Magnesium deficiency augments myocardial response to reactive oxygen species

Magnesium (Mg) deficiency and oxidative stress are independently implicated in the etiopathogenesis of various cardiovascular disorders. This study was undertaken to examine the hypothesis that Mg deficiency augments the myocardial response to oxidative stress. Electrically stimulated rat papillary muscle was used for recording the contractile variation. Biochemical variables of energy metabolism (adenosine triphosphate (ATP) and creatine phosphate) and markers of tissue injury (lactate dehydrogenase (LDH) release and lipidperoxidation), which can affect myocardial contractility, were assayed in Langendorff-perfused rat hearts. Hydrogen peroxide (100 micromol/L) was used as the source of reactive oxygen species. The negative inotropic response to H2O2 was significantly higher in Mg deficiency (0.48 mmol Mg/L) than in Mg sufficiency (1.2 mmol Mg/L). Low Mg levels did not affect ATP levels or tissue lipid peroxidation. However, H2O2 induced a decrease in ATP; enhanced lipid peroxidation and the release of LDH were augmented by Mg deficiency. Increased lipid peroxidation associated with a decrease in available energy might be responsible for the augmentation of the negative inotropic response to H2O2 in Mg deficiency. The observations from this study validate the hypothesis that myocardial response to oxidative stress is augmented by Mg deficiency. This observation has significance in ischemia-reperfusion injury, where Mg deficiency can have an additive effect on the debilitating consequences.     

Regional Levels of Lactate and Norepinephrine After Experimental Brain Injury

Abstract: The recently developed controlled cortical impact model of brain injury in rats may be an excellent tool by which to attempt to understand the neurochemical mechanisms mediating the pathophysiology of traumatic brain injury. In this study, rats were subjected to lateral controlled cortical impact brain injury of low grade severity; their brains were frozen in situ at various times after injury to measure regional levels of lactate, high energy phosphates, and norepinephrine. Tissue lactate concentration in the injury site left cortex was increased in injured animals by sixfold at 30 min and twofold at 2.5 h and 24 h after injury ( p < 0.05). At all postinjury times, lactate concentration was also increased in injured animals by about twofold in the cortex and hippocampus adjacent to the injury site ( p < 0.05). No significant changes occurred in the levels of ATP and phosphocreatine in most of the brain regions of injured animals. However, in the primary site of injury (left cortex), phosphocreatine concentration was decreased by 40% in injured animals at 30 min after injury ( p < 0.05). The norepinephrine concentration was decreased in the injury site left cortex of injured animals by 38% at 30 min, 29% at 2.5 h, and 30% at 24 h after injury ( p < 0.05). The level of norepinephrine was also reduced by ∼20% in the cortex adjacent to the injury site in injured animals. The present results suggest that controlled cortical impact brain injury produces disorder in the neuronal oxidative and norepinephrine metabolism.     

High-performance liquid chromatographic method for fingerprinting and quantitative determination of E- and Z-guggulsterones in Commiphora mukul resin and its products

A high-performance liquid chromatographic method has been developed and validated for the fingerprinting (profiling) and quantitative determination of E- and Z-guggulsterones, the hypolipidemic agents in the gum-resin exudate of Commiphora mukul, currently marketed worldwide as hypocholesterolemic. The method involves extraction of the guggul-resin from either the raw exudate or compounded tablets (or capsules) with ethyl acetate, concentration of the combined extracts and chromatography on a reversed-phase C₁₈ column using an acetonitrile–water gradient. The method has a validated quantitation range of 15–85 μg/ml for E-guggulsterone and 25–130 μg/ml for Z-guggulsterone with a precision of ±2% S.D. and a recovery of >99.5%. Standard curve correlation coefficients of 0.992 or greater were obtained during validation experiments. The method was applied to six commercial (OTC) products, all of which were found to contain significantly less (in most cases very little or none) of the claimed guggulsterones.     

Role of packing defects in the evolution of allostery and induced fit in human UDP-glucose dehydrogenase

Allosteric feedback inhibition is the mechanism by which metabolic end products regulate their own biosynthesis by binding to an upstream enzyme. Despite its importance in controlling metabolism, there are relatively few allosteric mechanisms understood in detail. This is because allostery does not have an identifiable structural motif, making the discovery of new allosteric enzymes a difficult process. The lack of a conserved motif implies that the evolution of each allosteric mechanism is unique. Here we describe an atypical allosteric mechanism in human UDP-α-d-glucose 6-dehydrogenase (hUGDH) based on an easily acquired and identifiable structural attribute: packing defects in the protein core. In contrast to classic allostery, the active and allosteric sites in hUGDH are present as a single, bifunctional site. Using two new crystal structures, we show that binding of the feedback inhibitor, UDP-α-d-xylose, elicits a distinct induced-fit response; a buried loop translates ～4 ? along and rotates ～180° about the main chain axis, requiring surrounding side chains to repack. This allosteric transition is facilitated by packing defects, which negate the steric conformational restraints normally imposed by the protein core. Sedimentation velocity studies show that this repacking favors the formation of an inactive hexameric complex with unusual symmetry. We present evidence that hUGDH and the unrelated enzyme dCTP deaminase have converged to very similar atypical allosteric mechanisms using the same adaptive strategy, the selection for packing defects. Thus, the selection for packing defects is a robust mechanism for the evolution of allostery and induced fit.     

