Hyperglycemia Increases the Production of Amyloid Beta-Peptide Leading to Decreased Endothelial Tight Junction.

AIMS: Amyloid beta-peptide (Aß), the main component of senile plaques in the Alzheimer's disease (AD) brains, is generated from sequential cleavage of amyloid precursor protein (APP) by ß- and γ-secretase. Hyperglycemia in diabetes may compromise barrier integrity in endothelial cells (ECs). However, the roles of endothelial APP in response to high glucose (HG) remain to be delineated. The aims of this study were to test whether HG may increase Aß secretion, thereby leading to heightened paracellular permeability in ECs. METHODS: We determined the effects of HG on production of Aß, expression of full-length APP, intercellular permeability, and expression levels of specific junctional proteins in human umbilical vein endothelial cells (HUVECs). RESULTS: HG at 30 mM significantly stimulated expression of full-length APP accompanied by heightened secretion of Aß1-42, increased paracellular permeability, and attenuated expression of zona occluden-1 (ZO-1), claudin-5, occludin, and junctional adhesion molecule (JAM)-C in HUVECs; all of which were abolished by the γ-secretase inhibitor BMS299897. Exogenous application of Aß1-42, but not the reverse peptide Aß42-1, was sufficient to downregulate the expression of the same junction proteins. CONCLUSION: Hyperglycemia enhances APP expression with increased Aß production, which downregulates junctional proteins causing increased intercellular permeability in ECs.

Engineered xylose utilization enhances bio-products productivity in the cyanobacterium Synechocystis sp. PCC 6803.

Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO2 fixation.

Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria.

Central carbon metabolism in cyanobacteria comprises the Calvin-Benson-Bassham (CBB) cycle, glycolysis, the pentose phosphate (PP) pathway and the tricarboxylic acid (TCA) cycle. Redundancy in this complex metabolic network renders the rational engineering of cyanobacterial metabolism for the generation of biomass, biofuels and chemicals a challenge. Here we report the presence of a functional phosphoketolase pathway, which splits xylulose-5-phosphate (or fructose-6-phosphate) to acetate precursor acetyl phosphate, in an engineered strain of the model cyanobacterium Synechocystis (ΔglgC/xylAB), in which glycogen synthesis is blocked, and xylose catabolism enabled through the introduction of xylose isomerase and xylulokinase. We show that this mutant strain is able to metabolise xylose to acetate on nitrogen starvation. To see whether acetate production in the mutant is linked to the activity of phosphoketolase, we disrupted a putative phosphoketolase gene (slr0453) in the ΔglgC/xylAB strain, and monitored metabolic flux using (13)C labelling; acetate and 2-oxoglutarate production was reduced in the light. A metabolic flux analysis, based on isotopic data, suggests that the phosphoketolase pathway metabolises over 30% of the carbon consumed by ΔglgC/xylAB during photomixotrophic growth on xylose and CO2. Disruption of the putative phosphoketolase gene in wild-type Synechocystis also led to a deficiency in acetate production in the dark, indicative of a contribution of the phosphoketolase pathway to heterotrophic metabolism. We suggest that the phosphoketolase pathway, previously uncharacterized in photosynthetic organisms, confers flexibility in energy and carbon metabolism in cyanobacteria, and could be exploited to increase the efficiency of cyanobacterial carbon metabolism and photosynthetic productivity.

Utilizing liver-specific microRNA-122 to modulate replication of dengue virus replicon.

microRNAs (miRNAs) are endogenous non-coding RNAs that spatiotemporally modulate mRNAs in a post-transcriptional manner. The engineering of viruses by insertion of a tissue-specific miRNA recognition element (MRE) into viral mRNA can restrict viral tissue tropism. In this study we employed dengue virus (DEN) replicons to investigate whether miRNAs are able to suppress flavivirus replication through the targeting of non-polyadenylated viral mRNA. Because liver infection by DEN may contribute to the virus pathogenesis, we inserted an MRE of hepatic-specific microRNA-122 (miR-122) into its 3'-untranslated region (3'-UTR) to test the feasibility of creating a liver-restricted DEN replicon. Our results demonstrate that incorporation of the miR-122-MRE confers upon the DEN replicon an inhibitory susceptibility to miR-122 targeting, suggesting that DEN can be engineered to exert the desired replication restriction effect to avoid infection of vital tissues/organs. This approach provides an additional layer of biosafety and thus has great potential for use in the rational development of safer flavivirus vaccines.

