The complete structure of the core carbohydrate backbone from the LPS of marine halophilic bacterium Pseudoalteromonas carrageenovora type strain IAM 12662T

The complete novel structure of the components of the core oligosaccharide fraction from the LOS of the halophilic marine bacterium Pseudoalteromonas carrageenovora was characterized. The fully de-acylated lipooligosaccharide was studied by means of compositional analysis, matrix-assisted laser desorption/ionization mass spectrometry and complete (1)H and (13)C and (31)P NMR spectroscopy. The core oligosaccharide is composed by a mixture of species differing for the length of the sugar chain and the phosphorylation pattern: [carbohydrate structure]; see text. All sugars are D-pyranoses. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked.     

Metabotropic glutamate receptors and neuroadaptation to antidepressants: imipramine-induced down-regulation of beta-adrenergic receptors in mice treated with metabotropic glutamate 2/3 receptor ligands

Antidepressant drugs have a clinical latency that correlates with the development of neuroadaptive changes, including down-regulation of beta-adrenergic receptors in different brain regions. The identification of drugs that shorten this latency will have a great impact on the treatment of major depressive disorders. We report that the time required for the antidepressant imipramine to reduce the expression of beta-adrenergic receptors in the hippocampus is reduced by a co-administration with centrally active ligands of type 2/3 metabotropic glutamate (mGlu2/3) receptors. Daily treatment of mice with imipramine alone (10 mg/kg, i.p.) reduced the expression of beta-adrenergic receptors in the hippocampus after 21 days, but not at shorter times, as assessed by western blot analysis of beta1-adrenergic receptors and by the amount of specifically bound [3H]CGP-12177, a selective beta-adrenergic receptor ligand. Down-regulation of beta-adrenergic receptors occurred at shorter times (i.e. after 14 days) when imipramine was combined with low doses (0.5 mg/kg, i.p.) of the selective mGlu2/3 receptor agonist LY379268, or with the preferential mGlu2/3 receptor antagonist LY341495 (1 mg/kg, i.p.). Higher doses of LY379268 (2 mg/kg, i.p.) were inactive. This intriguing finding suggests that neuroadaptation to imipramine--at least as assessed by changes in the expression of beta1-adrenergic receptors--is influenced by drugs that interact with mGlu2/3 receptors and stimulates further research aimed at establishing whether any of these drugs can shorten the clinical latency of classical antidepressants.     

Acetyl substitution of the O-specific polysaccharide caryophyllan from the phenol phase of Pseudomonas (Burkholderia) caryophylli

The intact O-specific caryophyllan polysaccharide of the lipopolysaccharide fraction from the bacterium Pseudomonas (Burkholderia) caryophylli was isolated for the first time. Its structure was clarified by means of chemical and spectroscopic analysis and consisted of a homopolymer of randomly acetylated caryophyllose units. The position of acetyl groups when present is not unique: all the hydroxyl-groups on the side chain of the sugar can be substituted with a slight preference for acetylation of the C-5-C-10 tail of this unusual monosaccharide     

Appearance of a class of cell-surface fucosyl-glycopeptides in differentiated muscle cells in culture

Fucosyl-glycopeptides synthesized in culture by duplicating myoblasts and multinucleated myotubes were partially resolved by gel-filtration on Sephadex G-50 in two main components with K_av of 0.3 and 0.6, respectively. DEAE-cellulose chromatography of fucosyl-glycopeptides resolved several components common both to myoblasts and myotubes; however an acidic component, eluted at 24 mM Na-phosphate, is present only in multinucleated myotubes. Neuraminidase treatment of this component abolished its affinity for DEAE-cellulose indicating that its anionic properties are due to the presence of sialic acid residues. Its location on the outer myotube plasma membrane is suggested by the observation that this acidic glycoconjugate was also found in the glycopeptide fraction released by mild trypsin treatment of intact cells in culture. This component appears heterogeneous since it was resolved on Sephadex G-50 into two main peaks corresponding to those obtained by gel-filtration of total glycopeptides. Differentiated postmitotic myoblasts, whose fusion has been inhibited by low Ca²⁺ concentration, synthesize the specific anionic glycopeptides whereas BrdU-treated myoblasts do not. Culture conditions have no effect on the synthesis of these glycopeptides, since myoblasts grown in conditioned medium, collected from myotube cultures, or myoblasts, grown at high cell density, do not synthesize this class of acidic glycopeptides.     

