Complex modulation of Cav3.1 T-type calcium channel by nickel

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni²⁺-binding site with critical histidine-191 (H191) within the extracellular IS3–IS4 domain of the most Ni²⁺-sensitive Ca_v3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni²⁺ effect and its dependence on PDC remain uncertain. Here we compared Ni²⁺ action on the wild-type “Ni²⁺-insensitive” Ca_v3.1_w/t channel and Ca_v3.1_Q172H mutant having glutamine (Q) equivalent to H191 of Ca_v3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni²⁺ blockade of Ca²⁺, Sr²⁺, or Ba²⁺ currents was assessed by electrophysiology. Inhibition of Ca_v3.1_w/t by Ni²⁺ conformed to two sites binding. Ni²⁺ binding with high-affinity site (IC₅₀ = 0.03–3 μM depending on PDC) produced maximal inhibition of 20–30 % and was voltage-dependent, consistent with its location within the channel’s pore. Most of the inhibition (70–80 %) was produced by Ni²⁺ binding with low-affinity site (IC₅₀ = 240–700 μM). Q172H-mutation mainly affected low-affinity binding (IC₅₀ = 120–160 μM). The IC₅₀ of Ni²⁺ binding with both sites in the Ca_v3.1_w/t and Ca_v3.1_Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca²⁺, Sr²⁺, or Ba²⁺ with Ni²⁺. We conclude that differential Ni²⁺-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni²⁺, may be occupied by PDC, influencing in turn the channel’s permeation.     

Comparison of glutamate dehydrogenase and glutamine synthetase activities in the roots and aerial organs of an obligate halophyte: Suaeda maritima var. macrocarpa and a glycophyte: Phaseolus vulgaris, grown in presence of different concentration of NaCl]

The glutamate dehydrogenase and glutamine synthetase activities of an obligate halophyte, Suaeda maritima var. macrocarpa and a glycophyte. Phaseolus vulgaris are compared in function of salinity (increasing concentrations of NaCl) of the culturing solution. In culture, addition of NaCl stimulates glutamine synthetase activity and lowers glutamine dehydrogenase activity in the aerial organs and in the roots of Suaeda as opposed to what is observed in the glycophyte. Hence the obligatory halophily of Suaeda is related to an increase of the glutamine synthetase activity in a sal-trich medium corresponding to the stimulation of nitrate reductase and proteogenesis.     

Protein levels of clusterin and glutathione synthetase in platelets allow for early detection of colorectal cancer

Colorectal cancer (CRC) is one of the most frequent malignancies in the Western world. Early tumor detection and intervention are important determinants on CRC patient survival. During early tumor proliferation, dissemination and angiogenesis, platelets store and segregate proteins actively and selectively. Hence, the platelet proteome is a potential source of biomarkers denoting early malignancy. By comparing protein profiles of platelets between healthy volunteers (n = 12) and patients with early- (n = 7) and late-stage (n = 5) CRCs using multiplex fluorescence two-dimensional gel electrophoresis (2D-DIGE), we aimed at identifying differentially regulated proteins within platelets. By inter-group comparisons, 94 differentially expressed protein spots were detected (p < 0.05) between healthy controls and patients with early- and late-stage CRCs and revealed distinct separations between all three groups in principal component analyses. 54 proteins of interest were identified by mass spectrometry and resulted in high-ranked Ingenuity Pathway Analysis networks associated with Cellular function and maintenance, Cellular assembly and organization, Developmental disorder and Organismal injury and abnormalities (p < 0.0001 to p = 0.0495). Target proteins were validated by multiplex fluorescence-based Western blot analyses using an additional, independent cohort of platelet protein samples [healthy controls (n = 15), early-stage CRCs (n = 15), late-stage CRCs (n = 15)]. Two proteins—clusterin and glutathione synthetase (GSH-S)—featured high impact and were subsequently validated in this independent clinical cohort distinguishing healthy controls from patients with early- and late-stage CRCs. Thus, the potential of clusterin and GSH-S as platelet biomarkers for early detection of CRC could improve existing screening modalities in clinical application and should be confirmed in a prospective multicenter trial.     

Sulindac sulfide suppresses 5-lipoxygenase at clinically relevant concentrations

Sulindac is a non-selective inhibitor of cyclooxygenases (COX) used to treat inflammation and pain. Additionally, non-COX targets may account for the drug’s chemo-preventive efficacy against colorectal cancer and reduced gastrointestinal toxicity. Here, we demonstrate that the pharmacologically active metabolite of sulindac, sulindac sulfide (SSi), targets 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes (LTs). SSi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human polymorphonuclear leukocytes (IC₅₀ ≈ 8–10 μM). Importantly, SSi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC₅₀ = 18.7 μM). SSi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone (SSo), failed to inhibit 5-LO. Mechanistic analysis demonstrated that SSi directly suppresses 5-LO with an IC₅₀ of 20 μM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy.     

