Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, of which only ϳ30% become stably engrafted. Rapid and adequate revascularization of transplanted islets is important for islet survival and function. Delayed and insufficient revascularization can deprive islets of oxygen and nutrients, resulting in islet cell death and early graft failure. To improve islet revascularization, we delivered human vascular endothelial growth factor (VEGF) cDNA to murine islets, followed by transplantation under the renal capsule in diabetic mice. Diabetic animals receiving a marginal mass of 300 islets that were pretransduced with a VEGF vector exhibited near normoglycemia. In contrast, diabetic mice receiving an equivalent number of islets that were transduced with a control vector remained hyperglycemic. Immunohistochemistry with anti-insulin and anti-CD31 antibodies revealed a relatively higher insulin content and greater degree of microvasculature in the VEGF vector-transduced islet grafts, which correlated with significantly improved blood glucose profiles and enhanced insulin secretion in response to glucose challenge in this group of diabetic recipient mice. These results demonstrate that VEGF production in islets stimulates graft angiogenesis and enhances islet revascularization. This mechanism might be explored as a novel strategy to accelerate islet revascularization and improve longterm survival of functional islet mass posttransplantation. Diabetes 53: [963][964][965][966][967][968][969][970] 2004

Dietary advanced glycosylation end products (AGEs) have been linked to insulin resistance in db/db(؉؉) mice. To test whether dietary AGEs play a role in the progression of insulin resistance in normal mice fed high-fat diets, normal C57/BL6 mice were randomly assigned to high-fat diets (35% g fat), either high (HAGE-HF group; 995.4 units/mg AGE) or low (by 2.4-fold LAGE-HF group; 329.6 units/mg AGE) in AGE content for 6 months. Age-matched C57/BL6 and db/db (؉؉) mice fed regular diet (5% g fat, 117.4 units/mg AGE) served as controls. After 6 months, 75% of HAGE-HF mice were diabetic and exhibited higher body weight (P < 0.001), fasting glucose (P < 0.001), insulin (P < 0.001), and serum AGEs (P < 0.01) than control mice, while none of the LAGE-HF mice were diabetic despite a similar rise in body weight and plasma lipids. The HAGE-HF group displayed markedly impaired glucose and insulin responses during glucose tolerance tests and euglycemic and hyperglycemic clamps and altered pancreatic islet structure and function compared with those of LAGE-HF mice, in which findings resembled those of control mice. The HAGE-HF group had more visceral fat (by two-and fourfold) and more AGE-modified fat (by two-and fivefold) than LAGE-HF and control mice, respectively. In the HAGE-HF group, plasma 8-isoprostane was higher (P < 0.01) and adiponectin lower (P < 0.001) than control mice, while in the LAGE-HF group, these were more modestly affected (P < 0.05). These results demonstrate that the development of insulin resistance and type 2 diabetes during prolonged high-fat feeding are linked to the excess AGEs/advanced lipoxidation end products inherent in fatty diets. Diabetes 54: 2314 -2319, 2005

Mitochondria-mediated production of reactive oxygen species (ROS) plays a key role in apoptosis. Mitochondrial phospholipid cardiolipin molecules are likely the main target of ROS because they are particularly rich in polyunsaturated fatty acids. They are also located in the inner mitochondrial membrane near the ROS-producing sites. Under physiological conditions mitochondria can repair peroxidative damage in part through a remodeling mechanism via the deacylation-reacylation cycle

Mitochondrial production of reactive oxygen species and oxidation of cardiolipin are key events in initiating apoptosis. We reported that group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) localizes in and protects beta-cell mitochondria from oxidative damage during staurosporine-induced apoptosis. Here, we used iPLA(2)beta-null (iPLA(2)beta(-/-)) mice to investigate the role of iPLA(2)beta in the repair of mitochondrial membranes. We show that islets isolated from iPLA(2)beta(-/-) mice are more sensitive to staurosporine-induced apoptosis than those from wild-type littermates and that 2 wk of daily ip administration of staurosporine to iPLA(2)beta(-/-) mice impairs both the animals' glucose tolerance and glucose-stimulated insulin secretion by their pancreatic islets. Moreover, the iPLA(2)beta inhibitor bromoenol lactone caused mitochondrial membrane peroxidation and cytochrome c release, and these effects were reversed by N-acetyl cysteine. The mitochondrial antioxidant N-t-butyl hydroxylamine blocked staurosporine-induced cytochrome c release and caspase-3 activation in iPLA(2)beta(-/-) islets. Furthermore, the collapse of mitochondrial membrane potential in INS-1 insulinoma cells caused by high glucose and fatty acid levels was attenuated by overexpressing iPLA(2)beta. Interestingly, iPLA(2)beta was expressed only at low levels in islet beta-cells from obesity- and diabetes-prone db/db mice. These findings support the hypothesis that iPLA(2)beta is important in repairing oxidized mitochondrial membrane components (e.g. cardiolipin) and that this prevents cytochrome c release in response to stimuli that otherwise induce apoptosis. The low iPLA(2)beta expression level in db/db mouse beta-cells may render them vulnerable to injury by reactive oxygen species.

Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a "1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.

Islet Ca2+-independent phospholipase A2 (iPLA2) is postulated to mediate insulin secretion by releasing arachidonic acid in response to insulin secretagogues. However, the significance of iPLA2 signaling in insulin secretion in vivo remains unexplored. Here we investigated the physiological role of iPLA2 in beta-cell lines, isolated islets, and mice. We showed that small interfering RNA-specific silencing of iPLA2 expression in INS-1 cells significantly reduced insulin-secretory responses of INS-1 cells to glucose. Immunohistochemical analysis revealed that mouse islet cells expressed significantly higher levels of iPLA2 than pancreatic exocrine acinar cells. Bromoenol lactone (BEL), a selective inhibitor of iPLA2, inhibited glucose-stimulated insulin secretion from isolated mouse islets; this inhibition was overcome by exogenous arachidonic acid. We also showed that iv BEL administration to mice resulted in sustained hyperglycemia and reduced insulin levels during glucose tolerance tests. Clamp experiments demonstrated that the impaired glucose tolerance was due to insufficient insulin secretion rather than decreased insulin sensitivity. Short-term administration of BEL to mice had no effect on fasting glucose levels and caused no apparent pathological changes of islets in pancreas sections. These results unambiguously demonstrate that iPLA2 signaling plays an important role in glucose-stimulated insulin secretion under physiological conditions.

The G1 phase of the cell cycle is characterized by a high rate of membrane phospholipid turnover. Cells regulate this turnover by coordinating the opposing actions of CTP:phosphocholine cytidylyltransferase and the group VI Ca2+-independent phospholipase A2 (iPLA2). However, little is known about how such turnover affects cell-cycle progression. Here, we show that G1-phase phospholipid turnover is essential for cell proliferation. Specific inhibition of iPLA2 arrested cells in the G1 phase of the cell cycle. This G1-phase arrest was associated with marked upregulation of the tumour suppressor p53 and the expression of cyclin-dependent kinase inhibitor p21cip1. Inactivation of iPLA2 failed to arrest p53-deficient HCT cells in the G1 phase and caused massive apoptosis of p21-deficient HCT cells, suggesting that this G1-phase arrest requires activation of p53 and expression of p21cip1. Furthermore, downregulation of p53 by siRNA in p21-deficient HCT cells reduced the cell death, indicating that inhibition of iPLA2 induced p53-dependent apoptosis in the absence of p21cip1. Thus, our study reveals hitherto unrecognized cooperation between p53 and iPLA2 to monitor membrane-phospholipid turnover in G1 phase. Disrupting the G1-phase phospholipid turnover by inhibition of iPLA2 activates the p53-p21cip1 checkpoint mechanism, thereby blocking the entry of G1-phase cells into S phase.

We determined the nucleotide and deduced amino acid sequence of the 5' terminus of the hepatitis G virus (HGV) genome from isolates of varied geographical origins. Our analysis showed that the putative 5' non-coding region (NCR) contains several blocks of highly conserved sequences that may be useful for the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of HGV RNA. Overall, the degree of conservation within the 669-nucleotide (nt) 5'terminal sequence was found to range from 99.5% to 86% sequence identity. We also showed that the HGV NCR from some isolates contained conserved insertions or deletions that altered the translational reading frames at the 5'-end of the genome, resulting in different sizes of predicted polyproteins encoded by genomes of individual isolates. Specifically, the insertions/deletions affected the size of the peptide preceding the putative first envelope (E1) protein. Phylogenetic analysis of the nucleotide sequences suggested that the isolates examined can be classified into distinct groups that may be useful for studying the molecular evolution of HGV and possible relationships between isolate sequence characteristics and infection patterns.