The extended relationship between child cardiovascular risks and academic performance measures

Objective: To examine the relationship between children's overweight status and other cardiovascular risk fitness factors and academic performance among fifth‐grade students. Research Methods and Procedures: Using a sample of 968 fifth‐grade students (50.7% boys; mean age = 10.6 years), children's cardiovascular risks (BMI, blood pressure, acanthosis nigricans) and fitness measures were compared with their mean group performance scores across four subscales (mathematics, reading/language arts, science, and social studies) of a statewide standardized academic performance test. Results: Of this sample, 39% were either at risk for being overweight or were already overweight; slightly over one half were of normal weight. Initial findings revealed a significant relationship between children's weight category and their reading/language arts, mathematics, and science test scores even after controlling for a proxy of socioeconomic status. When additional cardiovascular risk and fitness measures were included in the model, however, children's BMI status had no association. Instead, a composite fitness index, children's blood pressure, sex, and proxy of socioeconomic status were significantly associated with children's academic test scores. Discussion: This study expanded our understanding of the connection between children's overweight risks and academic performance by examining the impact of other cardiovascular risk factors such as high blood pressure and measures of fitness. These findings support the development and implementation of childhood cardiovascular risk surveillance programs that evaluate not only children's overweight risks but also their fitness, risk for type 2 diabetes, and/or high blood pressure by showing a relationship between some of these risks and children's academic test performance.     

The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis

Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted.     

A divalent major histocompatibility complex/IgG1 fusion protein induces antigen-specific T cell activation in vitro and in vivo

Activation of antigen-specific T cell clones in vivo might be possible by generating soluble MHC molecules; however, such molecules do not induce effective T cell responses unless cross-linked. As a first step in generating a soluble MHC molecule that could function as an antigen-specific immunostimulant, the extracellular domains of the murine H-2Kb MHC class I molecule were fused to the constant domains of a murine IgG1 heavy chain, resulting in a divalent molecule with both a TCR-reactive and an Fc receptor (FcR)-reactive moiety. The fusion protein can be loaded with peptide and can induce T cell activation in a peptide-specific, MHC-restricted manner following immobilization on plastic wells or following cross-linking by FcR+ spleen cells. The fusion protein induces partial T cell activation in vivo in a mouse transgenic for a TCR restricted to H-2Kb. This fusion protein molecule may be useful to study peptide-MHC interactions and may provide a strategy for boosting in vivo antigen-specific T cell responses, such as to viral or tumor antigens.     

A real-time PCR assay for DNA-methylation using methylation-specific blockers

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.     

Antikinetoplastid activity of 3-aryl-5-thiocyanatomethyl-1,2,4-oxadiazoles

A series of 5-thiocyanatomethyl- and 5-alkyl-3-aryl-1,2,4-oxadiazoles were synthesized and evaluated for their activity against kinetoplastid parasites. Formation of the oxadiazole ring was accomplished through the reaction of benzamidoximes with acyl chlorides, while the thiocyanate group was inserted by reacting the appropriate 5-halomethyl oxadiazole with ammonium thiocyanate. The thiocyanate-containing compounds possessed low micromolar activity against Leishmania donovani and Trypanosoma brucei, while the 5-alkyl oxadiazoles were less active against these parasites. 3-(4-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4b) displayed modest selectivity for L. donovani axenic amastigote-like parasites over J774 macrophages, PC3 prostate cancer cells, and Vero cells (6.4-fold, 3.8-fold, and 9.1-fold, respectively), while 3-(3,4-dichlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4 h) showed 30-fold selectivity against Vero cells but was not selective against PC3 cells. In a murine model of visceral leishmaniasis, compound 4b decreased liver parasitemia caused by L. donovani by 48% when given in five daily i.v. doses at 5mg/kg and by 61% when administered orally for 5 days at 50 mg/kg. These results indicate that aromatic thiocyanates hold promise for the treatment of leishmanial infections if the selectivity of these compounds can be improved.     

