Increase of regulatory T cells in the peripheral blood of dogs with metastatic tumors

It is well known that lymphocytes from patients with advanced-stage cancer have impaired immune responsiveness and that type1 T lymphocyte subsets in tumor bearing hosts are suppressed. Treg have been reported to comprise a subgroup which inhibits T cell mediated immune responses. In the present study, the percentage of Treg, Th1 and Tc1 in the peripheral blood of tumor bearing dogs with or without metastases was evaluated. The percentages of Th1 and Tc1 in dogs with metastatic tumor were significantly less, and that of Treg was significantly greater, than those of dogs without metastatic tumor. The percentage of Treg showed an inverse correlation with that of Th1 and Tc1 in tumor bearing dogs. It was concluded that an increase in Treg in the peripheral blood of dogs with metastatic tumor may induce suppression of tumor surveillance by the Type1 immune response and lead to metastasis of tumor[0][0].[0]     

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies

AimsInsulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice.Main methodsTo localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2^+/+ mice. IA-2^?/? mice served as a negative control.Key findingsWestern blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells.SignificanceThe IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.     

Vav1 acidic region tyrosine 174 is required for the formation of T cell receptor-induced microclusters and is essential in T cell development and activation

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.     

N-linked oligosaccharides on chondroitin 6-sulfotransferase-1 are required for production of the active enzyme, Golgi localization, and sulfotransferase activity toward keratan sulfate

We have shown previously that purified chondroitin 6-sulfotransferase-1 (C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however, functional roles of the N-glycans have remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, whereas deletion of the fifth site resulted in a marked loss of the KSST activity. Wild-type recombinant C6ST-1 showed a typical Golgi localization, whereas M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an endoplasmic reticulum-resident protein. Unlike wildtype recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi localization, thereby causing the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.     

Descriptions of two new endoparasitic cecidomyiids (Diptera: Cecidomyiidae) from Japan

Two endoparasitic species of Cecidomyiidae (Diptera) new to science are reported from Japan. Females of Endaphis psyllophaga sp. nov. lay their eggs on the wing of Calophya nigridorsalis (Hemiptera: Psylloidea: Calophyidae) on Rhus succedanea (Anacardiaceae), and newly hatched larvae bore into the adult body. The six nominal species of the genus Endaphis are endoparasitoids of aphids. The genus Endopsylla, which is morphologically similar to the genus Endaphis, consists of two species whose larvae attack psyllids or tingids. Females of Endaphis muraii sp. nov. lay their eggs near colonies of host aphids and newly hatched larvae bore into the body of aphids such as Macrosiphum euphorbiae and Aphis glycines (Hemiptera: Aphididae). The two new species are described, illustrated, and compared to known congeners, and information is given for the two species on their distribution, host range and ecological traits. Now, E. muraii is considered to be a potential biological control agent against aphids.     

Genome-wide identification of in vivo binding sites of GlxR, a cyclic AMP receptor protein-type regulator in Corynebacterium glutamicum

Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant.     

The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB-atpE dicistronic mRNAs in chloroplasts

The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the tobacco dicistronic mRNA, and that the efficiency of atpB translation is higher than that of atpE translation. When the atpB 5′-UTR was replaced with lower efficiency 5′-UTRs, atpE translation was higher than atpB translation. Removal of the entire atpB 5′-UTR arrested atpB translation but atpE translation still proceeded. Introduction of a premature stop codon in the atpB cistron did not abolish atpE translation. These results indicate that atpE translation is independent of atpB translation. Mutation analysis showed that the atpE cistron possesses its own cis-element(s) for translation, located ~25 nt upstream from the start codon.     

Metabolic engineering of 1,2-propanediol pathways in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We analyzed 1,2-propanediol (1,2-PD) production in metabolically engineered Corynebacterium glutamicum. Wild-type C. glutamicum produced 93 μM 1,2-PD after 132 h incubation under aerobic conditions. No gene encoding the methylglyoxal synthase (MGS) which catalyzes the first step of 1,2-PD synthesis from the glycolytic pathway was detected on the C. glutamicum genome, but several genes annotated as encoding putative aldo-keto reductases (AKRs) were present. AKR functions as a methylglyoxal reductase in the 1,2-PD synthesis pathway. Expressing Escherichia coli mgs gene in C. glutamicum increased 1,2-PD yield 100-fold, suggesting that wild-type C. glutamicum carries the genes downstream of MGS in the 1,2-PD synthesis pathway. Furthermore, simultaneous overexpression of mgs and cgR_2242, one of the genes annotated as AKRs, enhanced 1,2-PD production to 24 mM. This work establishes that 1,2-PD synthesis by C. glutamicum, previously unknown, is possible.