Structural biology of the purine biosynthetic pathway

Purine biosynthesis requires ten enzymatic transformations to generate inosine monophosphate. PurF, PurD, PurL, PurM, PurC, and PurB are common to all pathways, while PurN or PurT, PurK/PurE-I or PurE-II, PurH or PurP, and PurJ or PurO catalyze the same steps in different organisms. X-ray crystal structures are available for all 15 purine biosynthetic enzymes, including 7 ATP-dependent enzymes, 2 amidotransferases and 2 tetrahydrofolate-dependent enzymes. Here we summarize the structures of the purine biosynthetic enzymes, discuss similarities and differences, and present arguments for pathway evolution. Four of the ATP-dependent enzymes belong to the ATP-grasp superfamily and 2 to the PurM superfamily. The amidotransferases are unrelated, with one utilizing an N-terminal nucleophileglutaminase and the other utilizing a triad glutaminase. Likewise the tetrahydrofolate-dependent enzymes are unrelated. Ancestral proteins may have included a broad specificity enzyme instead of PurD, PurT, PurK, PurC, and PurP, and a separate enzyme instead of PurM and PurL. Received 26 May 2008; received after revision 30 June 2008; accepted 9 July 2008     

Interaction of extrinsic fluorescent probes with E. coli glutamine synthetase.

Binding of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) to adenylylated (E--11) glutamine synthetase is cooperative and time-dependent, with 3 dye sites per subunit. In fluorescence polarization experiments TNS and pyrene butyrate give normalized Perrin plots that indicate a symmetrical arrangement of dye excited state dipoles, relative to the rotational axis of the oblate ellipsoid of the dodecameric native enzyme.     

Glutamate synthase: a complex iron-sulfur flavoprotein

Glutamate synthase is a complex iron-sulfur flavoprotein that forms l-glutamate from l-glutamine and 2-oxoglutarate. It participates with glutamine synthetase in ammonia assimilation processes. The known structural and biochemical properties of glutamate synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of glutamate synthase in order to gain insight into the mechanism of the glutamate synthase reaction. Sequence analyses also revealed that the small subunit of bacterial glutamate synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain. Received 10 November 1998, received after revision 10 December 1998; accepted 10 December 1998     

Glutamine synthetase activity in subdivisions of brain of the shark, Squalus acanthias

Specific activity of glutamine synthetase in Squalus acanthias (spiny dogfish) central nervous system regions was highest in the cerebellum and lowest in the spinal cord. The levels of activity may relate to the excitability of each region by regulating the glutamate pool.     

Molybdenum cofactor biosynthesis and deficiency

The molybdenum cofactor (Moco) forms the active site of all molybdenum (Mo) enzymes, except nitrogenase. Mo enzymes catalyze important redox reactions in global metabolic cycles. Moco consists of Mo covalently bound to one or two dithiolates attached to a unique tricyclic pterin moiety commonly referred to as molybdopterin (MPT). Moco is synthesized by an ancient and conserved biosynthetic pathway that can be divided into four steps, according to the biosynthetic intermediates precursor Z (cyclic pyranopterin monophosphate), MPT and adenylated MPT. In a fifth step modifications such as attachment of nucleotides, sulfuration or bond formation between Mo and the protein result in different catalytic Mo centers. A defect in any of the steps of Moco biosynthesis results in the pleiotropic loss of all Mo enzyme activities. Human Moco deficiency is a hereditary metabolic disorder characterized by severe neurodegeneration resulting in early childhood death. Recently, a first substitution therapy was established. Received 17 June 2005; received after revision 18 August 2005; accepted 1 September 2005     

Inhibition of rat liver glutamine synthetase by phosphonic analogues of glutamic acid

Summary Analogues of glutamic acid, -methylglutamic acid and glutamine in which the -or -COOH groups are replaced by PO₃H₂ or P(O)(OH₃)OH functions competitively inhibit rat liver glutamine synthetase. The K₁ values are comparable to or lower than K_M for L-glutamate.This study was supported by grant R.1.9.     

Ultradian and other rhythms in human respiration rate

Summary Twelve male subjects had their respiration rate (RR) measured at 3 min intervals for 6 h. Although there were substantial individual differences, most subjects demonstrated a 90±15 min rhythm of RR, with several subjects also showing 60±10 min and 30±3 min rhythms.     

