Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule

Immunological Changes in Mesothelioma Patients and Their Experimental Detection

It is common knowledge that asbestos exposure causes asbestos-related diseases such as asbestosis, lung cancer and malignant mesothelioma (MM) not only in people who have handled asbestos in the work environment, but also in residents living near factories that handle asbestos. These facts have been an enormous medical and social problem in Japan since the summer of 2005. We focused on the immunological effects of asbestos and silica on the human immune system. In this brief review, we present immunological changes in patients with MM and outline their experimental detection. For example, there is over-expression of bcl-2 in CD4+ peripheral T-cells, high plasma concentrations of interleukin (IL)-10 and transforming growth factor (TGF)-ß, and multiple over-representation of T cell receptor (TcR)-Vß in peripheral CD3+ T-cells found in MM patients. We also detail an experimental long-term exposure T-cell model. Analysis of the immunological effects of asbestos may help our understanding of the biological effects of asbestos

Involvement of SIK3 in Glucose and Lipid Homeostasis in Mice

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3−/− mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3−/− mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3−/− mice. Lipid metabolism disorders in Sik3−/− mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice

SNAP23/25 and VAMP2 mediate exocytic event of transferrin receptor-containing recycling vesicles

We recently showed that Rab11 is involved not only in formation of recycling vesicles containing the transferrin (Tfn)–transferrin receptor (TfnR) complex at perinuclear recycling endosomes but also in tethering of recycling vesicles to the plasma membrane (PM) in concert with the exocyst tethering complex. We here aimed at identifying SNARE proteins responsible for fusion of Tfn–TfnR-containing recycling vesicles with the PM, downstream of the exocyst. We showed that exocyst subunits, Sec6 and Sec8, can interact with SNAP23 and SNAP25, both of which are PM-localizing Qbc-SNAREs, and that depletion of SNAP23 and/or SNAP25 in HeLa cells suppresses fusion of Tfn–TfnR-containing vesicles with the PM, leading to accumulation of the vesicles at the cell periphery. We also found that VAMP2, an R-SNARE, is colocalized with endocytosed Tfn on punctate endosomal structures, and that its depletion in HeLa cells suppresses recycling vesicle exocytosis. These observations indicate that fusion of recycling vesicles with the PM downstream of the exocyst is mediated by SNAP23/25 and VAMP2, and provide novel insight into non-neuronal roles of VAMP2 and SNAP25

Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis

正常な細胞分裂に不可欠なタンパク質の機能と構造を解明. 京都大学プレスリリース. 2012-05-31.A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin-like protein 1 (MKLP1), a Flemming body-localizing protein essential for cytokinesis. The crystal structure of the Arf6-MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended β-sheet composed of 22 β-strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure-based mutagenesis and siRNA-mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6-MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane

The Radiation-Induced Cell-Death Signaling Pathway is Activated by Concurrent Use of Cisplatin in Sequential Biopsy Specimens from Patients with Cervical Cancer.

Objective: To identify changes in gene expression related to the concurrent use of platinum compounds with radiotherapy, in the treatment of cervical cancer. Patients and Methods: Biopsy specimens were obtained from 39 patients with squamous cell carcinoma of the uterine cervix, before and during fractionated radiotherapy. Twenty patients were treated with radiotherapy (RT) alone, while 19 received the same radiotherapy plus concomitant chemotherapy with cisplatin (CRT). Changes in gene expression induced by treatment were investigated using single-color oligo-microarrays consisting of 44K human sequences. Paraffin-embedded samples were used to examine apoptosis and the expression of protein by treatment-responsive genes. Changes in mRNA expression were assessed for these genes by real-time reverse transcriptase-polymerase chain reaction. Aberrant genomic change (detected using microarray-based comparative genomic hybridization), human papillomavirus infection, and p53 status were also evaluated. Results: The expression of CDKN1A, BAX, TNFSF8, and RRM2B was consistently upregulated by CRT (9 Gy with a single administration of cisplatin). Similar expression changes were induced by RT (9 Gy) alone, although the variability between tumors was greater. Apoptotic cells were significantly increased in both groups. CRT significantly increased the numbers of cases with diffusely distributed CDKN1A-positive cells. Genetic losses at 2q33-ter and gains of 3q26-ter were detected in the samples with high frequency; 60% were positive for human papillomavirus DNA; and three tumors had deletions/mutations of the p53 gene. There was no difference in the incidence of these genomic changes between the groups, and no association was found with the changes in expression of CDKN1A, BAX, TNFSF8 or RRM2B. Conclusions: Using biopsy samples from pretreatment and midtreatment cervical tumors, we identified therapy-induced genes related to the cell death signaling pathway. CRT produced a homogenous pattern of changes in expression of known radiation-responsive genes

TNF-mediated cell-death signaling pathway and extracellular matrix pathway are activated by concurrent use of cisplatin with radiotherapy in sequential biopsy specimens from patients with cervical cancer.

Objective: To identify changes in gene expression related to the concurrent use of platinum compounds with radiotherapy, in the treatment of cervical cancer.Patients and Methods: Biopsy specimens were obtained from 53 patients with the uterine carcinoma, before and during fractionated radiotherapy. Twenty-five patients were treated with radiotherapy (RT) alone, while 28 received the same radiotherapy plus concomitant chemotherapy with cisplatin (CRT). Changes in gene expression induced by treatment were investigated using CodeLink single-color oligo-microarrays consisting of 44K human sequences. Paraffin-embedded samples were used to examine apoptosis and the expression of protein related with treatment-responsive genes. Changes in mRNA expression were assessed for these genes by real-time reverse transcriptase-polymerase chain reaction. Results: We found several tens of genes, including CDKN1A, BAX, TNFSF8, RRM2B, and FGF2, responded to CRT using microarray analysis, and suggested CRT activated TNF-mediated cell death pathway and extra-cellular matrix pathway related with cell death pathway. Apoptotic cells were significantly increased in both groups. In immunohistochemical study, CRT significantly increased the numbers of cases with diffusely distributed CDKN1A-positive cells (P < 0.002). Protein expression of ICAD was recognized weakly in nucleus of tumor cells in pre-treatment samples and decreased significantly after CRT (P < 0.05). BAK1 increased its intensity in half of cases at mid-treatment. The expression changes of FGF2 were revealed to be significantly different between good responders and poor responders (P < 0.05). Conclusions: CRT produced a homogenous pattern of changes in expression of known radiation-responsive genes. Our data suggest that concurrent use of cisplatin produced a radiosensitizing effect in most of these cervical cancer patients. Microarray analysis appeared a useful tool to get a comprehensive overview of the changes in gene expression after irradiation. In addition, transcriptional profiling of sequential biopsies after irradiation would improve our understanding of the biological effectiveness of radiotherapy, and provide information on potential targets for adjuvant therapy.The 13th International Congress of Radiation Researc