Nucleophilic participation of reduced flavin coenzyme in mechanism of UDP-galactopyranose mutase

UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(β) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).     

Risk of Cerebral Palsy and Childhood Epilepsy Related to Infections before or during Pregnancy

Background and Aim

Maternal infections during pregnancy have been associated with several neurological disorders in the offspring. However, given the lack of specificity for both the exposures and the outcomes, other factors related to infection such as impaired maternal immune function may be involved in the causal pathway. If impaired maternal immune function plays a role, we would expect infection before pregnancy to be associated with these neurological outcomes.

Methods/Principal Findings

The study population included all first-born singletons in Denmark between January 1 1982 and December 31 2004. We identified women who had hospital-recorded infections within the 5 year period before pregnancy, and women who had hospital-recorded infections during pregnancy. We grouped infections into either infections of the genitourinary system, or any other infections. Cox models were used to estimate adjusted hazard ratios (aHRs) with 95% confidence interval (CI). Maternal infection of the genitourinary system during pregnancy was associated with an increased risk of cerebral palsy (aHR = 1.63, 95% CI: 1.34–1.98) and epilepsy (aHR = 1.27, 95% CI: 1.13–1.42) in the children, compared to children of women without infections during pregnancy. Among women without hospital-recorded infections during pregnancy, maternal infection before pregnancy was associated with an increased risk of epilepsy (aHR = 1.35, 95% CI: 1.21–1.50 for infections of the genitourinary system, and HR = 1.12, 95% CI: 1.03–1.22 for any other infections) and a slightly higher risk of cerebral palsy (aHR = 1.20, 95% CI: 0.96–1.49 for infections of the genitourinary system, and HR = 1.23, 95% CI: 1.06–1.43 for any other infections) in the children, compared to children of women without infections before (and during) pregnancy.

Conclusions

These findings indicate that the maternal immune system, maternal infections, or factors related to maternal immune function play a role in the observed associations between maternal infections before pregnancy and cerebral diseases in the offspring.     

Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles

Background

Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.

Principal Finding

In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10⁶ the tissue culture''s infectious dose (TCID₅₀) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10⁻⁵ was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24–28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35–38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176–190.

Conclusion

These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.     

Rice Crinkly4 receptor-like kinase positively regulates culm elongation and amino acid K532 is not essential for its kinase activity

Receptor-like kinases (RLKs) play important roles in multiple aspects of plant growth and development. As a member of the TNFR-like RLK subfamily, rice Crinkly4 (OsCR4) functions mainly in epidermal cell differentiation in many organs. Here we show that in addition to its essential role in epidermal cell differentiation in the palea and lemma, OsCR4 positively regulates rice culm elongation, similar to maize CR4. Although OsCR4 is an active kinase, like CR4 in maize and ACR4 in Arabidopsis, the conserved amino acid K532 in OsCR4 is not essential for its kinase activity in vitro. Whether other conserved amino acids are required for its kinase activity and the relationship between its activity and function in plant development remain to be investigated.     

Candidate Markers That Associate with Chemotherapy Resistance in Breast Cancer through the Study on Taxotere-Induced Damage to Tumor Microenvironment and Gene Expression Profiling of Carcinoma-Associated Fibroblasts (CAFs)

Recently, emerging evidence has suggested that carcinoma-associated fibroblasts (CAFs) could contribute to chemotherapy resistances in breast cancer treatment. The aim of this study is to compare the gene expression profiling of CAFs before and after chemotherapy and pick up candidate genes that might associate with chemotherapy resistance and could be used as predictors of treatment response. CAFs were cultured from surgically resected primary breast cancers and identified with immunohistochemistry (IHC) and Flow cytometry (FCM). MDA-MB-231 cells were cultured as the breast cancer cell line. Cell adhesion assay, invasion assay, and proliferation assay (MTT) were performed to compare the function of MDA-MB-231 cells co-cultured with CAFs and MDA-MB-231 cells without co-culture, after chemotherapy. Totally 6 pairs of CAFs were prepared for microarray analysis. Each pair of CAFs were obtained from the same patient and classified into two groups. One group was treated with Taxotere (regarded as after chemotherapy) while the other group was not processed with Taxotere (regarded as before chemotherapy). According to our study, the primary-cultured CAFs exhibited characteristic phenotype. After chemotherapy, MDA-MB-231 cells co-cultured with CAFs displayed increasing adhesion, invasiveness and proliferation abilities, compared with MDA-MB-231 cells without CAFs. Moreover, 35 differentially expressed genes (absolute fold change >2) were identified between CAFs after chemotherapy and before chemotherapy, including 17 up-regulated genes and 18 down-regulated genes. CXCL2, MMP1, IL8, RARRES1, FGF1, and CXCR7 were picked up as the candidate markers, of which the differential expression in CAFs before and after chemotherapy was confirmed. The results indicate the changes of gene expression in CAFs induced by Taxotere treatment and propose the candidate markers that possibly associate with chemotherapy resistance in breast cancer.     

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.     