Altered brain cholinergic enzymes activity in the genetically obese rat

Summary Genetically obese male Zucker rats (fa/fa) and their lean littermates (Fa/-) were used in this experiment. Fourteen-week-old obese and lean littermates were sacrificed and choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) enzymes were assayed in specific brain regions. The assays of these enzymes indicate that obese animals and a significantly lower ChAT activity in the cerebellum, pons, and cerebral cortex and a significant increase in ChAT activity in the thalamus and hypothalamus. Meanwhile, the cerebral cortex, cerebellum, midbrain, thalamus and hypothalamus of the obese animals showed significantly higher AChE activity than their lean littermates. It was concluded from this study that obesity may be associated with changes in the enzymes of the brain cholinergic system.This work was supported by a grant from the National Aeronautics and Space Administration (NAG 2-411), a grant from the National Institutes of Health (NIH RR 0811), and a grant from the Division of Research Resources, National Institutes of Health (NIH Grant RR 03020).     

庆大霉素产生菌GntB基因的克隆与转化

根据小单孢菌产生庆大霉素的生物合成机理,利用基因克隆方法从棘孢小单孢菌（Micromonospora echinospora）基因组中扩增出庆大霉素生物合成的关键酶基因—2-脱氧青蟹肌糖合成酶基因（GntB）,并将其通过大肠杆菌／链霉菌穿梭质粒pIJ699转化原菌株,采用硫链丝菌素抗性基因启动子带动2-脱氧青蟹肌糖合成酶基因在棘孢小单孢菌细胞中实现了转化。     

Nucleolar localization and circadian regulation of Per2S,a novel splicing variant of the Period 2 gene

In this work, we show for the first time that a second splicing variant of the core clock gene Period 2 (Per2), Per2S, is expressed at both the mRNA and protein levels in human keratinocytes and that it localizes in the nucleoli. Moreover, we show that a reversible perturbation of the nucleolar structure acts as a resetting stimulus for the cellular clock. Per2S expression and periodic oscillation upon dexamethasone treatment were assessed by qRT-PCR using specific primers. Western blot (WB) analysis using an antibody against the recombinant human PER2 (abRc) displayed an intense band at a molecular weight of ~55 kDa, close to the predicted size of Per2S, and a weaker band at the expected size of Per2 (~140 kDa). The antibody raised against PER2 pS662 (abS662), an epitope absent in PER2S, detected only the higher band. Immunolocalization studies with abRc revealed a peculiar nucleolar signal colocalizing with the nucleolar marker nucleophosmin, whereas with abS662 the signal was predominantly diffuse all over the nucleus and partially colocalized with abRc in the nucleolus. The analysis of cell fractions by WB confirmed the enrichment of PER2S and the presence of PER2 in the nucleolar compartment. Finally, a pulse (1 h) of actinomycin D (0.01 μg/ml) induced reversible nucleolar disruption, PER2S de-localization and circadian synchronization of clock and Per2S genes. Our work represents the first evidence that the Per2S splicing isoform is a clock component expressed in human cells localizing in the nucleolus. These results suggest a critical role for the nucleolus in the process of circadian synchronization in human keratinocytes.     

Quantitative analysis of mucosal oxygenation using ex vivo imaging of healthy and inflamed mammalian colon tissue

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O₂ consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O₂ in freshly prepared samples of healthy and inflamed colon tissue. We developed protocols for efficient ex vivo staining of mouse distal colon mucosa with a cell-penetrating O₂ sensitive probe Pt-Glc and high-resolution imaging of O₂ concentration in live tissue by confocal phosphorescence lifetime-imaging microscopy (PLIM). Microscopy analysis revealed that Pt-Glc stained mostly the top 50–60 μm layer of the mucosa, with high phosphorescence intensity in epithelial cells. Measured O₂ values in normal mouse tissue ranged between 5 and 35 μM (4–28 Torr), tending to decrease in the deeper tissue areas. Four-day treatment with dextran sulphate sodium (DSS) triggered colon inflammation, as evidenced by an increase in local IL6 and mKC mRNA levels, but did not affect the gross architecture of colonic epithelium. We further observed an increase in oxygenation, partial activation of hypoxia inducible factor (HIF) 1 signalling, and negative trends in pyruvate dehydrogenase activity and O₂ consumption rate in the colitis mucosa, suggesting a decrease in mitochondrial respiration, which is known to be regulated via HIF-1 signalling and pyruvate oxidation rate. These results along with efficient staining with Pt-Glc of rat and human colonic mucosa reveal high potential of PLIM platform as a powerful tool for the high-resolution analysis of the intestinal tissue oxygenation in patients with inflammatory bowel disease and other pathologies, affecting tissue respiration.     

