OmpR (TCS response regulator) of Aeromonas veronii plays a major role in drug resistance, stress resistance and virulence by regulating biofilm formation.

Aeromonas veronii (A. veronii), a highly pathogenic bacteria with a wide range of hosts, widely exists in the environment of humans, animals and aquatic animals, and can cause a variety of diseases. In this study, the receptor regulator ompR in the envZ/ompR of two-component system was selected to construct a mutant strain (Δ ompR) and a complement strain (C-ompR) to explore the regulatory effect of ompR on the biological characteristics and virulence of TH0426. The results showed that the ability of biofilm formation and osmotic stress of TH0426 were significantly reduced (P < 0.001), the resistance to ceftriaxone and neomycin were slightly down-regulate when the ompR gene was deleted. At the same time, animal pathogenicity experiments showed that the virulence of TH0426 was significantly down-regulated (P < 0.001). These results indicated that ompR gene regulates the biofilm formation of TH0426, and regulates some biological characteristics of TH0426, including drug sensitivity, resistance to osmotic stress, and also affects its virulence.

Functional analysis of ascP in Aeromonas veronii TH0426 reveals a key role in the regulation of virulence.

Aeromonas veronii is a pathogen which can induce diseases in humans, animals and aquatic organisms, but its pathogenic mechanism and virulence factors are still elusive. In this study, we successfully constructed a mutant strain (ΔascP) by homologous recombination. The results showed that the deletion of the ascP gene significantly down-regulated the expression of associated effector proteins in A. veronii compared to its wild type. The adhesive and invasive abilities of ΔascP to EPC cells were 0.82-fold lower in contrast to the wild strain. The toxicity of ΔascP to cells was decreased by about 2.91-fold (1 h) and 1.74-fold (2 h). Furthermore, the LD50 of the mutant strain of crucian carp was reduced by 19.94-fold, and the virulence was considerably attenuated. In contrast to the wild strain, the ΔascP content in the liver and spleen was considerably lower. The titers of serum cytokines (IL-8, TNF-α, and IL-1ß) in crucian carp after the infection of the ΔascP strain were considerably lower in contrast to the wild strain. Hence, the ascP gene is essential for the etiopathogenesis of A. veronii TH0426.

The effects of dietary supplementation of ginseng stem and leaf saponins on the antioxidant capacity, immune response, and disease resistance of crucian carp, Carassius auratus.

This is the first study to explore the positive effects of ginseng stem and leaf saponins (GSLS) on antioxidant capability, immunity, and disease resistance of crucian carp. Seven hundred fifty crucian carps (initial body weight: 25 ± 0.15 g (mean ± SE)) were randomly allocated into five groups with three replicates each; five diets supplemented with the final concentration of 0, 1, 2, 4, and 8 g/kg GSLS were fed to crucian carp for 5 weeks. The results demonstrated that, at a concentration of 8 g/kg, the contents of IgM, C4, SOD, GSH-Px, and the activity of AKP in serum of crucian carp gradually increased at 7, 14, 21, 28, and 35 days, and the expression of immune-relative cytokine genes (TNF-α, IL-10, IFN-γ) in the liver, spleen, and the intestinal tract also had a significant up-regulation (P < 0.05), and which were significant difference compared with control (P < 0.05). The above results demonstrated that dietary GSLS showed enhancement effects on the antioxidant and anti-inflammatory capability, and innate immune response of crucian carp. The feed of 8 g/kg GSLS for 1 week could improve the survival rate 44% more than the control group when crucian carp infected Aeromonas hydrophila (A. hydrophila). In conclusion, the addition of GSLS at a concentration of 8 g/kg in the diet improve immune-related enzyme activity better, immune-relative cytokine expression, and disease resistance.

STEAP4 promoter methylation correlates with tumorigenesis of hepatocellular carcinoma.

