GTPase stimulation in shrimp Ras(Q(61)K) with geranylgeranyl pyrophosphate but not mammalian GAP.

BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2000). The GTPase-activating protein (GAP) in the cytosol fraction was significantly expressed and degraded, compared to untransformed cells on the western blot. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with GAP was investigated using GAP purified from mouse brains. SDS-polyacrylamide gel electrophoresis revealed the monomers of the purified GAP to have a relative mass of 65,000. Since the purified GAP was bound to the Ras conjugated affinity sepharose column with high affinity and its GTP hydolysis activity upon binding with tubulin was suppressed, the purified enzyme was concluded to be neurofibromin-like. The purified GAP enhanced the intrinsic GTPase activity of the S-Ras, to convert it into the inactive GDP-bound form, in agreement with findings for GTP-bound K(B)-Ras in vitro. To compare the effects between isoprenoids and GAP on the GTP-hydrolysis of Ras, we applied the GTP-locked shrimp mutant S-Ras(Q(61)K) and GTP-locked rat mutant K(B)-ras(Q(61)K). Radioassay studies showed that geranylgeranyl pyrophosphate at microg level catalyzed the GTP hydrolysis of S-Ras(Q(61)K) and K(B)-ras(Q(61)K) competently, but not farnesyl pyrophosphate or the purified GAP. The present study provides the view that the geranylgeranyl pyrophosphate at carboxyl terminal CAAX assists GTP hydrolysis to Ras proteins probably in a manner similar to the substrate assisted catalysis in GTPase mechanism.     

Search for the tumor-related proteins of transition cell carcinoma in Taiwan by proteomic analysis

To better understand the carcinogenesis of bladder cancer in Taiwan, we utilized the proteomic approach to search for potential biomarkers of transitional cell carcinoma (TCC). Analysis by 2-DE and MS/MS indicated that seven proteins are down-regulated and three proteins up-regulated in grade III samples as compared with those of grade II. Of these deregulated proteins, fatty acid binding proteins, annexin V, heat-shock protein 27, and lactate dehydrogenase have been shown to be associated with bladder cancer. Our studies also found altered expression of a group of proteins that have not been documented previously in bladder cancer, including annexin I, 15-hydroxyprostaglandin dehydrogenase, galectin-1, lysophospholipase and mitochondrial short-chain enoyl-coenzyme A hydratase 1 precursor. These results illustrate a pattern of differential protein expression between low- and high-grade tumors and it may be utilized as the molecular fingerprinting of a subset of bladder cancers. In addition, the present study provides a valuable resource in the study of pathological mechanisms in cancers of urothelial origin. The immunohistochemical staining of grade II and III TCC samples with antiserum to annexin I protein was utilized to confirm that the annexin I protein is up-regulated in grade III TCC.     

Remote Sensing of Climatic Anomalies and West Nile Virus Incidence in the Northern Great Plains of the United States

The northern Great Plains (NGP) of the United States has been a hotspot of West Nile virus (WNV) incidence since 2002. Mosquito ecology and the transmission of vector-borne disease are influenced by multiple environmental factors, and climatic variability is an important driver of inter-annual variation in WNV transmission risk. This study applied multiple environmental predictors including land surface temperature (LST), the normalized difference vegetation index (NDVI) and actual evapotranspiration (ETa) derived from Moderate-Resolution Imaging Spectroradiometer (MODIS) products to establish prediction models for WNV risk in the NGP. These environmental metrics are sensitive to seasonal and inter-annual fluctuations in temperature and precipitation, and are hypothesized to influence mosquito population dynamics and WNV transmission. Non-linear generalized additive models (GAMs) were used to evaluate the influences of deviations of cumulative LST, NDVI, and ETa on inter-annual variations of WNV incidence from 2004–2010. The models were sensitive to the timing of spring green up (measured with NDVI), temperature variability in early spring and summer (measured with LST), and moisture availability from late spring through early summer (measured with ETa), highlighting seasonal changes in the influences of climatic fluctuations on WNV transmission. Predictions based on these variables indicated a low WNV risk across the NGP in 2011, which is concordant with the low case reports in this year. Environmental monitoring using remote-sensed data can contribute to surveillance of WNV risk and prediction of future WNV outbreaks in space and time.     

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.     

The Impact of Hepatitis B Vaccination Status on the Risk of Diabetes,Implicating Diabetes Risk Reduction by Successful Vaccination

Background

The liver plays a key role in fuel metabolism. It is well established that liver disease is associated with an increased risk for diabetes mellitus. Hepatitis C virus infection has been known to increase the risk of diabetes. However, much less is known about the role of hepatitis B virus (HBV) infection in diabetes. We examined the association of diabetes based on the vaccination status for HBV.

Methods

In this cross-sectional study, we included adult subjects (≥20 y/o) with HBV serology available from the National Health and Nutrition Examination Survey 2005–2010. Diabetes was defined as established diabetes or fasting plasma glucose concentration ≥7.0 mmol/L, 2-hour plasma glucose concentration ≥11.1 mmol/L, or HbA1c ≥ 47.5 mmol/mol (6.5%). Vaccination was based on the reported history and immunization was determined by HBV serology. The odds ratio (OR) with 95% confidence intervals (95% CI) were calculated with consideration of the following covariates: age, gender, BMI, ethnic/racial group, current smoker, current alcohol consumption, family history of diabetes, poverty index, and education.

