JNK inhibitory kinase is up-regulated in retinal ganglion cells after axotomy and enhances BimEL expression level in neuronal cells

Optic nerve transection results in retinal ganglion cell (RGC) death in adult mammals, after the alteration of gene expression of RGCs. To elucidate the molecular mechanism by which axotomy induces RGC death, we isolated the molecules up-regulated after optic nerve transection. One of these, axotomy-related [corrected] gene (ARG)357, an 898-amino-acid [corrected] protein containing a complete serine-threonine kinase domain, was isolated from a subtraction library of the rat retina. The sequence showed that this gene was a rat homolog of human c-Jun N-terminal kinase (JNK) inhibitory kinase and so belonged to the germinal center kinase-VIII subfamily of Sterile20s protein kinase. We designated ARG357 as rat JNK inhibitory kinase (JIK). Rat JIK was expressed ubiquitously in various tissues and was highly expressed in the retina, with selective expression in RGCs. After axotomy, BimEL and Hrk, which are BH3-only proteins, and rat JIK were up-regulated in RGCs. Overexpression of rat JIK in neuronal cells up-regulated the expression of BimEL, but not that of Hrk. These results indicate that JIK may contribute to axotomy-induced RGC death by up-regulating the expression of BH3-only protein.     

Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli

cDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2.6.1.1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46,295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT cDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a beta-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic properties were comparable with those of cAspAT preparations purified from rat liver.     

Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes

The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin.     

The Interconversion between Variotin and Ketovariotin

The interconversion between variotin and ketovariotin is described.     

Natural OX40L expressed on human T cell leukemia virus type-I-immortalized T cell lines interferes with infection of activated peripheral blood mononuclear cells by CCR5-utilizing human immunodeficiency virus

Background

Food allergy has been reported increasingly around the world during the past several decades. Epstein-Barr virus (EBV), a common herpesvirus with high infection rate, is now suspected to be a risk or protective factor in food allergy. The aim of the study was to investigate the possible role of EBV infection in IgE-mediated food allergy.

Methods

34 patients with an egg allergy and 34 healthy controls participated in this study. Egg allergy was confirmed by open-food challenge. Serum anti-viral capsid antigen (VCA), anti-Epstein-Barr nuclear antigen 1 (EBNA-1) IgG and egg specific (yolk and white)-IgE levels were evaluated by enzyme linked immunosorbent assay (ELISA). At the same time, EBV DNA as well as viral miRNAs in these samples was quantified by real-time PCR.

Results

The results showed that serum anti EBNA-1 IgG and two viral miRNAs (miR-BART1-5p and miR-BART7) were highly expressed in patients with egg allergy compared with healthy controls (p < 0.05, < 0.001 and < 0.01, respectively). Moreover, the expressions of anti EBNA-1 specific IgG, miR-BART1-5p and miR-BART7 positively correlated with the level of egg-specific IgE (p < 0.05, < 0.01 and < 0.01, respectively). The differences in anti VCA IgG concentration and EBV DNA copy number between the allergy patients and control individuals were not statistically significant.

Conclusions

The high expression of EBV-specific antibody and miRNAs indicated that EBV infection might play a promoting role in IgE-mediated egg food allergy, and viral miRNAs-related immunomodulatory pathway was likely involved in this allergy process.     

Effects of Lipooligosaccharide Inner Core Truncation on Bile Resistance and Chick Colonization by Campylobacter jejuni

Campylobacter jejuni is the most common bacterium that causes diarrhea worldwide, and chickens are considered the main reservoir of this pathogen. This study investigated the effects of serial truncation of lipooligosaccharide (LOS), a major component of the outer membrane of C. jejuni, on its bile resistance and intestinal colonization ability in chickens. Genes encoding manno-heptose synthetases or glycosyltransferases were inactivated to generate isogenic mutants. Serial truncation of the LOS core oligosaccharide caused a stepwise increase in susceptibilities of two C. jejuni strains, NCTC 11168 and 81-176, to bile acids. Inactivation of hldE, hldD, or waaC caused severe truncation of the core oligosaccharide, which greatly increased the susceptibility to bile acids. Both wild-type strains grew normally in chicken intestinal extracts, whereas the mutants with severe oligosaccharide truncation were not detected 12 h after inoculation. These mutants attained viable bacterial counts in the bile acid-free extracts 24 h after inoculation. The wild-type strain 11-164 was present in the cecal contents at >10⁷ CFU/g on 5 days after challenge infection and after this time period, whereas its hldD mutant was present at <10³ CFU/g throughout the experimental period. Trans-complementation of the hldD mutant with the wild-type hldD allele completely restored the in vivo colonization level to that of the wild-type strain. Mutants with a shorter LOS had higher hydrophobicities. Thus, the length of the LOS core oligosaccharide affected the surface hydrophobicity and bile resistance of C. jejuni as well as its ability to colonize chicken intestines.     

Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica

A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.