BACKGROUND: The study was aimed to find promising targets for cancer therapy involved in the tumorigenesis of hepatocellular carcinoma (HCC). METHODS: Identification of STEAP4 in HCC between GSE54503 and TCGA datasets by performing RNA-seq. The STEAP4 mRNA expression level was determined by qRT-PCR. DNA methylation was measured by MSP and BSP. Besides, the effect of STEAP4 tumorigenesis was determined by in vivo experiments. The function of STEAP4 on methylation was further assessed by 5-Aza­dC, a demethylating agent. RESULTS: Reduced STEAP4 expression was found in HCC tissues. Promoter region methylation correlated with the downregulated expression of STEAP4. STEAP4 inhibited the proliferation and metastasis of HCC cells. Re-expression of STEAP4 was induced 5-Aza­dC. STEAP4 mediated the biological effects of HCC cells through PI3K/AKT/mTOR pathway inhibition. CONCLUSIONS: Our findings indicate that STEAP4 functions as a suppressor gene in HCC, and hypermethylation is a driving factor in cancer progression.

The Influence of Spiritual Leadership on Harmonious Passion: A Case Study of the Hotel Industry in China.

The hotel manager has the responsibility to stimulate the passion of the staff. The vision, hope/faith, and altruistic love advocated by spiritual leaders can meet the independent psychological needs of employees, thus enhancing their harmonious passion. This study is based on self-determination theory, intrinsic motivation theory and psychological capital theory, and explores the relationship between spiritual leadership and employees' harmonious passion. This study uses 260 employees of star hotels in Beijing, Shanghai, Hangzhou, Hefei, Huangshan, and other cities in China. Results show that spiritual leadership positively impacts employees' harmonious passion, and calling plays an mediation role between spiritual leadership and employees' harmonious passion. The results are helpful to clarify the formation mechanism of employees' harmonious passion from the perspective of self-determination theory, intrinsic motivation theory and psychological capital theory and show that spiritual leadership can drive employees' harmonious passion, especially when hotel vision and employee calling are consistent. Furthermore, the altruistic love of spiritual leaders for their followers also plays a key role in employee calling and promoting harmonious passion. Therefore, this study also emphasizes the importance of calling in improving the harmonious passion of employees. The theoretical and management implications that help to enhance the harmonious passion of employees are discussed in detail.

Role Stress and Prosocial Service Behavior of Hotel Employees: A Moderated Mediation Model of Job Satisfaction and Social Support.

Hotel employees' positive behavior is prone to increase customer satisfaction, and thus, exploring the influencing mechanism of role stress on prosocial service behavior is critical to relieving their stress and improving service quality and hotel performance. This study aims to develop and test a moderated mediation model that links hotel employees' role stress to prosocial service behavior. Based on the conservation of resources theory and job demands-resources model, this study suggests that the effect of role stress on prosocial service behavior is mediated by the level of job satisfaction, whereas the relationship between role stress and job satisfaction is moderated by social support. Data from 256 hotel employees in China largely support the hypotheses that role stress reduces job satisfaction, and that job dissatisfaction is related to low levels of prosocial service behavior. The data also show that job satisfaction partly mediates the relationship between role stress and prosocial service behavior, and social support weakens the relationship between role stress and job satisfaction. The results can help us understand the role of organization-level resources in the workplace and how role stress and job satisfaction affect prosocial service behavior.

A Comprehensive Review on Edge Caching from the Perspective of Total Process: Placement, Policy and Delivery.

With the explosive growth of smart devices and mobile applications, mobile core networks face the challenge of exponential growth in traffic and computing demand. Edge caching is one of the most promising solutions to the problem. The main purpose of edge caching is to place popular content that users need at the edge of the network, borrow free space to reduce user waiting time, and lighten the network load by reducing the amount of duplicate data. Due to the promising advantages of edge caching, there have been many efforts motivated by this topic. In this paper, we have done an extensive survey on the existing work from our own perspectives. Distinguished from the existing review articles, our work not only investigates the latest articles in this area, but more importantly, covers all the researches of the total process of edge caching from caching placement optimization, policy design, to the content delivery process. In particular, we discuss the benefits of caching placement optimization from the perspective of different stakeholders, detail the delivery process, and conduct in-depth discussions from the five phases, i.e., requested content analysis, user model analysis, content retrieval, delivery, and update. Finally, we put forward several challenges and potential future directions, and hope to bring some ideas for the follow-up researches in this area.

