Gad65 is recognized by t-cells, but not by antibodies from nod-mice

Since the 64kDa-protein glutamic acid decarboxylase (GAD) is one of the major autoantigens in T-cell mediated Type 1 diabetes, its relevance as a T-cell antigen needs to be clarified. After isolation of splenic T-cells from non-obese diabetic (NOD) mice, a useful model for human Type 1 diabetes, we found that these T-cells proliferate spontaneously when incubated with human GAD65, but only marginally after incubation with GAD67, both recombinated in the baculovirus system. No effect was observed with non-diabetic NOD mice or with T-cells from H-2 identical NON-NOD-H-2g7 control mice. It has been published previously that NOD mice develop autoantibodies against a 64kDa protein detected with mouse beta cells. In immunoprecipitation experiments with sera from the same NOD mice and 33S-methionine-labelled GAD, no autoantibody binding could be detected. We conclude firstly that GAD65 is an important T-cell antigen which is relevant early in the development of Type 1 diabetes and secondly that there is an antigenic epitope in the human GAD65 molecule recognized by NOD T-cells, but not by NOD autoantibodies precipitating conformational epitopes. Our results therefore provide further evidence that GAD65 is a T-cell antigen in NOD mice, being possibly also involved in very early processes leading to the development of human Type 1 diabetes

A Role for von Hippel-Lindau Protein in Pancreatic β-Cell Function

OBJECTIVE—The Vhlh gene codes for the von Hippel-Lindau protein (VHL), a tumor suppressor that is a key player in the cellular response to oxygen sensing. In humans, a germline mutation in the VHL gene leads to the von Hippel-Lindau disease, a familial syndrome characterized by benign and malignant tumors of the kidney, central nervous system, and pancreas

Direct Binding of pRb/E2F-2 to GATA-1 Regulates Maturation and Terminal Cell Division during Erythropoiesis

Cell differentiation is often coupled with cell cycle arrest. Here, we show that direct binding of the erythroid transcription factor GATA-1 to the retinoblastoma protein and the pRb/E2F transcription factor complex is critical for red blood cell formation

An siRNA Screen in Pancreatic Beta Cells Reveals a Role for Gpr27 in Insulin Production

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production

Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator

INTRODUCTION: Activated leukocyte cell adhesion molecule (ALCAM) (CD166) is an immunoglobulin molecule that has been implicated in cell migration. The present study examined the expression of ALCAM in human breast cancer and assessed its prognostic value. METHODS: The immunohistochemical distribution and location of ALCAM was assessed in normal breast tissue and carcinoma. The levels of ALCAM transcripts in frozen tissue (normal breast, n = 32; breast cancer, n = 120) were determined using real-time quantitative PCR. The results were then analyzed in relation to clinical data including the tumor type, the grade, the nodal involvement, distant metastases, the tumor, node, metastasis (TNM) stage, the Nottingham Prognostic Index (NPI), and survival over a 6-year follow-up period. RESULTS: Immunohistochemical staining on tissue sections in ducts/acini in normal breast and in breast carcinoma was ALCAM-positive. Differences in the number of ALCAM transcripts were found in different types of breast cancer. The level of ALCAM transcripts was lower (P = 0.05) in tumors from patients who had metastases to regional lymph nodes compared with those patients without, in higher grade tumors compared with Grade 1 tumors (P < 0.01), and in TNM Stage 3 tumors compared with TNM Stage 1 tumors (P < 0.01). Tumors from patients with poor prognosis (with NPI > 5.4) had significantly lower levels (P = 0.014) of ALCAM transcripts compared with patients with good prognosis (with NPI < 3.4), and tumors from patients with local recurrence had significantly lower levels than those patients without local recurrence or metastases (P = 0.04). Notably, tumors from patients who died of breast cancer had significantly lower levels of ALCAM transcripts (P = 0.0041) than those with primary tumors but no metastatic disease or local recurrence. Patients with low levels of ALCAM transcripts had significantly (P = 0.009) more incidents (metastasis, recurrence, death) compared with patients with primary breast tumors with high levels of ALCAM transcripts. CONCLUSIONS: In the present panel of breast cancer specimens, decreased levels of ALCAM correlated with the nodal involvement, the grade, the TNM stage, the NPI, and the clinical outcome (local recurrence and death). The data suggest that decreased ALCAM expression is of clinical significance in breast cancer, and that reduced expression indicates a more aggressive phenotype and poor prognosis