Importin alpha-mediated nuclear import of cytoplasmic poly(A) binding protein occurs as a direct consequence of cytoplasmic mRNA depletion

Recent studies have found the cytoplasmic poly(A) binding protein (PABPC) to have opposing effects on gene expression when concentrated in the cytoplasm versus in the nucleus. PABPC is predominantly cytoplasmic at steady state, where it enhances protein synthesis through simultaneous interactions with mRNA and translation factors. However, it accumulates dramatically within the nucleus in response to various pathogenic and nonpathogenic stresses, leading to an inhibition of mRNA export. The molecular events that trigger relocalization of PABPC and the mechanisms by which it translocates into the nucleus to block gene expression are not understood. Here, we reveal an RNA-based mechanism of retaining PABPC in the cytoplasm. Expression either of viral proteins that promote mRNA turnover or of a cytoplasmic deadenylase drives nuclear relocalization of PABPC in a manner dependent on the PABPC RNA recognition motifs (RRMs). Using multiple independent binding sites within its RRMs, PABPC interacts with importin α, a component of the classical import pathway. Finally, we demonstrate that the direct association of PABPC with importin α is antagonized by the presence of poly(A) RNA, supporting a model in which RNA binding masks nuclear import signals within the PABPC RRMs, thereby ensuring efficient cytoplasmic retention of this protein in normal cells. These findings further suggest that cells must carefully calibrate the ratio of PABPC to mRNA, as events that offset this balance can dramatically influence gene expression.     

Screening and molecular characterization of Serratia marcescens VITSD2: A strain producing optimum serratiopeptidase

The current work was attempted to isolate and characterize the serratiopeptidase producing Serratia sp. Among the 10 bacterial isolates 7 strains were identified as Serratia sp. Out of 7 strains one showed potent proteolytic activity and selected for further studies. Based on the morphological, biochemical and molecular characterization, the potent isolate （RH03） was identified as Serratia marcescens （GenBank accession number： KC961637） and the strain was designated as Serratia marcescens VITSD2. The production of serratiopeptidase was carried out in trypticase soya broth and the enzyme was partially purified using ammonium sulfate precipitation and dialysis. The specific activity was determined by casein hydrolysis assay and was found to be 12.00, 21.33, and 25.40 units/rag for crude, precipitated and dialysed samples. The molecular weight of the protease was determined by SDS-PAGE and it was found to be 50 kDa. The antibacterial activity of the produced serratiopeptidase showed moderate activity against Pseudomonas aeruginosa MTCC No. 4676 （12 mm） and Escherichia coli MTCC No. 1588 （15 mm）.     

Rhou maintains the epithelial architecture and facilitates differentiation of the foregut endoderm

Rhou encodes a Cdc42-related atypical Rho GTPase that influences actin organization in cultured cells. In mouse embryos at early-somite to early-organogenesis stages, Rhou is expressed in the columnar endoderm epithelium lining the lateral and ventral wall of the anterior intestinal portal. During foregut development, Rhou is downregulated in regions where the epithelium acquires a multilayered morphology heralding the budding of organ primordia. In embryos generated from Rhou knockdown embryonic stem (ES) cells, the embryonic foregut displays an abnormally flattened shape. The epithelial architecture of the endoderm is disrupted, the cells are depleted of microvilli and the phalloidin-stained F-actin content of their sub-apical cortical domain is reduced. Rhou-deficient cells in ES cell-derived embryos and embryoid bodies are less efficient in endoderm differentiation. Impaired endoderm differentiation of Rhou-deficient ES cells is accompanied by reduced expression of c-Jun/AP-1 target genes, consistent with a role for Rhou in regulating JNK activity. Downregulation of Rhou in individual endoderm cells results in a reduced ability of these cells to occupy the apical territory of the epithelium. Our findings highlight epithelial morphogenesis as a required intermediate step in the differentiation of endoderm progenitors. In vivo, Rhou activity maintains the epithelial architecture of the endoderm progenitors, and its downregulation accompanies the transition of the columnar epithelium in the embryonic foregut to a multilayered cell sheet during organ formation.     

Proteomic comparison of plastids from developing embryos and leaves of Brassica napus

Plastids are highly specialized organelles, responsible for photosynthesis and biosynthesis of various phytochemicals. To better understand plastid diversity and metabolism, a quantitative proteomic study of two plastid forms from Brassica napus (oilseed rape) was performed. Plastids were isolated from leaves (chloroplasts) of two-week-old plants and developing embryos (embryoplasts) three-weeks after flowering, using an approach avoiding protein storage vacuole contamination. Proteins from five different plastid preparations were prefractionated by SDS-PAGE and sectioned into multiple bands, and in-gel proteins were subjected to trypsin digestion. Tryptic peptides from each band were eluted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectra were searched against a comprehensive plant database. Proteins were quantified based on MS/MS spectral counting of unique, nonhomologous peptides. Functional classification and quantitative comparison of over 2000 redundant proteins (compiled to 675 nonredundant proteins) determined that light reaction proteins are more prominent in chloroplasts, while many Calvin cycle enzymes are more prominent in embryoplasts. Embryoplasts also contain a diversity of other metabolic enzymes undetected in chloroplasts. Many enzymes involved in de novo fatty acid and amino acid biosynthesis were detected in embryoplasts but not chloroplasts. Additionally, protein synthesis-related proteins were prominent in embryoplasts. Collectively, these results indicate that these two plastid types are distinct.     

YidC Occupies the Lateral Gate of the SecYEG Translocon and Is Sequentially Displaced by a Nascent Membrane Protein

Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements.