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]     

Phenethyl isothiocyanate induces cell cycle arrest and reduction of alpha- and beta-tubulin isotypes in human prostate cancer cells

This study was to investigate the effect of phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables, on the expression of α- and β-tubulins, which are the main components of microtubules in prostate cancer cells. Flow cytometry, light microscopy and western blot were used to study the cell cycle distribution, morphology changes and the expression of α- and β-tubulins in prostate cancer cells treated with PEITC. The results showed that PEITC-induced G2-M cell phase arrest and inhibited the expression of α- and β-tubulin proteins in a number of human prostatic carcinoma cell lines. Further, it is showed that this inhibitory effect could be reversed by antioxidant N-acetyl cysteine and proteasome inhibitor MG132. Finally, it is concluded that PEITC inhibited the expression of α- and β-tubulins in prostate cancer cells, which is at least related to the oxygen reaction species and protein degradation.     

Systematic genome-wide scanning for genes involved in ATP generation in Escherichia coli

Adenosine 5'-triphosphate (ATP) generation is an essential biological reaction for all living cells. Recently, we developed a Permeable Cell Assay for high-throughput measurement of cellular ATP synthetic activity, mainly resulting from glycolysis [Hara, K.Y., Mori, H., 2006. An efficient method for quantitative determination of cellular ATP synthetic activity. J. Biomol. Screen. 11, 310-317]. By using this method, we determined the cellular ATP synthetic activity in the stationary phase of a complete set of single-gene deletion strains of Escherichia coli. Their activities ranged from a minimum of 2% to a maximum of 445%, relative to parental strains. Deletions of metabolism-related genes frequently caused an increase in the rate of ATP synthetic activity, while activity was reduced by deletions of a variety of functional genes, including many poorly characterized genes. We also demonstrated that the deletion of the ptsG gene doubled ATP-driven glutathione synthesis and increased cellular ATP synthetic activity. Our study also indicated that it should be easy to obtain active strains for ATP synthesis from deletion strains. Overall, the data set of this study may be useful to improve E. coli strains for ATP-dependent industrial processes and, therefore, may be important for the design of so-called cell factories.     

N-Glycosylation of the I-like Domain of ��1 Integrin Is Essential for ��1 Integrin Expression and Biological Function: IDENTIFICATION OF THE MINIMAL N-GLYCOSYLATION REQUIREMENT FOR ��5��1*

N-Glycosylation of integrin α5β1 plays a crucial role in cell spreading, cell migration, ligand binding, and dimer formation, but the detailed mechanisms by which N-glycosylation mediates these functions remain unclear. In a previous study, we showed that three potential N-glycosylation sites (α5S3–5) on the β-propeller of the α5 subunit are essential to the functional expression of the subunit. In particular, site 5 (α5S5) is the most important for its expression on the cell surface. In this study, the function of the N-glycans on the integrin β1 subunit was investigated using sequential site-directed mutagenesis to remove the combined putative N-glycosylation sites. Removal of the N-glycosylation sites on the I-like domain of the β1 subunit (i.e. the Δ4-6 mutant) decreased both the level of expression and heterodimeric formation, resulting in inhibition of cell spreading. Interestingly, cell spreading was observed only when the β1 subunit possessed these three N-glycosylation sites (i.e. the S4-6 mutant). Furthermore, the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5 mutant of the α5 subunit. Taken together, the results of the present study reveal for the first time that N-glycosylation of the I-like domain of the β1 subunit is essential to both the heterodimer formation and biological function of the subunit. Moreover, because the α5S3-5/β1S4-6 mutant represents the minimal N-glycosylation required for functional expression of the β1 subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α and a β subunit (1). The interaction between integrin and the extracellular matrix is essential to both physiologic and pathologic events, such as cell migration, development, cell viability, immune homeostasis, and tumorigenesis (2, 3). Among the integrin superfamily, β1 integrin can combine with 12 distinct α subunits (α1–11, αv) to form heterodimers, thereby acquiring a wide variety of ligand specificity (1, 4). Integrins are thought to be regulated by inside-out signaling mechanisms that provoke conformational changes, which modulate the affinity of integrin for the ligand (5). However, an increasing body of evidence suggests that cell-surface carbohydrates mediate a variety of interactions between integrin and its extracellular environment, thereby affecting integrin activity and possibly tumor metastasis as well (6–8).Guo et al. (9) reported that an increase in β1–6-GlcNAc sugar chains on the integrin β1 subunit stimulated cell migration. In addition, elevated sialylation of the β1 subunit, because of Ras-induced STGal-I transferase activity, also induced cell migration (10, 11). Conversely, cell migration and spreading were reduced by the addition of a bisecting GlcNAc, which is a product of N-acetylglucosaminyltransferase III (GnT-III),² to the α5β1 and α3β1 integrins (12, 13). Alterations of N-glycans on integrins might also regulate their cis interactions with membrane-associated proteins, including the epidermal growth factor receptor, the galectin family, and the tetraspanin family of proteins (14–19).In addition to the positive and negative regulatory effects of N-glycan, several research groups have reported that N-glycans must be present on integrin α5β1 for the αβ heterodimer formation and proper integrin-matrix interactions. Consistent with this hypothesis, in the presence of the glycosylation inhibitor, tunicamycin, normal integrin-substrate binding and transport to the cell surface are inhibited (20). Moreover, treatment of purified integrin with N-glycosidase F blocked both the inherent association of the subunits and the interaction between integrin and fibronectin (FN) (21). These results suggest that N-glycosylation is essential to the functional expression of α5β1. However, because integrin α5β1 contains 26 potential N-linked glycosylation sites, 14 in the α subunit and 12 in the β subunit, identification of the sites that are essential to its biological functions is key to understanding the molecular mechanisms by which N-glycans alter integrin function. Recently, our group determined that N-glycosylation of the β-propeller domain on the α5 subunit is essential to both heterodimerization and biological functions of the subunit. Furthermore, we determined that sites 3–5 are the most important sites for α5 subunit-mediated cell spreading and migration on FN (22). The purpose of this study was to clarify the roles of N-glycosylation of the β1 subunit. Therefore, we performed combined substitutions in the putative N-glycosylation sites by replacement of asparagine residues with glutamine residues. We subsequently introduced these mutated genes into β1-deficient epithelial cells (GE11). The results of these mutation experiments revealed that the N-glycosylation sites on the I-like domain of the β1 subunit, sites number 4–6 (S4-6), are essential to both heterodimer formation and biological functions, such as cell spreading.     

Comparative Analysis of argK-tox Clusters and Their Flanking Regions in Phaseolotoxin-Producing Pseudomonas syringae Pathovars

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181–10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064–11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named “tox islands.” The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437–457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

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.