High Glucose Suppresses Human Islet Insulin Biosynthesis by Inducing miR-133a Leading to Decreased Polypyrimidine Tract Binding Protein-Expression

BACKGROUND: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates. METHODOLOGY/PRINCIPAL FINDINGS: Human islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose. CONCLUSION: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction

ALCAM Regulates Motility, Invasiveness, and Adherens Junction Formation in Uveal Melanoma Cells

ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM’s role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves

Erythroid-Specific Transcriptional Changes in PBMCs from Pulmonary Hypertension Patients

Gene expression profiling of peripheral blood mononuclear cells (PBMCs) is a powerful tool for the identification of surrogate markers involved in disease processes. The hypothesis tested in this study was that chronic exposure of PBMCs to a hypertensive environment in remodeled pulmonary vessels would be reflected by specific transcriptional changes in these cells.The transcript profiles of PBMCs from 30 idiopathic pulmonary arterial hypertension patients (IPAH), 19 patients with systemic sclerosis without pulmonary hypertension (SSc), 42 scleroderma-associated pulmonary arterial hypertensio patients (SSc-PAH), and 8 patients with SSc complicated by interstitial lung disease and pulmonary hypertension (SSc-PH-ILD) were compared to the gene expression profiles of PBMCs from 41 healthy individuals. Multiple gene expression signatures were identified which could distinguish various disease groups from controls. One of these signatures, specific for erythrocyte maturation, is enriched specifically in patients with PH. This association was validated in multiple published datasets. The erythropoiesis signature was strongly correlated with hemodynamic measures of increasing disease severity in IPAH patients. No significant correlation of the same type was noted for SSc-PAH patients, this despite a clear signature enrichment within this group overall. These findings suggest an association of the erythropoiesis signature in PBMCs from patients with PH with a variable presentation among different subtypes of disease.In PH, the expansion of immature red blood cell precursors may constitute a response to the increasingly hypoxic conditions prevalent in this syndrome. A correlation of this erythrocyte signature with more severe hypertension cases may provide an important biomarker of disease progression

A Novel Pathogenicity Gene Is Required in the Rice Blast Fungus to Suppress the Basal Defenses of the Host

For successful colonization and further reproduction in host plants, pathogens need to overcome the innate defenses of the plant. We demonstrate that a novel pathogenicity gene, DES1, in Magnaporthe oryzae regulates counter-defenses against host basal resistance. The DES1 gene was identified by screening for pathogenicity-defective mutants in a T-DNA insertional mutant library. Bioinformatic analysis revealed that this gene encodes a serine-rich protein that has unknown biochemical properties, and its homologs are strictly conserved in filamentous Ascomycetes. Targeted gene deletion of DES1 had no apparent effect on developmental morphogenesis, including vegetative growth, conidial germination, appressorium formation, and appressorium-mediated penetration. Conidial size of the mutant became smaller than that of the wild type, but the mutant displayed no defects on cell wall integrity. The Δdes1 mutant was hypersensitive to exogenous oxidative stress and the activity and transcription level of extracellular enzymes including peroxidases and laccases were severely decreased in the mutant. In addition, ferrous ion leakage was observed in the Δdes1 mutant. In the interaction with a susceptible rice cultivar, rice cells inoculated with the Δdes1 mutant exhibited strong defense responses accompanied by brown granules in primary infected cells, the accumulation of reactive oxygen species (ROS), the generation of autofluorescent materials, and PR gene induction in neighboring tissues. The Δdes1 mutant displayed a significant reduction in infectious hyphal extension, which caused a decrease in pathogenicity. Notably, the suppression of ROS generation by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, resulted in a significant reduction in the defense responses in plant tissues challenged with the Δdes1 mutant. Furthermore, the Δdes1 mutant recovered its normal infectious growth in DPI-treated plant tissues. These results suggest that DES1 functions as a novel pathogenicity gene that regulates the activity of fungal proteins, compromising ROS-mediated plant defense