Kinetic comparisons of amniotic fluid inactive renin and renal renin using synthetic and human renin substrates

Inactive renin has been isolated from pooled amniotic fluid and purified ~642-fold. Prior to activation the isolates has ~4% of the activity found after activation. The observation is similar to that reported for inactive renin from chorionic cell culture and suggests a placental origin of amniotic fluid inactive renin.Using plasma from an estrogen-treated woman, renin substrate was recovered free of renin and inactive renin and a portion was separated into NMW and HMW components. The NMW form constituted ~93% and the HMW form ~7% of the renin substrate.Amniotic fluid inactive renin was used for determinations of enzyme-substrate kinetics with the pooled, NMW, and HMW plasma substrate and tetradecapeptide synthetic substrate, and the results were compared to similar determinations using standard renal renin. Using synthetic substrate, the kinetics of renal renin and amniotic fluid inactive renin before and after activation were similar. The kinetics of renal renin with pooled, NMW, and HMW plasma substrate were also similar.Amniotic fluid inactive renin had a lower Km with pooled than with NMW substrate, however, which resulted from a significantly smaller Km with HMW component. Although the affinity constants with pooled substrate were not different for renin and inactive renin, the Km of inactive renin was significantly less with the HMW component of plasma renin substrate.The observation are compatible with a role for placental inactive renin in normal pregnancy and suggest the possibility of a further role in hypertensive pregnancy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25955/1/0000021.pd

Influenza A (H3N2) Outbreak, Nepal

Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain

Alterations in the neuropeptide galanin system in major depressive disorder involve levels of transcripts, methylation, and peptide

Major depressive disorder (MDD) is a substantial burden to patients, families, and society, but many patients cannot be treated adequately. Rodent experiments suggest that the neuropeptide galanin (GAL) and its three G protein-coupled receptors, GAL1–3, are involved in mood regulation. To explore the translational potential of these results, we assessed the transcript levels (by quantitative PCR), DNA methylation status (by bisulfite pyrosequencing), and GAL peptide by RIA of the GAL system in postmortem brains from depressed persons who had committed suicide and controls. Transcripts for all four members were detected and showed marked regional variations, GAL and galanin receptor 1 (GALR1) being most abundant. Striking increases in GAL and GALR3 mRNA levels, especially in the noradrenergic locus coeruleus and the dorsal raphe nucleus, in parallel with decreased DNA methylation, were found in both male and female suicide subjects as compared with controls. In contrast, GAL and GALR3 transcript levels were decreased, GALR1 was increased, and DNA methylation was increased in the dorsolateral prefrontal cortex of male suicide subjects, however, there were no changes in the anterior cingulate cortex. Thus, GAL and its receptor GALR3 are differentially methylated and expressed in brains of MDD subjects in a region- and sex- specific manner. Such an epigenetic modification in GALR3, a hyperpolarizing receptor, might contribute to the dysregulation of noradrenergic and serotonergic neurons implicated in the pathogenesis of MDD. Thus, one may speculate that a GAL3 antagonist could have antidepressant properties by disinhibiting the firing of these neurons, resulting in increased release of noradrenaline and serotonin in forebrain areas involved in mood regulation

Integral Membrane Protein Fragment Recombination after Transfer from Nanolipoprotein Particles to Bicelles

A new method for the measurement of membrane protein oligomer association is described. Two engineered fragments of bacteriorhodopsin, which are known to spontaneously associate in bicelles, were expressed in nanolipoprotein particles (NLPs or nanodiscs) using an <i>Escherichia coli</i> S30 cell-free synthesis system. When separately prepared NLPs containing the fragments were mixed, fragment association did not occur, indicating that the apolipoprotein edge blocks transfer between NLPs. However, when bicelles were added to this mixture, fragment association was detected by disulfide cross-linking. The rate of cross-linking was consistent with previously published equilibrium and kinetic parameters. Characterization of the NLP/bicelle mixture by dynamic light scattering and fluorescence spectroscopy indicates that the NLP bilayer transfers to bicelles in a simple reversal of the synthesis of NLPs from bicelles. These experiments validate using cell-free synthesis of membrane proteins in NLPs, followed by treatment with bicelles, as a method for measuring oligomerization of integral membrane protein subunits in a bilayer-like environment