We have characterized a phosphoserine binding domain in the coactivator CREB-binding protein (CBP) which interacts with kinase A-phosphorylated, and hence activated, form of cyclic AMP-responsive factor CREB. The CREB domain, referred to as KIX, is alpha helical binds an unstructured kinase-inducible following phosphorylation at Ser-133. Phospho-Ser-133 forms direct contacts residues these are further stabilized by hydrophobic flank phospho-Ser-133. Like src homology 2 (SH2) domains bind...

Background Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus extensive study. DNA sequences that regulate transcription majority regulatory factors have already identified. However, only recently other components investigated, in this study we examine role methylation regulation mouse human expression. Methodology/Principal Findings Genomic samples from several tissues were bisulfite-treated sequenced which revealed...

The endocrine pancreas consists of several differentiated cell types that are distinguished by their selective expression peptide hormones such as insulin, glucagon, and somatostatin. Although a number homeobox-type factors have been proposed key regulators individual genes in the pancreas, cellular distribution relative abundance remain uncharacterized. Also, overlapping DNA binding specificities further obscured regulatory functions these perform during development. In this report we...

Bone marrow-derived mesenchymal stem cells (MSCs) are known to produce vascular endothelial growth factor. We hypothesize that co-transplantation of MSCs and islets promotes revascularization improves islet graft function.Lewis rat were infused into the liver streptozotocin-diabetic syngeneic recipients or transplanted under renal capsule nonobese diabetic severe combined immunodeficiency (NOD SCID) mice with isolated from Lewis bone marrow expanded in culture.Co-transplantation 500 107...

cAMP regulates the expression of a number genes through protein kinase A-mediated phosphorylation CREB at Ser-133. The effects Ser-133 appear to be primarily transmitted modulatory kinase-inducible domain in that functions cooperatively with 120-amino acid glutamine-rich region (Q2) stimulate transcription. Indeed, activity alone is not sufficient sustain transcriptional response as illustrated by CREM family repressors, which contain but lack Q2 region. Here we demonstrate potent...

The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction the majority pancreatic insulin-producing beta cells. We previously demonstrated that DNA encoding insulin gene is uniquely unmethylated these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating cell streptozotocin-treated mice prior rise blood glucose. current study extends non-obese diabetic (NOD) humans, showing NOD death six weeks before sugar coincides...

AbstractThe homeobox protein STF-1 appears to function as a master control switch for expression of the pancreatic program during development. Here we characterize composite enhancer which directs islet cells via two functional elements that recognize nuclear factors HNF-3β and BETA-2. In keeping with their inhibitory effects on cell maturation, glucocorticoids were found repress gene by interfering activity islet-specific enhancer. Overexpression suppressed glucocorticoid receptor-mediated...

Standardized assessment of islet quality is imperative for clinical transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (∆OCRglc) can predict vivo efficacy transplantation mice. To further evaluate approach, we studied three factors: specificity, composition and agreement between results obtained different groups. Equivalent perifusion systems were set up at City Hope University Washington values ∆OCRglc both institutions compared....

Label-free optical detection of insulin would allow in vitro assessment pancreatic cell functions their natural state and expedite diabetes-related clinical research treatment; however, no existing method has met these criteria at physiological concentrations. Using spatially uniform 3D gold-nanoparticle sensors, we have demonstrated surface-enhanced Raman sensing the secretions from human islets under low high glucose environments without use labels such as antibodies or aptamers....

Epidemiological studies support strong links between obesity, diabetes, and pancreatic disorders including pancreatitis adenocarcinoma (PDAC). Type 2 diabetes (T2DM) is associated with insulin resistance, hyperglycemia, hyperinsulinemia, the latter due to increased secretion by beta-cells. We reported that high-fat diet-induced PDAC progression in mice activation of stellate cells (PaSC). investigated here effects high concentrations glucose on mouse human PaSC growth fibrosing responses....

Silymarin is a polyphenolic flavonoid that has strong antioxidant activity and exhibits anticarcinogenic, antiinflammatory, cytoprotective effects. Although its hepatoprotective effect been well documented, the of silymarin on pancreatic beta-cells largely unknown. In this study, IL-1beta and/or interferon (IFN)-gamma-induced beta-cell damage was investigated using RINm5F cells human islets. IFN-gamma induced cell death in time-dependent manner cells. The increase cytokine-induced appeared...

DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of methylation. We previously reported that the mouse insulin-2 (Ins2) promoter has three potential (CpG) sites, all are unmethylated in insulin-producing but methylated other tissues. In this study we examined Ins2 exon 2 found similar tissue-specific pattern. These can differentiate between from beta hypothesized damaged release their into circulation at onset type 1...

Objectives: Islet transplantation is hampered by the shortage of donor tissues. Our objective was to generate islet-like cell clusters (ICCs) from cultures non-islet pancreatic cells. Methods: The starting cultured cells came fractions human pancreases after enzymatic digestion and purification for purpose islet isolation. Initially, these expanded in monolayer became confluent on collagen-coated flasks. After trypsination suspension a defined differentiation medium, aggregated form ICCs....

We evaluated three commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well quality post-isolation.Retrospectively compared and analyzed isolations from pancreata using different enzyme groups: Liberase HI (n=63), Collagenase NB1/Neutral Protease (NP) (n=43), Mammalian Tissue Free Collagenase/Thermolysin (MTF C/T) (n=115). A standardized purification method was used. Islet assessment carried out count, viability, in vitro...

The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury several organs used for transplantation and also play a significant role in primary islet graft nonfunction. Activation MAPK may occur during cryopreservation thawing. In this study, inhibitor (p38IH) was applied human improve yield quality after Under serum-free conditions, islets were cryopreserved, thawed cultured using our standard procedures. Three types solutions tested:...

Background: Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus extensive study.DNA sequences that regulate transcription majority regulatory factors have already identified.However, only recently other components investigated, in this study we examine role DNA methylation regulation mouse human expression.Methodology/Principal Findings: Genomic samples from several tissues were bisulfite-treated sequenced which revealed...

The aim of this study was to investigate the effects elevated donor HbA1c levels (type 2 diabetes, T2D) on islet yield and functionality postisolation. In retrospective analysis, donors for isolations were classified into two groups: T2D group (HbA1c ≥ 6.5%, n = 18) normal < 308). Optimum pancreas digestion time (switch time) significantly higher in compared (13.7 ± 1.2 vs. 11.7 0.1 min, respectively, p 0.005). Islet yields lower control (T2D control): equivalent (IEQ)/g (prepurification...

Autoimmune diseases such as type 1 diabetes (T1D) involve the loss of regulatory mechanisms resulting in increased tissue-specific cytotoxicity. The result is destruction pancreatic insulin-producing β-cells and glucose homeostasis. We are developing a novel oral vaccine using live attenuated Salmonella to deliver TGFβ, IL10, diabetic autoantigen preproinsulin combined with low-doses anti-CD3 mAb. Here we show that administration Salmonella-based mAb therapy reverses new-onset T1D non-obese...

Background. The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury donor organs. islet isolation process, from pancreas procurement through collection, may activate p38MAPK leading to cytokine release and damage. This damage be prevented by treating pancreata with a inhibitor (p38IH) before preservation. Methods. Pancreata removed Beagle dogs were infused University Wisconsin solution containing the p38IH, SB203580, Pefabloc (n=6) or...

Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes transplantation therapy. Parameters such as percent β-cell apoptosis and composition are found vary markedly between different preparations may serve markers of quality. We have developed fluorescence-based assays using laser scanning cytometry on serial sections intact isolated islets.Isolated islets were fixed in formalin embedded paraffin. Serial...