Results

This study included 15,316 subjects. Among them, 2,320 subjects was immunized based the HBV serology. Among 4,063 subjects who received HBV vaccination, successful vaccination was only noted in 39% of subjects. The HBV vaccination was not associated with diabetes (OR: 1.08, 95%CI: 0.96–1.23). Serology evidence of HBV immunization was associated with a reduced OR of diabetes (0.75, 95%CI: 0.62–0.90). Successful HBV vaccination was also associated with a reduced OR of diabetes (0.67, 95%CI: 0.52–0.84).

Conclusions

Although our study shows the association of HBV vaccination with the reduced odds of diabetes by 33%, a prospective study is warranted to confirm and examine the impact of HBV vaccination in prevention of diabetes.     

Regional assignment of two genes of the human branched-chain alpha-keto acid dehydrogenase complex: the E1 beta gene (BCKDHB) to chromosome 6p21-22 and the E2 gene (DBT) to chromosome 1p31

Maple syrup urine disease (MSUD) is caused by the deficiency of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The multienzyme complex is a macromolecule (Mr 4 X 10(6] consisting of at least six distinct subunits. In this study, the human E1 beta gene (BCKDHB) has been localized to human chromosome 6 by hybrid somatic cell analysis, and regionally assigned to chromosome bands 6p21-22 by in situ hybridization. The E2 gene (DBT), which was previously localized to chromosome 1, is regionally assigned to the chromosome band 1p31 also by in situ hybridization. Localization of the E1 beta gene to chromosome 6p21-22 assigns another major human disease locus to a region that contains several important genes, including the major histocompatability complex, tumor necrosis factor, and heat-shock protein HSP70. Mapping of the E1 beta and the E2 genes may provide information for the linkage analysis of MSUD families with mutations in these two loci.     

The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel

TRPM8, a member of the transient receptor potential family of ion channels, depolarizes somatosensory neurons in response to cold. TRPM8 is also activated by the cooling agents menthol and icilin. When exposed to menthol or cold, TRPM8 behaves like many ligand-gated channels, exhibiting rapid activation followed by moderate Ca(2+)-dependent adaptation. In contrast, icilin activates TRPM8 with extremely variable latency followed by extensive desensitization, provided that calcium is present. Here, we show that, to achieve full efficacy, icilin requires simultaneous elevation of cytosolic Ca2+, either via permeation through TRPM8 channels or by release from intracellular stores. Thus, two stimuli must be paired to elicit full channel activation, illustrating the potential for coincidence detection by TRP channels. Determinants of icilin sensitivity map to a region of TRPM8 that corresponds to the capsaicin binding site on the noxious heat receptor TRPV1, suggesting a conserved molecular logic for gating of these thermosensitive channels by chemical agonists.     

Dengue virus-induced autoantibodies bind to plasminogen and enhance its activation

Dengue virus infection can lead to life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) in patients. Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks associated with DHF/DSS patients. However, the mechanisms that cause pathology in DHF/DSS patients are still unclear. Because conversion of plasminogen (Plg) to plasmin (Plm) is the first step in the activation of fibrinolysis, Abs against Plg found in DHF/DSS patients may be important. Therefore, to investigate the specificity, function, and possible origin of these Abs, we generated several Plg cross-reactive mAbs from DENV-immunized mice. An IgG mAb, 6H11, which recognizes an epitope associated with a dengue envelope protein, demonstrated a high level of cross-reactivity with Plg. The 6H11 Ab was further characterized with regard to its effect on Plg activation. Using Plm-specific chromogenic substrate S-2251, we found that mAb 6H11 demonstrated serine protease activity and could convert Plg directly to Plm. The serine protease activity of mAb 6H11 was further confirmed using serine protease chromogenic substrate S-2288. In addition, we found several Plg cross-reactive mAbs that could enhance urokinase-induced Plg activation. Lastly, mAb 6H11 could induce Plm activity and increase the level of D-dimer (a fibrin degradation product) in both human and mouse platelet-poor plasma. Taken together, these data suggest DENV-induced Plg cross-reactive Abs may enhance Plg conversion to Plm, which would be expected to contribute to hyperfibrinolysis in DHF/DSS patients.     

Monoclonal antibody-based quantitation of poly(ethylene glycol)-derivatized proteins, liposomes, and nanoparticles

Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.     

Structural determinants for cross-talk between pyruvate dehydrogenase kinase 3 and lipoyl domain 2 of the human pyruvate dehydrogenase complex

Pyruvate dehydrogenase kinase isoforms (PDK1-4) are the molecular switch that down-regulates activity of the human pyruvate dehydrogenase complex through reversible phosphorylation. We showed previously that binding of the lipoyl domain 2 (L2) of the pyruvate dehydrogenase complex to PDK3 induces a "cross-tail" conformation in PDK3, resulting in an opening of the active site cleft and the stimulation of kinase activity. In the present study, we report that alanine substitutions of Leu-140, Glu-170, and Glu-179 in L2 markedly reduce binding affinities of these L2 mutants for PDK3. Unlike wildtype L2, binding of these L2 mutants to PDK3 does not preferentially reduce the affinity of PDK3 for ADP over ATP. The inefficient removal of product inhibition associated with ADP accounts for the decreased stimulation of PDK3 activity by these L2 variants. Serial truncations of the PDK3 C-terminal tail region either impede or abolish the binding of wild-type L2 to the PDK3 mutants, resulting in the reduction or absence of L2-enhanced kinase activity. Alanine substitutions of residues Leu-27, Phe-32, Phe-35, and Phe-48 in the lipoyl-binding pocket of PDK3 similarly nullify L2 binding and L2-stimulated PDK3 activity. Our results indicate that the above residues in L2 and residues in the C-terminal region and the lipoyl-binding pocket of PDK3 are critical determinants for the cross-talk between L2 and PDK3, which up-regulates PDK3 activity.