In vitro non-enzymatic glycosylation of myofibrillar proteins

• 1.1. Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered.
• 2.2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered.
• 3.3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated.
• 4.4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation.
• 5.5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.

     

Comparison of 5′-nucleotidase activities of isolated plasma membranes of two ascites cell variants

• 1.1. The glycogen-containing ascites cell line was found to have a 3–5 times higher 5′-nucleotidase specific activity than the glycogen-free variant, resulting in different substrate affinity constants of K_m = 0.14mM and K_m = 0.69mM respectively.
• 2.2. These activity differences were due to true 5′-nucleotidase as shown by its inactivation through specific inhibitors such as concanavalin A and α,β-methylene adenosine diphosphate.
• 3.3. Substrate specificity of the enzyme was similar in both cell lines, but differences were observed with respect to the pH optimum and stability.

     

Combined effect of adenosine,alpha adrenergic and adenosine antagonists on serum insulin and insulin secretion from rat pancreatic islets

• 1.1. The effect of adenosine separately or in combination with alpha-1 adrenergic antagonist prazosin and alpha-2 adrenergic antagonist yohimbine as well as adenosine antagonists 8-phenyltheophylline and xanthine amine conjugate on glucose-induced insulin secretion from isolated rat pancreatic islets was studied.
• 2.2. Their in vivo effects on serum glucose and insulin levels were also investigated. Adenosine at 10 and 100 μM inhibited significantly, insulin secretion from the isolated islets whereas at 10 mM slightly increased the secretion of insulin.
• 3.3. Prazosin used at 100 μM inhibited insulin secretion. When it combined with adenosine (10 μM) it augmented the inhibitory effect of adenosine.
• 4.4. In vivo prazosin (21 mg/kg bodywt) caused a hyperglycaemia which was accompanied by hypoinsulinaemia.
• 5.5. Concurrent administration of this drug with adenosine neither affect the hyperglycaemic nor the hypoinsulinaemic effects of adenosine.
• 6.6. On the other hand, yohimbine (100 μM) has no effect neither separately nor in combination with adenosine (10 μM) in modulating the inhibitory effect of adenosine on insulin secretion.
• 7.7. When Yohimbine administered at 19.5 mg/kg body wt it did not alter serum glucose but it markedly increased the serum insulin level. Its combined administration with adenosine reduced the hyperglycaemic effect of adenosine with a remarkable increase in serum insulin.
• 8.8. Both adenosine-antagonists were ineffective in alteration of insulin secretion.
• 9.9. However, combination of 8-phenyltheophylline with adenosine (10 μM) totally blocked the inhibitory effect of adenosine on insulin secretion while xanthine amine conjugate failed to prevent this effect of adenosine.
• 10.10. These results indicate that the inhibitory effect of adenosine on insulin secretion is neither mediated via alpha-1 nor alpha-2 adrenoceptors. It might be via activation of specific adenosine receptors on rat islets which are sensitive to blockade by 8-phenyltheophylline.

     

Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni

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Highlights

• •Protein N-glycosylation is essential for nitrate reductase (Nap) activity in C. jejuni.
• •Removal of N-glycosylation results in a metabolic switch from Asp to Pro uptake.
• •N-glycosylation is required for optimal chemotaxis towards several substrates.
• •Loss of N-glycosylation reduces survival following temperature and osmotic shock.

     

The inhibitory effect of tetramethylethylene diamine on water soluble and membrane bound acetylcholinesterase activity

• 1.1. The inhibitory effect of N,N,N′,N′-tetramethylethylene diamine (TEMED) on water soluble (WSAChE) and membrane bound (MBAChE) acetylcholinesterase was investigated.
• 2.2. TEMED (0.5–4.0 mM) reversibly inhibited WSAChE activity (18–62%) and MBAChE (20–61%) in a concentration dependent manner.
• 3.3. The IC₅₀ being about 2.8 mM for WSAChE and 2.6 mM for MBAChE.
• 4.4. Lineweaver-Burk plots indicated that the nature of inhibition is noncompetitive for both water soluble and membrane bound acetylcholinesterase, with K_m values 68 μM and 123 μM respectively.
• 5.5. An Arrhenius plot showed that the transition temperature (TT) is unaffected in the presence of TEMED.
• 6.6. The activation energy was increased below and above TT in the case of WSAChE only.
• 7.7. On the basis of this behaviour of TEMED with AChE. it can be proposed that it can be used as an eluting agent for the bounded AChE to affinity ligand and may have beneficial action on the reactivatability of irreversibly-inhibited AChE due to its structure.
• 8.8. Moreover there is a possibility that it can be used as a therapeutic agent for the treatment of Alzheimer's disease, myasthenia gravia and glaucoma like some other inhibitors of AChE.