氨氯地平联合依那普利治疗原发性高血压的临床疗效及对患者左心室肥厚的影响

摘要目的：探讨氨氯地平联合依那普利治疗原发性高血压的临床效果,观察联合用药对左心室肥厚的影响。方法：选择本院收治的原发性高血压患者92例,随机分为观察组和对照组,各46例,对照组给予苯磺酸左旋氨氯地平5mg,1次／d,口服;观察组在对照组基础上加用马来酸依那普利10mg,2次／d,口服,疗程均为24周。观察两组治疗前后血压变化,应用超声心动图测量两组左心室厚度变化。结果：治疗后,观察组总有效率为91_3％;对照组总有效率为73．9％,观察组总有效率高于对照组（P〈0．05）。治疗前两组心率、血压比较无统计学差异（P〉0．05）,治疗后两组血压均明显降低,观察组收缩压、舒张压明显低于对照组（P〈O．05）;观察组心率明显低于对照组（P〈0．01）。治疗前两组左心室舒张末期室间隔厚度（Leaventricularend—diastolicventricularseptalthickness,IVST）、左心室后壁厚度（1eftventricularposteriorwallthickness,U,PwT）和左室射血分数（Leftventricularejectionfxaction,LVEF）比较无统计学差异（P〉0．05）;治疗后观察组IVST、L、,PwT明显低于对照组,LVEF明显高于对照组（P〈0．05）。结论：氨氯地平联合依那普利治疗原发性高血压能有效扭转左心室肥厚,降压效果较单独应用氨氯地平更佳。     

重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.     

Induction of the Wnt antagonist Dickkopf-1 is involved in stress-induced hippocampal damage

The identification of mechanisms that mediate stress-induced hippocampal damage may shed new light into the pathophysiology of depressive disorders and provide new targets for therapeutic intervention. We focused on the secreted glycoprotein Dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway, involved in neurodegeneration. Mice exposed to mild restraint stress showed increased hippocampal levels of Dkk-1 and reduced expression of β-catenin, an intracellular protein positively regulated by the canonical Wnt signalling pathway. In adrenalectomized mice, Dkk-1 was induced by corticosterone injection, but not by exposure to stress. Corticosterone also induced Dkk-1 in mouse organotypic hippocampal cultures and primary cultures of hippocampal neurons and, at least in the latter model, the action of corticosterone was reversed by the type-2 glucocorticoid receptor antagonist mifepristone. To examine whether induction of Dkk-1 was causally related to stress-induced hippocampal damage, we used doubleridge mice, which are characterized by a defective induction of Dkk-1. As compared to control mice, doubleridge mice showed a paradoxical increase in basal hippocampal Dkk-1 levels, but no Dkk-1 induction in response to stress. In contrast, stress reduced Dkk-1 levels in doubleridge mice. In control mice, chronic stress induced a reduction in hippocampal volume associated with neuronal loss and dendritic atrophy in the CA1 region, and a reduced neurogenesis in the dentate gyrus. Doubleridge mice were resistant to the detrimental effect of chronic stress and, instead, responded to stress with increases in dendritic arborisation and neurogenesis. Thus, the outcome of chronic stress was tightly related to changes in Dkk-1 expression in the hippocampus. These data indicate that induction of Dkk-1 is causally related to stress-induced hippocampal damage and provide the first evidence that Dkk-1 expression is regulated by corticosteroids in the central nervous system. Drugs that rescue the canonical Wnt pathway may attenuate hippocampal damage in major depression and other stress-related disorders.