Molecular pathogenesis of Gilbert's syndrome: decreased TATA-binding protein binding affinity of UGT1A1 gene promoter.

OBJECTIVES: Gilbert's syndrome is a congenital, nonhemolytic, unconjugated hyperbilirubinemia. The most common genotype of Gilbert's syndrome is the homozygous polymorphism, A(TA)7TAA, in the promoter of the gene for UDP-glucuronosyltransferase 1A1 (UGT1A1), with a thymine adenine insertion in the TATA-box-like sequence, which results in a decrease in UGT1A1 activity. The mechanism responsible for this decrease in UGT1A1 activity, however, has not been elucidated. To clarify the mechanism underlying this deficiency in UGT1A1 activity in patients with Gilbert's syndrome. METHODS: The promoter activity assay using the wild-type A(TA)6TAA or the mutant A(TA)7TAA promoter and a luciferase reporter was performed in two different hepatoma cell lines. The binding affinity for a nuclear protein complex or for TATA-binding protein was evaluated by a competitive electophoretic mobility shift assay using wild-type or mutant TATA-box-like oligonucleotide probes and nuclear extract or TATA-binding protein. The formation of complexes between TATA-binding protein and wild-type or mutant oligonucleotide probes was also studied by a quantitive electophoretic mobility shift assay. RESULTS: A TA insertion in the TATA-box-like sequence of the promoter activity of UGT1A1 gene. A competitive electrophoretic mobility shift assay showed a decrease in nuclear protein complex binding affinity and TATA-binding protein binding affinity of the mutant TATA-box-like sequence A(TA)7TAA. When the mutants A(TA)5TAA and A(TA)8TAA were also compared, quantitative electrophoretic mobility shift assay demonstrated that the TATA-binding protein binding affinity progressively decreased as the number of TA repeats in the TATA-box-like sequence increased. CONCLUSION: TA insertion in the TATA-box-like sequence of the UGT1A1 promoter affected its binding affinity for TATA-binding protein, causing a decrease in its activity. This explains the pathogenesis of Gilbert's syndrome.

The prevalence and risk factors analysis of serum antibody to hepatitis C virus in the elders in northeast Taiwan.

BACKGROUND: Hepatitis C virus (HCV) infection will result in liver cirrhosis and hepatocellular carcinoma, which are the leading causes of death in Taiwan. The prevalence of antibody to HCV (anti-HCV) was 2%-3% in Taipei city. However, it can be as high as 20% to 60% in Central and Southern part of Taiwan. In I-Lan, a county located in northern-east Taiwan, there is no large-scale investigation yet. The objective of this research is to evaluate the prevelance and risk factors of anti-HCV positivity in three towns of I-Lan county. METHODS: Blood sampled from people in San-Shing, Tou-Cheng and Tong-Shan was collected from October 1999 to June 2000. Totally, 1,316 persons (607 male, 790 female, mean age: 62 +/- 12 years old) were enrolled. Anti-HCV was measured by a second-generation enzyme immunoassay. Risk factors analysis was performed in anti-HCV positive subjects and age-sex matched anti-HCV negative controls. RESULTS: Sixty-seven persons (5.1%) had positive serum anti-HCV. The prevalence rate of anti-HCV increased with age. San-Shing and Tong-Shan showed a higher anti-HCV prevalence rate than Tou-Cheng (6.0% and 8.3% vs. 3.2%, p = 0.007). Risk factors analysis showed that people with positive serum anti-HCV had a significantly high rate of surgical history, usage of nondisposable needles injection, frequent nondisposable needles injection, frequent dental therapy, and a lower level of education than the anti-HCV negative counterpart (p < 0.05). Multivariate logistic regression analysis revealed that age > 60 years, surgical history and frequent nondisposable needles injection were significantly independent risk factors of positive anti-HCV (p < 0.05). CONCLUSIONS: The prevalence rate of anti-HCV in I-Lan county was 5.1%. Age > 60 years, surgical history and frequent nondisposable needles injection were the significant risk factors of HCV infection in I-Lan area.