Measurement of Methionine-Enkephalin [Arg6,Phe7] in Rat Brain by Specific Radioimmunoassay Directed at Methionine Sulphoxide Enkephalin[Arg6,Phe7]

Abstract: A specific and sensitive radioimmunoassay procedure for Metenkephalin[Arg⁶,Phe⁷] which allows its measurement in regions of the rat brain is described. The antiserum was raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized ¹²⁵I-labelled Met(O)-enkephalin[Arg⁶,Phe⁷]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg⁶,Phe⁷]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg⁶,Phe⁷]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.     

Punicalagin Targets Atherosclerosis: Gene Expression Profiling of THP-1 Macrophages Treated with Punicalagin and Molecular Docking

Atherosclerosis is an important cause of cardiovascular disorders worldwide. Natural botanical drugs have attracted attention due to their antioxidant, anti-inflammatory, and antiatherogenic properties in the treatment of atherosclerosis. Punicalagin is the major bioactive component of pomegranate peel, and has been shown to have antioxidant, anti-inflammatory, antiviral, anti proliferation, and anticancer properties. To explore its antiatherogenic properties at a molecular level, we investigated the genome-wide expression changes that occur in differentiated THP1 cells following treatment with a non-toxic dose of punicalagin. We also conducted a molecular docking simulation study to identify the molecular targets of punicalagin.     

Deoxyribonucleases of non-pathogenic corynebacteria

Fourteen species of non-pathogenic corynebacteria have been examined for the presence of DNases. Seven of the species were found to be DNase positive when assayed in Toluidine Blue-DNA-containing agar whereas only one, Corynebacterium callunae, could be detectable as DNase positive when the test was performed in DNA-methyl green-containing agar. Electrophoretic patterns obtained from sodium dodecyl sulfate-polyacrylamide gels containing DNA showed the presence of one or two bands of nucleolytic activity in two species of Brevibacterium and of several bands in C. callunae. Degradation of DNases by cellular proteases seem to occur in this species.     

Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts

Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism.     

Notch3 expression in capillary pericytes predicts worse graft outcome in human renal grafts with antibody‐mediated rejection

Microvasculature consisting of endothelial cells and pericytes is the main site of injury during antibody‐mediated rejection (ABMR) of renal grafts. Little is known about the mechanisms of activation of pericytes in this pathology. We have found recently that activation of Notch3, a mediator of vascular smooth muscle cell proliferation and dedifferentiation, promotes renal inflammation and fibrosis and aggravates progression of renal disease. Therefore, we studied the pericyte expression of Notch3 in 49 non‐selected renal graft biopsies (32 for clinical cause, 17 for graft surveillance). We analysed its relationship with patients’ clinical and morphological data, and compared with the expression of partial endothelial mesenchymal transition (pEndMT) markers, known to reflect endothelial activation during ABMR. Notch3 was de novo expressed in pericytes of grafts with ABMR, and was significantly correlated with the microcirculation inflammation scores of peritubular capillaritis and glomerulitis and with the expression of pEndMT markers. Notch3 expression was also associated with graft dysfunction and proteinuria at the time of biopsy and in the long term. Multivariate analysis confirmed pericyte expression of Notch3 as an independent risk factor predicting graft loss. These data suggest that Notch3 is activated in the pericytes of renal grafts with ABMR and is associated with poor graft outcome.