Laser-driven optothermal microactuator operated in water.

This paper proposes and studies the characteristics of a laser-driven optothermal microactuator (OTMA) directly operated in water. A theoretical model of optothermal temperature rise and expansion is established, and simulations on a 1000 µm long OTMA are conducted, revealing that its arm is able to expand and contract in response to the laser pulses in a water environment. Microactuating experiments are further carried out using a microfabricated OTMA. The results demonstrate that the OTMA can be practically actuated in water by a 650 nm laser beam and that the OTMA's deflection amplitude increases linearly with laser power. When irradiated by laser pulses with 9.9 mW power and 0.9-25.6 Hz frequencies, the OTMA achieves deflection amplitude ranging from 3.9 to 3.2 µm, respectively. The experimental results match well with theoretical model when taking the damping effect of water into account. This research may be conducive to developing particular micro-electromechanical systems or micro-optoelectromechanical devices such as underwater optothermal micromotors, micro-pumps, micro-robots, and other underwater microactuators.

Microscopic research on the properties of optothermal microactuators with different lever ratios.

This paper presents microscopic research on properties of asymmetric optothermal microactuator (OTMA) with different lever ratios. A theoretical model is established on the basis of thermal flux theorem to describe the increase in temperature induced by laser irradiation and thermal expansion of OTMAs' expansion arms. The increases in temperature of three asymmetric OTMAs with total lengths/lever ratios of 540 µm/7.2, 1,080 µm/7.2, 1,080 µm/14.4 were simulated under irradiation of 2.8 mW laser beam, which revealed that a similar increase in temperature will distribute on three expansion arms, with maximum the temperature increase of 82.73, 87.67, and 88.03°C, respectively. Due to these increases in temperature, the arms expand longitudinally and thus the OTMAs are capable of deflecting laterally with enlarged deflection amplitudes. To obtain optimized deflecting properties, three OTMAs with aforementioned lever ratios are further microfabricated and experimented using an optical microscopic observation and measuring system combined with a charge-coupled device. The experimental results show that these OTMAs can be directly actuated by laser beam and acquire maximum deflections of 6.8, 7.2, and 11.4 µm when only 2.8 mW laser power is employed. In addition, it is demonstrated that, although doubling the lever ratio of OTMA does not correspondingly generate twofold deflection, the deflection amplitude can significantly increase with the increase in the lever ratio. With their characteristic elegances, the OTMAs are expected to be practically applied in the micro-electromechanical system and the micro-opto-electromechanical system.

WiFi-controlled portable atomic force microscope.

This article proposes to develop a WiFi-controlled portable atomic force microscope (AFM). The AFM consists of a horizontal probe, controlling circuits, digital to analog (D/A) and analog to digital (A/D) interfaces, a microcomputer (Raspberry Pi, RPi), and a laptop. The proposed AFM uses a pocket-size power supply to drive the controlling circuits, the D/A and A/D interfaces, as well as the RPi that constructs network hotspots and generates scanning signals. With special design and integration of the whole system, both of the AFM probe and electronic controlling system are portable. At a distance of 50 m from the proposed AFM, experiments in the constant height mode and the constant force mode are conducted to evaluate its performance. The results show that this WiFi-controlled AFM has a maximum scan range of 3.6 × 3.6 µm2 with nanometer order resolution. Meanwhile, it achieves satisfactory image contrast, stability, and repeatability. Compared with conventional AFMs, the AFM proposed in this paper no longer relies on commercial AC mains supply or high-voltage DC power supply, and realizes WiFi-controlled AFM scanning and imaging in 50 m or farther without wire or network cable connection to a laptop or a desktop computer. Given credits to these features, WiFi-controlled AFMs are expected to own a wider range of application, especially in isolated environments, outdoor researches, or even fieldwork investigations.

Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

G6PC2: a negative regulator of basal glucose-stimulated insulin secretion.

Elevated fasting blood glucose (FBG) is associated with increased risk for the development of type 2 diabetes and cardiovascular-associated mortality. Genome-wide association studies (GWAS) have linked polymorphisms in G6PC2 with variations in FBG and body fat, although not insulin sensitivity or glucose tolerance. G6PC2 encodes an islet-specific, endoplasmic reticulum-resident glucose-6-phosphatase catalytic subunit. A combination of in situ perfused pancreas, in vitro isolated islet, and in vivo analyses were used to explore the function of G6pc2 in mice. G6pc2 deletion had little effect on insulin sensitivity and glucose tolerance, whereas body fat was reduced in female G6pc2 knockout (KO) mice on both a chow and high-fat diet, observations that are all consistent with human GWAS data. G6pc2 deletion resulted in a leftward shift in the dose-response curve for glucose-stimulated insulin secretion (GSIS). As a consequence, under fasting conditions in which plasma insulin levels were identical, blood glucose levels were reduced in G6pc2 KO mice, again consistent with human GWAS data. Glucose-6-phosphatase activity was reduced, whereas basal cytoplasmic calcium levels were elevated in islets isolated from G6pc2 KO mice. These data suggest that G6pc2 represents a novel, negative regulator of basal GSIS that acts by hydrolyzing glucose-6-phosphate, thereby reducing glycolytic flux.

Deletion of the G6pc2 gene encoding the islet-specific glucose-6-phosphatase catalytic subunit-related protein does not affect the progression or incidence of type 1 diabetes in NOD/ShiLtJ mice.

OBJECTIVE: Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), now known as G6PC2, is a major target of autoreactive T cells implicated in the pathogenesis of type 1 diabetes in both mice and humans. This study aimed to determine whether suppression of G6p2 gene expression might therefore prevent or delay disease progression. RESEARCH DESIGN AND METHODS: G6pc2(-/-) mice were generated on the NOD/ShiLtJ genetic background, and glycemia was monitored weekly up to 35 weeks of age to determine the onset and incidence of diabetes. The antigen specificity of CD8(+) T cells infiltrating islets from NOD/ShiLtJ G6pc2(+/+) and G6pc2(-/-) mice at 12 weeks was determined in parallel. RESULTS: The absence of G6pc2 did not affect the time of onset, incidence, or sex bias of type 1 diabetes in NOD/ShiLtJ mice. Insulitis was prominent in both groups, but whereas NOD/ShiLtJ G6pc2(+/+) islets contained CD8(+) T cells reactive to the G6pc2 NRP peptide, G6pc2 NRP-reactive T cells were absent in NOD/ShiLtJ G6pc2(-/-) islets. CONCLUSIONS: These results demonstrate that G6pc2 is an important driver for the selection and expansion of islet-reactive CD8(+) T cells infiltrating NOD/ShiLtJ islets. However, autoreactivity to G6pc2 is not essential for the emergence of autoimmune diabetes. The results remain consistent with previous studies indicating that insulin may be the primary autoimmune target, at least in NOD/ShiLtJ mice.

Characterization of the human SLC30A8 promoter and intronic enhancer.

