the only species whose abundance was negatively correlated with change in body weight and fat mass. This species decreased drastically in response to HFD, favouring propionate/acetate producing bacterial species whose abundance was strongly correlated with adiposity and deterioration of metabolic factors. Our observations suggest that these species may play a key role in the development of obesity in response to a HFD.     

Development of genetic markers for Ochradenus arabicus (Resedaceae), an endemic medicinal plant of Saudi Arabia

Some species of the genus Ochradenus are difficult to identify based on morphological markers. Similar limitations are found for biochemical markers. We developed genetic markers based on DNA sequences for Ochradenus arabicus, which is an endemic plant to Saudi Arabia, locally utilized as a medicinal shrub. The internal transcribed spacer sequence of nuclear ribosomal DNA and chloroplast (rpoB and rpoC1) markers were more informative than other chloroplast DNA markers. Based on these markers, we were able to discriminate this species from another species of the same genus (O. baccatus) that is widely distributed in Saudi Arabia, despite a high degree of morphological similarity. These genetic markers facilitate its identification, even when acquired in a dried state from local markets.     

Structure of the archaeal translation initiation factor aIF2 beta from Methanobacterium thermoautotrophicum: implications for translation initiation

aIF2 beta is the archaeal homolog of eIF2 beta, a member of the eIF2 heterotrimeric complex, implicated in the delivery of Met-tRNA(i)(Met) to the 40S ribosomal subunit. We have determined the solution structure of the intact beta-subunit of aIF2 from Methanobacterium thermoautotrophicum. aIF2 beta is composed of an unfolded N terminus, a mixed alpha/beta core domain and a C-terminal zinc finger. NMR data shows the two folded domains display restricted mobility with respect to each other. Analysis of the aIF2 gamma structure docked to tRNA allowed the identification of a putative binding site for the beta-subunit in the ternary translation complex. Based on structural similarity and biochemical data, a role for the different secondary structure elements is suggested.     

Perinatal transmission of SHIV-SF162P3 in Macaca nemestrina

We developed a SHIV/macaque model of transmission from infected dams to their infants. Ten pregnant dams were infected intravenously with 100 MID(50) of macaque-titered SHIV-SF162P3 during the second trimester. Nine infants were born; the seven surviving beyond day of birth suckled for 6 months. Four of nine infants were infected (transmission rate = 44.4%), with one infection in utero, and three intrapartum and/or immediately post-birth via suckling. Varying levels of binding and neutralizing antibodies were transplacentally transferred to infants. Passive antibodies were detected in plasma on the day of birth and persisted for 5 weeks. Infants infected at or after birth controlled acute and post-acute viremia. Exposure to maternal SHIV-SF162P3 during birth and suckling in the presence of autologous maternal neutralizing antibodies may have affected transmission or pathogenesis in the infants. This transmission model can allow investigation of key parameters involved in perinatal transmission of HIV.     

Angiotensin-(1–7) Via the Mas Receptor Alleviates the Diabetes-Induced Decrease in GFAP and GAP-43 Immunoreactivity with Concomitant Reduction in the COX-2 in Hippocampal Formation: An Immunohistochemical Study

We have previously shown that chronic treatment with angiotensin-(1?C7) [Ang-(1?C7)] can prevent diabetes-induced cardiovascular dysfunction. However, effect of Ang-(1?C7) treatment on diabetes-induced alterations in the CNS is unknown. The aim of this study was to test the hypothesis that treatment with Ang-(1?C7) can produce protection against diabetes-induced CNS changes. We examined the effect of Ang-(1?C7) on the number of cyclooxygenase-2 (COX-2) immunoreactive neurons and the glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and assessed the changes in the neuronal growth-associated protein-43 (GAP-43) of the hippocampal formation in streptozotocin-induced diabetes in rats. Animals were sacrificed 30?days after induction of diabetes and/or treatment with Ang-(1?C7). Ang-(1?C7) treatment significantly prevented diabetes-induced decrease in the number of GFAP immunoreactive astrocytes and GAP-43 positive neurons in all hippocampal regions. Co-administration of A779, a selective Ang-(1?C7) receptor antagonist, inhibited Ang-(1?C7)-mediated protective effects indicating that Ang-(1?C7) produces its effects through activation of receptor Mas. Further, Ang-(1?C7) treatment through activation of Mas significantly prevented diabetes-induced increase in the number of the COX-2 immunolabeled neurons in all sub-regions of the hippocampus examined. These results show that Ang-(1?C7) has a protective role against diabetes-induced changes in the CNS.     

Synthesis of a New Tri-Branched PEG-IFNα2 and Its Impact on Anti Viral Bioactivity

Efficacy of proteins can be enhanced by using polyethylene glycol (PEG) conjugation (PEGylation) to the protein molecules. Mobile non-toxic PEG chains conjugated to bio-therapeutics increase their hydrodynamic volume and in turn can prolong their plasma retention time and increase their solubility. An important aspect of PEGylation is the selection of PEG molecule with suitable structure and molecular weight. In this study, conceiving the idea that branched PEG-conjugates show superior efficacy over the linear PEG-conjugates, a tri-branched PEG-interferon (mPEG₃L₂-IFN) was synthesized by reacting a 30 KDa tri-branched mPEG₃L₂-NHS reagent with IFN to improve its pharmacokinetic properties and reduce the loss of in vitro bioactivity (which is generally exhibited by PEGylation) of the conjugated protein to some extent. The PEGylation procedure was optimized in terms of concentration and molar ratios of reactants, reaction time, temperature and pH conditions of the reaction mix. The conjugate was purified by cation exchange chromatography and characterized by SDS-PAGE and SE-HPLC. Trypsin digestion of mPEG₃L₂-IFN indicated a single site specificity of PEGylation. Anti viral bioactivity of mPEG₃L₂-IFN was found to be 2.38 × 10⁷ IU/mg which is approximately 9.52% of native IFNα2 (2.5 × 10⁸ IU/mg) and better than PEGasys from Roche Pharma. Therefore, it is reported that the tri-branched mPEG₃L₂-NHS reagent has the potential to be used to conjugate proteins for the promising therapeutic results.     

sli-3 negatively regulates the LET-23/epidermal growth factor receptor-mediated vulval induction pathway in Caenorhabditis elegans

The LIN-3-LET-23-mediated inductive signaling pathway plays a major role during vulval development in C. elegans. Studies on the components of this pathway have revealed positive as well as negative regulators that function to modulate the strength and specificity of the signal transduction cascade. We have carried out genetic screens to identify new regulators of this pathway by screening for suppressors of lin-3 vulvaless phenotype. The screens recovered three loci including alleles of gap-1 and a new gene represented by sli-3. Our genetic epistasis experiments suggest that sli-3 functions either downstream or in parallel to nuclear factors lin-1 and sur-2. sli-3 synergistically interacts with the previously identified negative regulators of the let-23 signaling pathway and causes excessive cell proliferation. However, in the absence of any other mutation sli-3 mutant animals display wild-type vulval induction and morphology. We propose that sli-3 functions as a negative regulator of vulval induction and defines a branch of the inductive signaling pathway. We provide evidence that sli-3 interacts with the EGF signaling pathway components during vulval induction but not during viability and ovulation processes. Thus, sli-3 helps define specificity of the EGF signaling to induce the vulva.