     

A very stable and potent fibrinolytic enzyme found in earthworm Lumbricus rubellus autolysate

• 1.1. The autolysate of earthworms was found to exhibit powerful fibrin and thrombin substrate hydrolyzing activity.
• 2.2. It also showed a clot-forming activity in the fibrinogen- or plasma-added system.
• 3.3. Zymography revealed that there were three active components with mol. wts of 40,000, 21,000 and 15,000 in the autolysate.
• 4.4. The major form with a mol. wt 35,500 (by SDS-PAGE) was further purified. The N-terminal amino acid sequence of this enzyme (16 residues) was similar to that of the swine pancreatic proelastase.

     

Isolation of allantoin and adenosine from the marine sponge Tethya aurantia

• 1.1. Propanol extracts of the sponge Tethya aurantia (Demospongiae) were fractionated, guided by bioassay, for a component with negative chronotropic and inotropic activity on isolated guinea pig atria.
• 2.2. The bioactive component was found to be adenosine. These extracts also contained allantoin.
• 3.3. This intermediate in the sequence of degradation of purines was unexpected, since it has been reported only once before to occur in marine invertebrate animals.

     

The distribution of monosaccharides and hexosamines in the oviduct of Salamandra salamandra (L.) (Amphibia,Urodela)

• 1.1. Biochemical analysis of the different segments of the oviduct in the ovoviviparous salamander, Salamandra salamandra, reveals the monosaccharides glucose, galactose, fucose, mannose, ribose and the hexosamines glucosamine and mannosamine.
• 2.2. In segment 1 (pars recta) ribose and mannose are absent, and in segments 2 (p. convoluta I) and 5 (p. convoluta IV, uterus) mannose is not detectable; fucose is absent in the uterus. Segments 3 (p. convoluta II) and 4 (p. convoluta II) contain all sugars identified.
• 3.3. The main hexoses present in the glandular segments are galactose, fucose and glucose.

     

Adenosine and deoxyadenosine metabolism in lymphocytes from horses,Equis cabalus

• 1.1. The toxic effects of adenosine and deoxyadenosine on lymphocytes from horses were evaluated.
• 2.2. Mitogen-stimulated peripheral blood lymphocytes (PBL) were found to be more sensitive to the inhibitory effects of adenosine than were lymphocytes from spleen, lymph node and thymus.
• 3.3. Adenosine deaminase activity was approximately 10 times lower in horse lymphoid tissue in comparison to that found in human lymphoid tissue. In horse, the highest activity was in spleen while the lowest activity was in thymus.
• 4.4. Adenosine inhibition of mitogenesis in PBL was prevented by uridine or cytidine, suggesting pyrimidine starvation as the major mechanism for adenosine toxicity.
• 5.5. Deoxyadenosine inhibition of mitogenesis in PBL was not prevented by the addition of various ribo- or deoxyribonucleosides. This and the finding that treated cells show no increase in deoxyATP suggest that some other mechanism for deoxyadenosine toxicity other than deoxyATP inhibition of ribonucleotide reductase operates in horse PBL.

     

In vivo effect of Berenil on rat liver polyamine metabolism

• 1.1. Berenil, administered to rats in vivo, promoted a decrease in liver SAMDC activity, but an increase in ODC and SAT activity.
• 2.2. Its effect on ODC was completely prevented by cycloheximide, that on SAT only partially.
• 3.3. Berenil had no effect on ODC activity in adrenalectomized rats. Adrenergic antagonists counteracted the effect of Berenil on ODC activity.
• 4.4. Polyamine content was increased. The maximum modification was observed for putrescine and N¹-acetylspermidine.