Susceptibility of a native and an exotic lady beetle (Coleoptera: Coccinellidae) to Beauveria bassiana

The exotic multicolored Asian lady beetle, Harmonia axyridis, became established and recently spread across much of North America and southern Canada. In a habitat now used by both the invading H. axyridis and a native lady beetle, Olla v-nigrum, we discovered that the native lady beetle was commonly infected by Beauveria bassiana; whereas, the exotic H. axyridis, was not. Laboratory assays revealed that B. bassiana isolates collected from naturally infected O. v-nigrum were pathogenic to adult O. v-nigrum but not to adult H. axyridis. In contrast, the GHA strain of B. bassiana was not significantly pathogenic to O. v-nigrum nor H. axyridis. Late-season field collections revealed significantly higher B. bassiana infection of O. v-nigrum than H. axyridis. Our results lead us to hypothesize that low susceptibility of H. axyridis to B. bassiana (found to infect O. v-nigrum) may provide an intraguild advantage to H. axyridis over O. v-nigrum; this may also occur with other species of native lady beetles and other endemic entomopathogens in different habitats and regions.     

Effect of recovery interval on multiple-bout sprint cycling performance after acute creatine supplementation

The purpose of this study was to examine the effect of varying recovery intervals on multiple-bout, short-duration, high-intensity cycling efforts of adult men supplemented with creatine (Cr) or a placebo (Pl). Thirty subjects underwent 3 trials of a maximal cycling protocol (T(0), T(1), T(2)). T(0) included V(O)2 max testing and familiarization with the sprint cycling protocol. T(1) consisted of 8 15-second bouts of sprint cycling exercise. Subjects were randomly assigned to recovery interval groups (1 minute, 3 minutes, 6 minutes), and Cr or Pl groups (0.3 g x kg(-1) x d(-1)). Posttesting (T(2)) took place 7 days after T(1) and consisted of an identical protocol as during T(1). Changes in mean power (MP), peak power (PP), and fatigue index (FI) were compared between trials. MP was significantly increased in Cr 1-minute, Cr 3-minute, and Pl 6-minute groups (p < 0.05). Significant PP increases were demonstrated in Cr 1-minute and Pl 6-minute groups (p < 0.05), and FI significantly increased in Pl 1-minute group (p < 0.05). Results indicate that Cr supplementation is effective in improving recovery from repeated sprint cycling performances when the recovery interval is of a short (<6 minutes) duration.     

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.     

Patterns in Abundance,Cell Size and Pigment Content of Aerobic Anoxygenic Phototrophic Bacteria along Environmental Gradients in Northern Lakes

There is now evidence that aerobic anoxygenic phototrophic (AAP) bacteria are widespread across aquatic systems, yet the factors that determine their abundance and activity are still not well understood, particularly in freshwaters. Here we describe the patterns in AAP abundance, cell size and pigment content across wide environmental gradients in 43 temperate and boreal lakes of Québec. AAP bacterial abundance varied from 1.51 to 5.49 x 10⁵ cells mL^-1, representing <1 to 37% of total bacterial abundance. AAP bacteria were present year-round, including the ice-cover period, but their abundance relative to total bacterial abundance was significantly lower in winter than in summer (2.6% and 7.7%, respectively). AAP bacterial cells were on average two-fold larger than the average bacterial cell size, thus AAP cells made a greater relative contribution to biomass than to abundance. Bacteriochlorophyll a (BChla) concentration varied widely across lakes, and was not related to AAP bacterial abundance, suggesting a large intrinsic variability in the cellular pigment content. Absolute and relative AAP bacterial abundance increased with dissolved organic carbon (DOC), whereas cell-specific BChla content was negatively related to chlorophyll a (Chla). As a result, both the contribution of AAP bacteria to total prokaryotic abundance, and the cell-specific BChla pigment content were positively correlated with the DOC:Chla ratio, both peaking in highly colored, low-chlorophyll lakes. Our results suggest that photoheterotrophy might represent a significant ecological advantage in highly colored, low-chlorophyll lakes, where DOC pool is chemically and structurally more complex.