Genome-wide association studies have shown that a polymorphic variant in SLC30A8, which encodes zinc transporter-8, is associated with altered susceptibility to type 2 diabetes (T2D). This association is consistent with the observation that glucose-stimulated insulin secretion is decreased in islets isolated from Slc30a8 knockout mice. In this study, immunohistochemical staining was first used to show that SLC30A8 is expressed specifically in pancreatic islets. Fusion gene studies were then used to examine the molecular basis for the islet-specific expression of SLC30A8. The analysis of SLC30A8-luciferase expression in ßTC-3 cells revealed that the proximal promoter region, located between -6154 and -1, relative to the translation start site, was only active in stable but not transient transfections. VISTA analyses identified three regions in the SLC30A8 promoter and a region in SLC30A8 intron 2 that are conserved in the mouse Slc30a8 gene. Additional fusion gene experiments demonstrated that none of these Slc30a8 promoter regions exhibited enhancer activity when ligated to a heterologous promoter whereas the conserved region in SLC30A8 intron 2 conferred elevated reporter gene expression selectively in ßTC-3 but not in αTC-6 cells. Finally, the functional effects of a single nucleotide polymorphism (SNP), rs62510556, in this conserved intron 2 enhancer were investigated. Gel retardation studies showed that rs62510556 affects the binding of an unknown transcription factor and fusion gene analyses showed that it modulates enhancer activity. However, genetic analyses suggest that this SNP is not a causal variant that contributes to the association between SLC30A8 and T2D, at least in Europeans.

The pancreatic islet ß-cell-enriched transcription factor Pdx-1 regulates Slc30a8 gene transcription through an intronic enhancer.

The SLC30A8 gene encodes the zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Genome-wide association studies have shown that a polymorphic variant in SLC30A8 is associated with altered susceptibility to Type 2 diabetes and we recently reported that glucose-stimulated insulin secretion is decreased in islets isolated from Slc30a8-knockout mice. The present study examines the molecular basis for the islet-specific expression of Slc30a8. VISTA analyses identified two conserved regions in Slc30a8 introns 2 and 3, designated enhancers A and B respectively. Transfection experiments demonstrated that enhancer B confers elevated fusion gene expression in both ßTC-3 cells and αTC-6 cells. In contrast, enhancer A confers elevated fusion gene expression selectively in ßTC-3 and not αTC-6 cells. These data suggest that enhancer A is an islet ß-cell-specific enhancer and that the mechanisms controlling Slc30a8 expression in α- and ß-cells are overlapping, but distinct. Gel retardation and ChIP (chromatin immunoprecipitation) assays revealed that the islet-enriched transcription factor Pdx-1 binds enhancer A in vitro and in situ respectively. Mutation of two Pdx-1-binding sites in enhancer A markedly reduces fusion gene expression suggesting that this factor contributes to Slc30a8 expression in ß-cells, a conclusion consistent with developmental studies showing that restriction of Pdx-1 to pancreatic islet ß-cells correlates with the induction of Slc30a8 gene expression and ZnT-8 protein expression in vivo.

Deletion of the mouse Slc30a8 gene encoding zinc transporter-8 results in impaired insulin secretion.

The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid residue 325 of human ZnT-8 are associated with altered susceptibility to Type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in Type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analysed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioural changes or differences in body weight were observed between wild-type and ZnT-8-/- mice, and ZnT-8-/- mouse islets were indistinguishable from wild-type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8-/- mouse islets, as evaluated both by Timm's histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16-week-old, 6 h fasted animals of either gender; however, plasma insulin concentrations were reduced in both female (approximately 31%) and male (approximately 47%) ZnT-8-/- mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8-/- mice, but glucose-stimulated insulin secretion from isolated islets was reduced approximately 33% relative to wild-type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism.

Insulin and epidermal growth factor suppress basal glucose-6-phosphatase catalytic subunit gene transcription through overlapping but distinct mechanisms.

