Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Stimulation of gastrin secretion from the perfused rat stomach by somatostatin antiserum

The effect of a high capacity somatostatin antiserum on antral gastrin secretion was examined in an isolated vascularly perfused rat stomach preparation. Infusion of somatostatin antiserum diluted 1:1 and 1:9 with Krebs buffer solution produced significant increases in gastrin secretion throughout the period of infusion. Neither infusion of somatostatin antiserum diluted 1:99 nor infusion of control rabbit serum had any effect on gastrin secretion. The data indicate that antral somatostatin excercises a continous restraint on gastrin secretion in the basal state.     

Delay of Membrane Lipid Degradation by Calcium Treatment during Cabbage Leaf Senescence

Cabbage leaf discs (Brassica oleracea L., Capitata group) were floated adaxial side up in 0, 0.05, or 0.25 m CaCl₂ solutions at 15°C for 14 d in the dark. To assess whether the delay of senescence by calcium treatment involved protection of membrane lipids, chlorophyll and protein content and the lipid composition of the membranes were determined during incubation. Chlorophyll and protein content decreased with time, in correlation with a reduction in the amount of phospholipids. The degree of unsaturation of phospholipids and free fatty acids decreased, whereas the ratio of sterol to phospholipid increased. The proportions of phospholipid classes did not change during senescence. The catabolism of phospholipids was delayed by 0.05 m calcium, but accelerated by 0.25 m, as compared to the untreated control. Based on the levels of the lipid intermediates, phospholipase D, phosphatidic acid phosphatase, lipolytic acyl hydrolase, and lipoxygenase appeared to be involved in the breakdown of phospholipids during senescence. Phospholipase D and phosphatidic acid phosphatase may be directly influenced by calcium. The calcium treatment apparently did not affect the activity of acyl hydrolase. Lipoxygenase, responsible for the peroxidation of the polyunsaturated fatty acids, was probably indirectly influenced by calcium. We conclude that the delay of senescence of cabbage leaf discs by calcium treatment involved protection of membrane lipids from degradation.     

Melatonin ingestion after exhaustive late-evening exercise attenuate muscle damage,oxidative stress,and inflammation during intense short term effort in the following day in teenage athletes

ABSTRACT

The present study aimed to investigate whether nocturnal melatonin (MEL) ingestion has beneficial effects against exercise-induced oxidative stress and muscle damage in young athletes. Fourteen healthy-trained teenagers performed two-test sessions separated by at least, 1 week. During each session, participants completed the Running-Based Anaerobic Sprint Test (RAST) at 20:00 h. Then, they ingested a single 10-mg tablet of MEL or Placebo (PLA) in a double-blind randomized order at 22:00 h. The following morning (i.e., 07:30 h), participants performed the same test as the previous night. Blood samples were taken before and after exercise. MEL intake increased the peak power (P_peak) (p < .01), mean power (P_mean) (p < .001) and decreased the total time (TT) (p < .001) and the fatigue index (FI) (p < .05). Furthermore, MEL ingestion attenuated the hematologic parameters before and after exercise (White Blood Cells (WBC: p < .001 and p < .001, respectively); Neutrophiles (NE: p < .001 and p < .001, respectively); Lymphocytes (LY: p < .001 and p < .001, respectively)) and the ultra-sensitive C-reactive protein (us-CRP: p < .001 and p < .001; respectively) compared to PLA. Also, MEL reduced muscle and hepatic damage enzymes before and after exercise (creatine kinase (CK: p < .001 and p < .001; respectively), lactate dehydrogenase (LDH: p < .05 and p < .01; respectively), aspartate aminotransferase (ASAT: p < .01 and p < .001; respectively)), Malondialdehyde (MDA: p < .001 and p < .001; respectively) and Homocysteine (Hcy: p < .001 and p < .001; respectively)) from placebo. Plasma lactate [La] and glucose (GL) remained unchangeable during the two conditions. In summary, acute MEL ingestion after strenuous late-evening exercise attenuated transient leucocytosis and protected against lipid peroxidation and muscle damage induced by strenuous exercise the following morning in healthy male teenage athletes.     