Associations between retinol-binding protein 4 and cardiometabolic risk factors and subclinical atherosclerosis in recently postmenopausal women: Cross-sectional analyses from the KEEPS Study

ABSTRACT: BACKGROUND: The published literature regarding the relationships between retinol-binding protein 4 (RBP4) and cardiometabolic risk factors and subclinical atherosclerosis is conflicting, likely due, in part, to limitations of frequently used RBP4 assays. Prior large studies have not utilized the gold-standard Western Blot analysis of RBP4 levels. METHODS: Full-length serum RBP4 levels were measured by Western Blot in 709 postmenopausal women screened for the Kronos Early Estrogen Prevention Study. Cross-sectional analyses related RBP4 levels to cardiometabolic risk factors, carotid artery intima-media thickness (CIMT), and coronary artery calcification (CAC). RESULTS: The mean age of women was 52.9 (+/- 2.6) years, and the median RBP4 level was 49.0 (interquartile range 36.9-61.5) ug/mL. Higher RBP4 levels were weakly associated with higher triglycerides (age, race, and smoking-adjusted partial Spearman correlation coefficient= 0.10; P=0.01), but were unrelated to blood pressure, cholesterol, C-reactive protein, glucose, insulin, and CIMT levels (all partial Spearman correlation coefficients [less than or equal to]0.06, P>0.05). Results suggested a curvilinear association between RBP4 levels and CAC, with women in the bottom and upper quartiles of RBP4 having higher odds of CAC (odds ratio [95% confidence interval] 2.10 [1.07-4.09], 2.00 [1.02-3.92], 1.64 [0.82-3.27] for the 1st, 3rd, and 4th RBP4 quartiles vs. the 2nd quartile). However, a squared RBP4 term in regression modeling was non-significant (P=0.10). CONCLUSIONS: In these healthy, recently postmenopausal women, higher RBP4 levels were weakly associated with elevations in triglycerides and with CAC, but not with other risk factors or CIMT. These data using the gold standard of RBP4 methodology only weakly support the possibility that perturbations in RBP4 homeostasis may be an additional risk factor for subclinical coronary atherosclerosis. Trial Registration: ClinicalTrials.gov number NCT00154180.     

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan''s crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.     

EFFECTS OF LIGHT ON PHOTOSYNTHESIS,GRAZING, AND POPULATION DYNAMICS OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

In studying how environmental factors control the population dynamics of Pfiesteria piscicida Steidinger et Burkholder, we examined the influence of light regime on kleptoplastidic photosynthesis, growth, and grazing. Prey (Rhodomonas sp.)‐saturated growth rate of P. piscicida increased (0.67 ± 0.03 d^?1 to 0.91 ± 0.11 d^?1) with light intensity varying from 0 to 200 μmol photons·m^?2·s^?1. No significant effect was observed on grazing, excluding the possibility that light enhanced P. piscicida growth through stimulating grazing. Light‐grown P. piscicida exhibited a higher gross growth efficiency (0.78 ± 0.10) than P. piscicida incubated in the dark (0.32 ± 0.16), and photosynthetic inhibitors significantly decreased growth of recently fed populations. These results demonstrate a role of kleptoplastidic photosynthesis in enhancing growth in P. piscicida. However, when the prey alga R. sp. was depleted, light's stimulating effect on P. piscicida growth diminished quickly, coinciding with rapid disappearance of Rhodomonas‐derived pigments and RUBISCO from P. piscicida cells. Furthermore, the effect of light on growth was reversed after extended starvation, and starved light‐grown P. piscicida declined at a rate significantly greater than dark‐incubated cultures. The observed difference in rates of decline appeared to be attributable to light‐dependent cannibalism. Using a 5‐chloromethylfluorescein diacetate staining technique, cannibalistic grazing was observed after 7 days of starvation, at a rate four times greater under illumination than in the dark. The results from this study suggest that kleptoplastidy enhances growth of P. piscicida only in the presence of algal prey. When prey is absent, P. piscicida populations may become vulnerable to light‐stimulated cannibalism.     

Saccharide-mediated antagonistic effects of bark beetle fungal associates on larvae

Bark beetles are among the most destructive of pine forest pests and they form close symbiotic relationships with ophiostomatoid fungi. Although some fungi are considered to be mutualistic symbionts of bark beetles with respect to the supply of nutrients, detrimental effects of fungal symbionts on larval growth have also been frequently reported. The mechanisms of such antagonistic effects are hypothesized to be a decrease in nutritional resources caused by competition for saccharides by the fungi. Here, we provide experimental evidence that three beetle-associated fungi modify the nutritional content of an artificial phloem diet, leading to a detrimental effect on the growth of Dendroctonus valens larvae. When larvae were fed a diet of pine phloem in agar medium colonized with any of these fungi, feeding activity was not affected but weight significantly decreased. Additional analysis showed that fungi depleted the fructose and glucose concentrations in the phloem media. Furthermore, these detrimental effects were neutralized by supplementing the media with fructose or glucose, suggesting that fungi may affect larval growth by modifying diet saccharide contents. These data indicate that fungus-induced nutritional changes in bark beetle diet can affect larval growth, and that the mechanism involves fungus-induced saccharide depletion from the larval diet.