The G6Pase (glucose-6-phosphatase catalytic subunit) catalyses the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate to glucose. We show here that, in HepG2 hepatoma cells, EGF (epidermal growth factor) inhibits basal mouse G6Pase fusion gene transcription. Several studies have shown that insulin represses basal mouse G6Pase fusion gene transcription through FOXO1 (forkhead box O1), but Stoffel and colleagues have recently suggested that insulin can also regulate gene transcription through FOXA2 (forkhead box A2) [Wolfrum, Asilmaz, Luca, Friedman and Stoffel (2003) Proc. Natl. Acad. Sci. 100, 11624-11629]. A combined GR (glucocorticoid receptor)-FOXA2 binding site is located between -185 and -174 in the mouse G6Pase promoter overlapping two FOXO1 binding sites located between (-188 and -182) and (-174 and -168). Selective mutation of the FOXO1 binding sites reduced the effect of insulin, whereas mutation of the GR/FOXA2 binding site had no effect on the insulin response. In contrast, selective mutation of the FOXO1 and GR/FOXA2 binding sites both reduced the effect of EGF. The effect of these mutations was additive, since the combined mutation of both FOXO1 and GR/FOXA2 binding sites reduced the effect of EGF to a greater extent than the individual mutations. These results suggest that, in HepG2 cells, GR and/or FOXA2 are required for the inhibition of basal G6Pase gene transcription by EGF but not insulin. EGF also inhibits hepatic G6Pase gene expression in vivo, but in cultured hepatocytes EGF has the opposite effect of stimulating expression, an observation that may be explained by a switch in ErbB receptor sub-type expression following hepatocyte isolation.

Foxa2 and MafA regulate islet-specific glucose-6-phosphatase catalytic subunit-related protein gene expression.

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP/G6PC2) is a major autoantigen in both mouse and human type 1 diabetes. IGRP is selectively expressed in islet beta cells and polymorphisms in the IGRP gene have recently been associated with variations in fasting blood glucose levels and cardiovascular-associated mortality in humans. Chromatin immunoprecipitation (ChIP) assays have shown that the IGRP promoter binds the islet-enriched transcription factors Pax-6 and BETA2. We show here, again using ChIP assays, that the IGRP promoter also binds the islet-enriched transcription factors MafA and Foxa2. Single binding sites for these factors were identified in the proximal IGRP promoter, mutation of which resulted in decreased IGRP fusion gene expression in betaTC-3, Hamster insulinoma tumor (HIT), and Min6 cells. ChiP assays have shown that the islet-enriched transcription factor Pdx-1 also binds the IGRP promoter, but mutational analysis of four Pdx-1 binding sites in the proximal IGRP promoter revealed surprisingly little effect of Pdx-1 binding on IGRP fusion gene expression in betaTC-3 cells. In contrast, in both HIT and Min6 cells mutation of these four Pdx-1 binding sites resulted in a approximately 50% reduction in fusion gene expression. These data suggest that the same group of islet-enriched transcription factors, namely Pdx-1, Pax-6, MafA, BETA2, and Foxa2, directly or indirectly regulate expression of the two major autoantigens in type 1 diabetes.

Long-range enhancers are required to maintain expression of the autoantigen islet-specific glucose-6-phosphatase catalytic subunit-related protein in adult mouse islets in vivo.

OBJECTIVE: Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet beta-cells and is a major autoantigen in both mouse and human type 1 diabetes. This study describes the use of a combination of transgenic and transfection approaches to characterize the gene regions that confer the islet-specific expression of IGRP. RESEARCH DESIGN AND METHODS: Transgenic mice were generated containing the IGRP promoter sequence from -306, -911, or -3911 to +3 ligated to a LacZ reporter gene. Transgene expression was monitored by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of pancreatic tissue. RESULTS: In all the transgenic mice, robust LacZ expression was detected in newborn mouse islets, but expression became mosaic as animals aged, suggesting that additional elements are required for the maintenance of IGRP gene expression. VISTA analyses identified two conserved regions in the distal IGRP promoter and one in the third intron. Transfection experiments demonstrated that all three regions confer enhanced luciferase reporter gene expression in beta TC-3 cells when ligated to a minimal IGRP promoter. A transgene containing all three conserved regions was generated by using a bacterial recombination strategy to insert a LacZ cassette into exon 5 of the IGRP gene. Transgenic mice containing a 15-kbp fragment of the IGRP gene were then generated. This transgene conferred LacZ expression in newborn mouse islets; however, expression was still suppressed as animals aged. CONCLUSIONS: The data suggest that long-range enhancers 5' or 3' of the IGRP gene are required for the maintenance of IGRP gene expression in adult mice.

Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex.

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.