High glucose accelerates MCP-1 production via p38 MAPK in vascular endothelial cells

In diabetes mellitus (DM), hyperglycemia causes cardiovascular lesions through endothelial dysfunction. Monocyte chemoattractant protein-1 (MCP-1) is implicated in the pathogenesis of cardiovascular lesions. By using human umbilical vein endothelial cells, we investigated the effect of hyperglycemia on MCP-1 production and its signaling pathways. Chronic incubation with high glucose increased mRNA expression and production rate of MCP-1 in a time (1-7 days)- and concentration (10-35 mM)-dependent manner. Chronic exposure to high glucose resulted in enhancement of generation of reactive oxygen species (ROS), as determined by increasing level of 2,7-dichlorofluorescein (DCF), and subsequent activation of p38 mitogen-activated protein kinase (MAPK). Neither c-Jun NH(2)-terminal kinase nor extracellular signal-regulated kinase1/2 was affected. SB203580 or FR167653, p38 MAPK specific inhibitors, completely suppressed MCP-1 expression. Catalase suppressed p38 MAPK phosphorylation and MCP-1 expression. These results indicate that hyperglycemia can accelerate MCP-1 production through the mechanism involving p38 MAPK, ROS-sensitive signaling pathway, in vascular endothelial cells.     

A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1

Defects in kinetochore proteins often lead to aneuploidy and cancer. Mis12-Mtw1 is a conserved, essential kinetochore protein family. Here, we show that a Mis12 core complex exists in Schizosaccharomyces pombe and human cells. Nine polypeptides bind to human hMis12; two of these, HEC1 and Zwint-1, are authentic kinetochore proteins. Four other human proteins of unknown function (c20orf172, DC8, PMF1 and KIAA1570) correspond to yeast Mis12-Mtw1 complex components and are shown to be required for chromosome segregation in HeLa cells using RNA interference (RNAi). Surprisingly, hMis12 also forms a stable complex with the centromeric heterochromatin components HP1alpha and HP1gamma. Double HP1 RNAi abolishes kinetochore localization of hMis12 and DC8. Therefore, centromeric HP1 may be the base to anchor the hMis12 core complex that is enriched with coiled coils and extends to outer Zwint-1 during mitosis.     

Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization

The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7-10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.     

Expression, purification, and inhibitory activities of mouse cytotoxic T-lymphocyte antigen-2alpha

Cytotoxic T-lymphocyte antigen-2 (CTLA-2) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregion of mouse cathepsin L. Here, we report the expression, purification, and characterization of recombinant CTLA-2 (CTLA-2alpha). CTLA-2alpha was cloned into the pET16b vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) pLysS. The recombinant CTLA-2alpha was highly expressed and purified by His-Bind affinity chromatography, Factor Xa digestion, and hydrophobic chromatography. Throughout these procedures, 3mg recombinant CTLA-2alpha was obtained from 450 ml of bacterial culture medium. The purified protein exhibited inhibitory activities towards certain cysteine proteinases and was properly refolded, as indicated by circular dichroism spectroscopy. Recombinant CTLA-2alpha fully inhibited Bombyx cysteine proteinase (BCP) (overall Kd (Ki*) = 0.23 nM) and and cathepsin L (overall Kd (Ki*) = 0.38 nM). Inhibition of cathepsin H ( Ki = 86 nM) and papain ( Ki = 560 nM) was much weaker, while inhibition of cathepsin B was negligible ( Ki > 1 microM). Our results indicate that mouse CTLA-2alpha is a selective inhibitor of the cathepsin L-like cysteine proteinases.     

Analysis of the interaction between hyaluronan and hyaluronan-binding proteins by capillary affinity electrophoresis: significance of hyaluronan molecular size on binding reaction

We developed a method for the analysis of the interaction between hyaluronan (HA) oligosaccharides and hyaluronan-binding proteins (HABPs) using capillary affinity electrophoresis (CAE). The method is based on high-resolution separation of fluorescent-labeled HA molecules in the presence of hyaluronan-binding proteins at different concentrations by capillary electrophoresis (CE) with laser-induced fluorescent detection. Hyaluronan-binding protein from bovine nasal cartilage interacts strongly with HA decasaccharide or larger oligosaccharides. Effect of the molecular size of HA oligomers clearly showed that longer carbohydrate chains than decasaccharide were required for recognition by HA binding protein. Interestingly, the interaction did not cause retardation of HA oligomers as observed in many binding reactions such as the interaction between pharmaceuticals and serum albumin, but showed disappearance of the oligomer peak. Although we cannot explain the accurate mechanism on the interaction, disappearance is probably due to low equilibrium rate between free and conjugate states. The present technique will be useful to compare the relative binding affinity, and to understand the mechanism on the interaction between hyaluronan and hyaluronan-binding proteins.     