     

Preparation and characterization of biotinylated probes for the β-interferon receptor

• 1.1. Recently we described the isolation of the β-interferon receptor [Zhang et al. (1986) J. biol. Chem. 261, 8017–8021]. A highly purified product was obtained but in low quantities.
• 2.2. The use ofbiotinylated β-interferon as a ligand represents an alternate approach to receptor isolation.
• 3.3. We have prepared and characterized the derivatives N-(biotinyl)- and N-(biotinyl-ϵ-aminocaproyl)-recombinant human [Ser¹⁷-interferon β (B- and BC-recHulFNβ).
• 4.4. Biotin incorporation does not result in any loss of antiviral activity, demonstrating the recognition of the derivative by the cell receptor.
• 5.5. The biotinylated recHuIFNβ binds specifically and reversibly to succinoylavidin or guanidine thiocyanate-stripped succinoylavidin linked to a Sepharose matrix.
• 6.6. Comparison of the competition curves obtained with [¹⁴C]biotin and [³H]biotinyl recHuIFN, in the presence of increasing concentrations of biotin suggests that the IFN moiety of the derivative has little effect on the affinity of biotin for avidin.
• 7.7. Biotinylated recHuIFNβ derivatives represent useful probes for the β-IFN receptor.

     

Multi-isotype Glycoproteomic Characterization of Serum Antibody Heavy Chains Reveals Isotype- and Subclass-Specific N-Glycosylation Profiles

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Highlights

• •nLC-MS/MS method to analyze immunoglobulin (Ig) N-glycopeptides from human serum.
• •Multi-isotype, site-specific characterization of immunoglobulin N-glycosylation.
• •IgA2 sequence and glycosylation-site variant analyses.
• •Platform to define disease-specific N-glycan signatures for different Ig isotypes.

     

Locust vitellogenin receptor: an acidic glycoprotein with N- and O-linked oligosaccharides

• 1.1. The locust vitellogenin (VTG) receptor which is embedded in oocyte plasma membranes is a glycoprotein.
• 2.2. With various lectins oligosaccharide units have been identified, among them neuraminic acid linked to Gal or GalNAc, mannose chains, Gal linked to GalNAc or GlcNAc and fucose linked to GlcNAc.
• 3.3. With specific enzymes it could be shown that mannose and most other oligosaccharides are O-linked while others like fucose are N-linked.
• 4.4. Enzymatic removal of all O-linked carbohydrates resulted in a drop of the molecular mass of the receptor protein from 200,000 to 110,000.
• 5.5. A total of N- and O-linked oligosaccharides of 54% was calculated.
• 6.6. The isoelectric point of the receptor was found to be at pH 3.4 increasing slightly after removal of neuraminic acid.
• 7.7. Removal of neuraminic acids destroyed the binding ability for VTG.

     

Functional properties of the two major hemoglobin components from Leporinus friderici (Pisces)

• 1.1. The hemoglobins of Leporinus friderici were separated by liquid chromatography on DEAE-Sepharose in order to isolate the two major electrophoretic components.
• 2.2. The chromatographic fraction I (electrophoretically slow anodic) showed no Bohr effect and no nucleoside triphosphate modulation.
• 3.3. The chromatographic fraction III (electrophoretically fast anodic) showed a normal Bohr effect and addition of nucleoside triphosphate decreased oxygen affinity but did not alter the Bohr effect.
• 4.4. The whole hemolysate showed a normal Bohr effect and phosphate modulation altered both Bohr effect and oxygen affinity.
• 5.5. No or little difference between the effect of adenosine or guanosine triphosphates on hemoglobin function was observed.