Heterologous desensitization mediated by G protein-specific binding to caveolin

We examined the notion that sequestration of G protein subunits by binding to caveolin impedes G protein reassociation and leads to transient, G protein-specific desensitization of response in dispersed smooth muscle cells. Cholecystokinin octapeptide (CCK-8) and substance P (SP) were used to activate G(q/11), cyclopentyl adenosine (CPA) was used to activate G(i3), and acetylcholine (ACh) was used to activate both G(q/11) and G(i3) via m3 and m2 receptors, respectively. CCK-8 and SP increased only Galpha(q/11), and CPA increased only Galpha(i3) in caveolin immunoprecipitates; caveolin and other G proteins were not increased. ACh increased both Galpha(q/11) and Galpha(i3) in a time- and concentration-dependent fashion: only Galpha(q/11) was increased in the presence of an m2 antagonist, and only Galpha(i3) was increased in the presence of an m3 antagonist. To determine whether transient G protein binding to caveolin affected subsequent responses mediated by the same G protein, PLC-beta activity was measured in cells stimulated sequentially with two different agonists that activate either the same or a different G protein. After treatment of the cells with ACh and an m2 antagonist, the phospholipase C-beta (PLC-beta) response to CCK-8 and SP, but not CPA, was decreased; conversely, after treatment of the cells with ACh and an m3 antagonist, the PLC-beta response to CPA, but not CCK-8 or SP, was decreased. Similarly, after treatment with CCK-8 or SP, the PLC-beta response mediated by G(q/11) only was decreased, whereas after treatment with CPA, the PLC-beta response mediated by G(i3) only was decreased. A caveolin-binding Galpha(q/11) fragment blocked the binding of activated Galpha(q/11) but not Galpha(i3) to caveolin-3 and prevented desensitization of the PLC-beta response mediated only by other G(q/11)-coupled receptors. A caveolin-binding Galpha(i3) fragment had the reverse effect. Thus, transient binding of receptor-activated G protein subunits to caveolin impedes reassociation of the heterotrimeric species and leads to desensitization of response mediated by other receptors coupled to the same G protein.     

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2)

This study examined the ability of protein kinase C (PKC) toinduce heterologous desensitization by targeting specific G proteinsand limiting their ability to transduce signals in smooth muscle.Activation of PKC by pretreatment of intestinal smooth muscle cellswith phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, orthe phosphatase 1 and phosphatase 2A inhibitor, calyculin A,selectively phosphorylated G_i-1 and G_i-2,but not G_i-3 or G_o, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to G_i-1 and opioid receptors coupled toG_i-2, but not by muscarinic M₂ and adenosineA₁ receptors coupled to G_i-3. Phosphorylationof G_i-1 and G_i-2 and blockade of cyclaseinhibition were reversed by calphostin C and bisindolylmaleimide, andadditively by selective inhibitors of PKC and PKC. Blockade ofinhibition was prevented by downregulation of PKC. Phosphorylation ofG-subunits by PKC also affected responses mediated by-subunits. Pretreatment of muscle cells withcANP-(4-23), a selective agonist of the natriureticpeptide clearance receptor, NPR-C, which activates phospholipase C(PLC)-3 via the -subunits of G_i-1 andG_i-2, inhibited the PLC- response to somatostatin and[D-Pen^2,5]enkephalin. The inhibition waspartly reversed by calphostin C. Short-term activation of PKC had noeffect on receptor binding or effector enzyme (adenylyl cyclase orPLC-) activity. We conclude that selective phosphorylation ofG_i-1 and G_i-2 by PKC partly accounts forheterologous desensitization of responses mediated by the - and-subunits of both G proteins. The desensitization reflects adecrease in reassociation and thus availability of heterotrimeric G proteins.

     

Increased expression of costimulatory markers CD134 and CD80 on interleukin-17 producing T cells in patients with systemic lupus erythematosus

Introduction  

There is growing evidence that interleukin 17 (IL-17) producing T cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies showed that increased percentages of T-cell subsets expressing the costimulatory molecules CD80 and CD134 are associated with disease activity and renal involvement in SLE. The aim of this study was to investigate the distribution and phenotypical characteristics of IL-17 producing T-cells in SLE, in particular in patients with lupus nephritis, with emphasis on the expression of CD80 and CD134.     

Linking X chromosome inactivation to pluripotency: Necessity or fate?

Silencing one X chromosome is essential for the development of female mammals, but the regulation of this process appears to vary between species. In the mouse, which has thus far been the leading model system in the field, X chromosome inactivation (XCI) is tightly coupled to pluripotency and the underlying mechanisms have just begun to be deciphered. However, mechanistic aspects of XCI regulation in other species have yet to be thoroughly investigated. Here we review current knowledge of the developmental regulation of XCI in mice and humans and discuss the extent to which the intimate link between XCI and pluripotency extends beyond rodents.