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Conversion of factor X to factor Xa results in release of a heavily glycosylated activation peptide. Analysis of protease-digested glycopeptides derived from the activation peptides of bovine and human blood coagulation factor X allowed the identification of sites of the 0-linked oligosaccharide chains in these peptides. Glycopeptides were prepared from the activation peptides by digestion with chymotrypsin or StaphyZococcus aureus V8 protease. By combined analysis of amino acid sequence and sialic acid content, we found that bovine factor X had an 0-linked oligosaccharide chain linked to Thr26, and human factor X had four carbohydrate-attachment sites, namely, 0-glycosidic linkages to Thrl7 and Thr29, respectively, and N-glycosidic linkages to Am39 and Asn49, respectively, in their activation peptides. The 0-linked carbohydrate-attachment sites were identified since the yields of phenylthiohydantoin derivatives of amino acids that corresponded to their residues were increased during amino acid sequencing after deglycosylation of the glycopeptides with sialidase and 0-glycanase. The effect of deglycosylation of bovine factor XI was investigated with factor-X-activating enzyme from Russell's viper venom or extrinsic Xase (factor VIId tissue factor/phosholipid) by examining the activation rates of derivatives of factor X prepared using 0-glycanase, sialidase, and/or N-glycanase. The removal of 0-linked carbohydrate resulted in a decrease in the rate of activation. It appears that carbohydrate residues in factor X play an important role in the activation of the zymogen.Blood coagulation factor X is a vitamin-K-dependent protein that circulates in blood as the precursor of a serine protease. Factor X is a glycoprotein composed of a heavy chain and a light chain which are held together by a single disulfide bond. The complete amino acid sequence [l, 21 and the cDNA sequence [3] of bovine factor X and the cDNA sequence [4] of human factor X have been reported. The glycoprotein is synthesized in hepatocytes and undergoes specific post-translational modifications prior to secretion. The modifications include vitamin-K-dependent y-carboxylation of glutamic acid residues in the amino-terminal region of the light chain [5-71, /?-hydroxylation of an aspartic acid residue located in the first epidermal-growth-factor-like domain [7], and glycosylation. It was previously reported that oligosaccharide chains are linked to Asn36 and Thr3OO of Correspondence to T. Morita, Department of Biochemistry, Meiji College of Pharmacy, Tanashi, Tokyo, Japan 188Abbreviations. AP, activation peptide; AP, and AP,, the activation peptides (residues 1-51 of the heavy chain) of bovine factor X, and X,, respectively ; Pth, phenylthiohydantoin; RVV-XCP, the factor-X-activating enzyme from Russell's viper venom; Boc, tertbutoxycarbonyl ; NH-Np, p-nitroanilide.Enzymes. Coagulation factor Xa (EC 3.4.21.6) ; peptide N-glycosidase F (EC 3.5.1.52) ; endo-a-N-acetylgalactosaminidase (EC 3.2.1.97); sialidase (EC 3.2.1.18); chymotrypsin (EC 3.4.21.1); Stap...

Carp myofibrillar proteins were glycosylated with glucose through the Maillard reaction (the early stage), and their solubility in various ionic strength media was investigated. To react with glucose, lyophilized myofibrils mixed with glucose (at weight ratio of 1:9) were kept at 40 °C and 65% relative humidity for 0−48 h. By glycosylation, myosin and actin became solubilized in low ionic strength media. The total solubility of myofibrillar proteins in 0.01−0.16 M NaCl increased with the progress of glycosylation and was almost equal to the solubility of 0.5 M NaCl. When 17% lysine residue in myofibrillar proteins was reacted with glucose, 61% myosin and 82% actin were solubilized in 0.1 M NaCl. These results indicate that water-soluble myofibrillar proteins can be prepared by glycosylation with the Maillard reaction. Keywords: Fish; myofibrillar proteins; neoglycoprotein; glycosylation; Maillard reaction; solubility

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ObjectivesAngiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes.Methodology/Principal FindingsAdenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver.ConclusionANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance.

Effective treatment of bacterial infection relies on timely diagnosis and proper prescription of antibiotic drugs. The antimicrobial susceptibility test (AST) is one of the most crucial experimental procedures, providing the baseline information for choosing effective antibiotic agents and their dosages. Conventional methods, however, require long incubation times or significant instrumentation costs to obtain test results. We propose a lab-on-a-chip approach to perform AST in a simple, economic, and rapid manner. Our assay platform miniaturizes the standard broth microdilution method on a microfluidic device (20 × 20 mm) that generates an antibiotic concentration gradient and delivers antibiotic-containing culture media to eight 30-nL chambers for cell culture. When tested with 20 μL samples of a model bacterial strain (E. coli ATCC 25922) treated with ampicillin or streptomycin, our method allows for the determination of minimum inhibitory concentrations consistent with the microdilution test in three hours, which is almost a factor of ten more rapid than the standard method.

Our results suggest that pneumoperitoneum increases sympathetic cardiac activity. The choice of general anesthetic did not seem to have a major influence on the change in the cardiac autonomic nervous system after induction of pneumoperitoneum for laparoscopic cholecystectomy.

To test the hypothesis that elderly patients who have surgery for femoral neck fractures may have delirium not only after surgery but before surgery, we prospectively investigated the perioperative temporal profile of cognitive function in such patients. We performed the Abbreviated Mental Test (AMT) six times in each patient (on the day of admission, 3 days after admission, on the day before surgery, 2 days after surgery, 7 days after surgery, and on the day of discharge). Patients were given no premedication and were anesthetized with spinal anesthesia using 0.2% hypobaric tetracaine. Of the 68 patients who were admitted because of a diagnosis of fractured neck of the femur and were scheduled to have surgery, 27 patients were subjected to analysis. Four and three patients showed a significant decrease (3 or more points) in AMT score 2 and 7 days after surgery, respectively. We conclude that surgery may have a stronger impact on cognitive function than environmental change shortly after admission in elderly patients with femoral neck fractures.