Increased serum HO-1 in hemophagocytic syndrome and adult-onset Still's disease: use in the differential diagnosis of hyperferritinemia

Heme oxygenase-1 (HO-1), an inducible heme-degrading enzyme, is expressed by macrophages and endothelial cells in response to various stresses. Because ferritin synthesis is stimulated by Fe²⁺, which is a product of heme degradation, we examined the relation between HO-1 and ferritin levels in the serum of patients with hemophagocytic syndrome (HPS), adult-onset Still's disease (ASD), and other diseases that may cause hyperferritinemia. Seven patients with HPS, 10 with ASD, 73 with other rheumatic diseases, 20 with liver diseases, 10 recipients of repeated blood transfusion because of hematological disorders, and 22 healthy volunteers were enrolled. Serum HO-1 and ferritin levels were determined by ELISA. Expression of HO-1 mRNA and protein by peripheral blood mononuclear cells (PBMCs) was determined by real-time PCR and immunocytochemical techniques, respectively. Serum levels of HO-1 were significantly higher in patients with active HPS and ASD than in the other groups (P < 0.01). HO-1 levels were not elevated in patients with other causes of hyperferritinemia but were moderately elevated in patients with dermatomyositis/polymyositis. Among patients with HPS and ASD, serum HO-1 levels correlated closely with serum ferritin levels, and the levels of both returned to normal after therapy had induced remission. Increased expression of HO-1 mRNA was confirmed in PBMCs from some patients with HPS and ASD. Hyperferritinemia correlated closely with increased serum HO-1 in patients with HPS and ASD but not other conditions, indicating that measurement of serum HO-1 and ferritin levels would be useful in the differential diagnosis of hyperferritinemia and perhaps also in monitoring disease activity in HPS and ASD.     

Insertion of unmarked DNA sequences in multiple loci of the Bacillus subtilis 168 genome: an efficient selection method

The construction process of Bacillus subtilis strain with multiple mutations is accelerated by simultaneous use of a positive selection method. All the strains selected by neomycin acquired two simultaneous mutations at different genomic loci. The extremely low rate of false positives was accounted for by employing the improved method.     

Elevated expression of N-acetylglucosaminyltransferase V in first trimester human placenta

In early pregnancy, placental trophoblast cells rapidly grow and invade into maternal uterine tissue. N-Acetylglucosaminyltransferase V (GnT-V) and its product, beta1-6-GlcNAc branching glycan, are known to correlate with tumor invasion and metastasis. Since the placentation process resembles invasion of cancer cells, we examined the expression of beta1-6-GlcNAc branching glycan and GnT-V in human placenta. Placentas derived from the first trimester contained a larger amount of beta1-6-GlcNAc branching glycan, detected by leukoagglutinating phytohemagglutinin lectin blotting, than those at term. Immunohistochemical study revealed that beta1-6-GlcNAc branching glycans and GnT-V protein were localized in the trophoblast layer. Both protein expression and the enzyme activity of GnT-V in first trimester placentas were higher than those at term. These results suggest that GnT-V would contribute to placentation in the early phase of pregnancy, possibly regulating the process of invasion of trophoblast cells.     

Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis

Two lactose-binding lectins (PFL-1 and -2) were identified in the skin mucus of ponyfish, Leiognathus nuchalis. The molecular masses of PFL-1 and -2 were estimated to be 24 and 30kDa, respectively. Cloning of the PFL-1 cDNA demonstrated its unique tandem repeat structure composed of two homologous domains with 41.7% internal identity. Furthermore, PFL-1 exhibited homology with L-rhamnose-binding lectins previously purified from the eggs of fish and sea urchins. The N-terminal amino acid sequence of PFL-2 was similar to that of PFL-1, suggesting that this protein is an isotype of PFL-1. The PFL-1 gene was expressed in the skin, an important line of defense against pathogens in fish, but was not expressed in any of the other tissues tested here. PFL-1 is the fourth type of fish skin mucus lectin to be identified, suggesting that different species of fish express different types of lectin in their skin mucus.     

Synthesis of phosphatidylcholine: an improved method without using the cadmium chloride complex of sn-glycero-3-phosphocholine

An improved safe method that does not contaminate the environment with cadmium chloride, a toxic heavy metal salt, was developed for the synthesis of phosphatidylcholine (PC). PC was synthesized from sn-glycero-3-phosphocholine (GPC) and fatty acid in one step under mild conditions without the use of cadmium chloride. GPC was prepared from egg yolk PC and adsorbed by kieselguhr in a Teflon vessel. The GPC on kieselguhr was acylated with fatty acid in the presence of two reagents, dicyclohexylcarbodiimide for synthesis of fatty acid anhydride and 4-dimethylaminopyridine as an acylating catalyst, at 30 degrees C overnight. The PC thus produced was purified by silica gel column chromatography. The yield of dioleoyl PC was 90% based on the starting material, GPC.