     

Thrombocyte aggregation in rainbow trout

• 1.1. Thrombocytes from mature rainbow trout (Sahmo gairdneri) aggregated in vitro after addition of ADP, ATP, collagen, epinephrine, 5-hydroxytryptamine or thrombin to thrombocyte-rich plasma.
• 2.2. Thrombocyte aggregation was dose dependent and could be inhibited by preincubating the thrombocyte-rich plasma with adenosine, acetylsalicylic acid or prostaglandin E₁.
• 3.3. Thrombocytes released ADP and ATP when aggregated by 10 μM epinephrine. This release was blocked by 1 mM adenosine.
• 4.4. Electron microscopic observations of thrombocytes revealed them to contain numerous microtubules and electron-dense vesicles. In addition a possible shape change associated with thrombocyte aggregation was noted.

     

Isolation of dna and rna from Streptococcus sobrinus OMZ176 using CsTFA gradients

• 1.1. A simple procedure for isolation of high molecular weight genomic DNA, and RNA, from Streptococcus sobrinus OMZ176 is described.
• 2.2. Cell cultures were grown aerobically for 10 hr.
• 3.3. Spheroplast formation and lysis was achieved by mutanolysin/lysozyme-dependent digestion of the cell wall, followed by N-lauroylsarcosinate-mediated lysis.
• 4.4. Nucleic acids were isolated directly from cell-lysates using cesium-trifluoroacetate (CsTFA) densitygradient centrifugation.
• 5.5. Three different centrifugation regimes were tested: self-generated density gradients in a fixed angle rotor; self-generated density-gradients in a swinging-bucket rotor; pre-formed density-gradients in a swinging-bucket rotor.
• 6.6. Genomic DNA isolated by the CsTFA-procedure was found to have higher purity as compared to genomic DNA isolated in a conventional CsCl gradient.
• 7.7. Isolated DNA was shown to be of a quality suitable for applications in molecular biology.

     

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme

• 1.1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage.
• 2.2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT.
• 3.3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA.
• 4.4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the K_m for glycerol phosphate.
• 5.5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

     

Purine metabolism in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) Seedlings

• 1.1. The metabolism of purine bases and nucleosides in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) was studied.
• 2.2. A large portion of absorbed [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]adenosine was salvaged in nucleotide and nucleic acid synthesis.
• 3.3. Most of the radioactivity of [8-¹⁴C]hypoxanthine and [8-¹⁴C]inosine was incorporated into allantoin and allantoic acid.
• 4.4. Activity of adenine phosphoribosyltransferase in enzyme extracts was much higher than that of hypoxanthme and guanine phosphoribosyltransferase(s).
• 5.5. Apparent activity of adenosine kinase was higher than that of inosine kinase. 6. NAD⁺-dependent xan thine dehydrogenase was detected in both cotyledons and embryonic axes of the seedlings.
• 6.7. The capacity of purine salvage was higher m 24 hr old cotyledons than 24 and 48 hr old embryonic axes. The reverse was observed concerning that of purine degradation.

     

Xenobiotic metabolizing enzymes in rainbow trout exposed to river water

• 1.1. Juvenile rainbow trout were exposed to river water in a flow-through system. After 15 days of exposure, hepatic biotransformation activities and related parameters were measured and compared to those of the control group organisms that were maintained in tap water under identical experimental conditions.
• 2.2. Liver somatic index (LSI), microsomal protein and cytochrome P-450 contents, benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD) and UDP glucuronyl transferase activities were not significantly affected.
• 3.3. Aminopyrine-N-demethylase (APD) activity showed a slight yet significant increase in exposed trout.

     

Evidence for extracellular deamination of adenosine in the rat heart

• 1.1. In rat heart perfused with adenosine (10⁻⁶M), dilazep (10⁻⁴M) inhibited incorporation of adenosine into nucleotides (an index of nucleoside transport and phosphorylation) to a greater extent (70%) than metabolism to inosine and uric acid (40%) and actually increased the recovery of inosine to 30% of the adenosine infused.
• 2.2. Extrapolating for complete inhibition of transport suggested that 60% of adenosine metabolism was intracellular and 40% extracellular.
• 3.3. Static incubations of atria also gave an estimate for extracellular metabolism of 40%.
• 4.4. Adenosine deaminase was localised by immunocytochemistry to the extracellular surface of endothelial cells of small coronary arteries.
• 5.5. Extracellular deamination may explain the lack of effect of nucleoside transport inhibitors on responses to adenosine in rat heart.