Gene structure and function of the 2'-5'-oligoadenylate synthetase family

2'-5'-Oligoadenylate synthetase was among the first interferon-induced antiviral enzymes to be discovered. This family of enzymes plays an important role in the mechanisms of action of interferon antiviral activity, but is also involved in other cellular processes such as apoptosis and growth control. We have reviewed the function and genomic structure of this class of at least nine proteins. By studying the recently available data in the human genome database and the human Expressed Sequence Tag database, we have been able to build a comprehensive picture of the 2'-5'-oligoadenylate synthetase gene family and its precise location on chromosome 12. Chromosomal localization as well as the intron/exon structure of all four genes has been established and an overview of the splice variant forms of the 2'-5'-oligoadenylate synthetases arising from expression of the four genes is presented. Alignments of the human 2'-5'-oligoadenylate synthetase sequences with non-human 2'-5'-oligoadenylate synthetase sequences suggest that the exon structure and several amino acid sequence motifs have been conserved during evolution.     

Purification and properties of glutamine synthetase I fromRhizobium sp. UMKL 20

Summary Glutamine synthetase I was purified fromRhizobium sp. UMKL 20 following polyethylene glycol precipitation. The enzyme had a subunit molecular weight of 58 kd. Apparent K_m values for ammonia and glutamate were 5.6 and 15.2 mM, respectively. Glutamine synthetase I activity was inhibited by several end products of glutamine metabolism. The purified enzyme was highly adenylylated (E _n ^– =8.5).Acknowledgment. I would like to thank Mr J. C. Lai for technical assistance. This work was carried out with the support of Vote F 153/79 from the University of Malaya.     

Diffusion of d-glucose measured in the cytosol of a single astrocyte

Astrocytes interact with neurons and endothelial cells and may mediate exchange of metabolites between capillaries and nerve terminals. In the present study, we investigated intracellular glucose diffusion in purified astrocytes after local glucose uptake. We used a fluorescence resonance energy transfer (FRET)-based nano sensor to monitor the time dependence of the intracellular glucose concentration at specific positions within the cell. We observed a delay in onset and kinetics in regions away from the glucose uptake compared with the region where we locally super-fused astrocytes with the d-glucose-rich solution. We propose a mathematical model of glucose diffusion in astrocytes. The analysis showed that after gradual uptake of glucose, the locally increased intracellular glucose concentration is rapidly spread throughout the cytosol with an apparent diffusion coefficient (D _app) of (2.38 ± 0.41) × 10^?10 m² s^?1 (at 22–24 °C). Considering that the diffusion coefficient of d-glucose in water is D = 6.7 × 10^?10 m² s^?1 (at 24 °C), D _app determined in astrocytes indicates that the cytosolic tortuosity, which hinders glucose molecules, is approximately three times higher than in aqueous solution. We conclude that the value of D _app for glucose measured in purified rat astrocytes is consistent with the view that cytosolic diffusion may allow glucose and glucose metabolites to traverse from the endothelial cells at the blood–brain barrier to neurons and neighboring astrocytes.     

Glutamine synthetase activity in subdivisions of brain of the shark,Squalus acanthias

Summary Specific activity of glutamine synthetase inSqualus acanthias (spiny dogfish) central nervous system regions was highest in the cerebellum and lowest in the spinal cord. The levels of activity may relate to the excitability of each region by regulating the glutamate pool.Publication No. 14 from the Laboratory of Biochemical Ecology. Contribution No. 527, College of Fisheries, University of Washington. This work was supported in part by a fellowship from the national Wildlife Federation and a Pacific Fisheries Biologists' scholarship to J.T.W.     

Glutamine stimulates translation of uncoupling protein 2mRNA

Uncoupling protein 2 (UCP2) belongs to a family of transporters/exchangers of the mitochondrial inner membrane. Using cell lines representing natural sites of UCP2 expression (macrophages, colonocytes, pancreatic beta cells), we show that UCP2 expression is stimulated by glutamine at physiological concentrations. This control is exerted at the translational level. We demonstrate that the upstream open reading frame (ORF1) in the 5’ untranslated region (5’UTR) of the UCP2 mRNA is required for this stimulation to take place. Cloning of the 5’ UTR of the UCP2 mRNA in front of a GFP cDNA resulted in a reporter gene with which GFP expression could be induced by glutamine. An effect of glutamine on translation of a given mRNA has not been identified before, and this is the first evidence for a link between UCP2 and glutamine, an amino acid oxidized by immune cells or intestinal epithelium and playing a role in the control of insulin secretion. Received 26 January 2007; received after revision 16 April 2007; accepted 8 May 2007 C. Hurtaud, C. Gelly: These authors contributed equally to this work.