Aldolase and Actin Protect Rabbit Muscle Lactate Dehydrogenase from Ascorbate Inhibition

Muscle-type LDH (LDH-m4) activity is critical for efficient anaerobic glycolysis. The results here show that rabbit LDH-M4 is inhibited by concentrations of ascorbate normally found in tissues. Aldolase and muscle G-actin were found to protect and to reverse inhibitions of LDH-m4 by ascorbate. G-actins showed some species specificity. Myosin, tropomyosin and troponin from rabbit muscle and muscle proteins from other animal sources had no affect on the inhibitions by ascorbate. The substrate inhibition of LDH-m4 by pyruvate is partially relieved by the presence of aldolase and lowers the Km without affecting the Vm. G-actin under similar conditions has no affect. It is believed that these studies reflect some of the resting properties of glycolytic enzymes that bind and unbind to contractile elements. It is proposed that ascorbate facilitates the storage of glycogen in muscle at rest by inhibiting glycolysis.     

Infant Feeding and School Attainment in Five Cohorts from Low- and Middle-Income Countries

Background

Performance in intelligence tests tends to be higher among individuals breastfed as infants, but little is known about the association between breastfeeding and achieved schooling. We assessed the association of infant feeding with school achievement in five cohorts from low- and middle-income countries. Unlike high-income country settings where most previous studies come from, breastfeeding is not positively associated with socioeconomic position in our cohorts, thus reducing the likelihood of a spurious positive association.

Methodology and Principal Findings

Participants included 10,082 young adults from five birth cohorts (Brazil, India, Guatemala, the Philippines, and South Africa). The exposures variables were whether the subject was ever breastfed, total duration of breastfeeding, and age at introduction of complementary foods. We adjusted the estimates for age at follow up, sex, maternal age, smoking during pregnancy, birthweight and socioeconomic position at birth. The key outcome was the highest grade achieved at school. In unadjusted analyses, the association between ever breastfeeding and schooling was positive in Brazil, inverse in the Philippines, and null in South Africa; in adjusted analyses, these associations were attenuated. In Brazil, schooling was highest among individuals breastfed for 3–12 months whereas in the Philippines duration of breastfeeding was inversely associated with schooling; and null associations were observed in South Africa and Guatemala. These associations were attenuated in adjusted models. Late introduction of solid foods was associated with lower schooling achievement in Brazil and South Africa.

Conclusion

Measures of breastfeeding are not consistently related to schooling achievement in contemporary cohorts of young adults in lower and middle-income countries.     

Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.     

Mucosal and systemic immune responses of mice to tetanus toxoid coadministered nasally with AFCo1

Mucosal immune responses are an early and important line of defense against pathogens. The current understanding of the mucosal immune system allows us to consider the use of nasal immunization for induction of antigen-specific immune responses at the mucosal surface and the systemic compartment. Mucosal adjuvants are key for developing novel mucosal vaccines and represent 1 approach to improving mucosal and systemic immunity. However, few mucosal vaccine adjuvants are currently approved for human use. Neisseria meningitidis B proteoliposome-derived cochleate (AFCo1 - Adjuvant Finlay Cochleate 1) has been demonstrated to be a potent mucosal adjuvant. The present work demonstrates that intranasal immunization of 3 doses of tetanus toxoid (TT) coadministered with AFCo1 in mice promotes high systemic and mucosal responses. The anti-TT IgG serum titers and the mucosal anti-TT IgA in saliva and vaginal wash were significantly higher than TT alone. The analysis of antibody subclasses showed that intranasal administration of AFCo1 + TT induced not only IgG1 but also IgG2a anti-TT antibodies at levels comparable to those obtained with TT vaccine (vax-TET). These data support the fact that AFCo1 is a potent mucosal adjuvant in nasal immunization to a coadministered protein antigen.     

Neutrophil elastase treated dendritic cells promote the generation of CD4(+)FOXP3(+) regulatory T cells in vitro

We have previously shown that neutrophilic elastase converts human immature dendritic cells (DCs) into TGF-β secreting cells and reduces its allostimulatory ability. Since TGF-β has been involved in regulatory T cells (Tregs) induction we analyzed whether elastase or neutrophil-derived culture supernatant treated DCs induce CD4⁺FOXP3⁺ Tregs in a mixed lymphocyte reaction (MLR). We found that elastase or neutrophil-derived culture supernatant treated DCs increased TGF-β and decreased IL-6 production. Together with this pattern of cytokines, we observed a higher number of CD4⁺FOXP3⁺ cells in the MLR cultures induced by elastase or neutrophil-derived culture supernatant treated DCs but not with untreated DCs. The higher number of CD4⁺FOXP3⁺ T cell population was not observed when the enzymatic activity of elastase was inhibited with an elastase specific inhibitor and also when a TGF-β1 blocking antibody was added during the MLR culture. The increased number of CD4⁺ that express FOXP3 was also seen when CD4⁺CD25^- purified T cells were cocultured with the TGF-β producing DCs. Furthermore, these FOXP3⁺ T cells showed suppressive activity in vitro.These results identify a novel mechanism by which the tolerogenic DCs generated by elastase exposure contribute to the immune regulation and may be relevant in the pathogenesis of several lung diseases where the inflammatory infiltrate contains high numbers of neutrophils and high elastase concentrations.     

Acute High Fat Diet Consumption Activates the Mesolimbic Circuit and Requires Orexin Signaling in a Mouse Model

Overconsumption of palatable energy-dense foods has negative health implications and it is associated with obesity and several eating disorders. Currently, little is known about the neuronal circuitries activated by the acute ingestion of a rewarding stimulus. Here, we used a combination of immunohistochemistry, pharmacology and neuronal tracing analyses to examine the role of the mesolimbic system in general, and the orexin neurons in particular, in a simple experimental test in which naïve mice are allowed to spontaneously eat a pellet of a high fat diet (HFD) for 2 h. We found that acute HFD activates c-Fos expression in several reward-related brain areas, including the ventral tegmental area (VTA), nucleus accumbens, central amygdala and lateral hypothalamic area. We also found that: i- HFD-mediated orosensory stimulation was required for the mesolimbic pathway activation, ii- acute HFD differentially activates dopamine neurons of the paranigral, parabrachial pigmented and interfascicular sub-regions of the VTA, and iii- orexin neurons of the lateral hypothalamic area are responsive to acute HFD. Moreover, orexin signaling blockade, with the orexin 1 receptor antagonist SB-334867, reduces acute HFD consumption and c-Fos induction in the VTA but not in the other mesolimbic nuclei under study. Finally, we found that most orexin neurons responsive to acute HFD innervate the VTA. Our results show that acute HFD consumption recruits the mesolimbic system and that the full manifestation of this eating behavior requires the activation of orexin signaling.     

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2^nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2^nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2^nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.     

Improved pharmacological properties for superoxide dismutase modified with β-cyclodextrin–carboxymethylcellulose polymer

Superoxide dismutase was glycosidated with cyclodextrin-branched carboxymethylcellulose. The modified enzyme contained 1.4 mol polymer per mol protein and retained 87% of the initial activity. The anti-inflammatory activity of superoxide dismutase was 2.2-times increased after conjugation and its plasma half-life time was prolonged from 4.8 min to 7.2 h.     

Chitosan-whey protein edible films produced in the absence or presence of transglutaminase: analysis of their mechanical and barrier properties

Chitosan-whey protein edible films with different protein concentrations were prepared in the absence or presence of microbial transglutaminase as cross-linking agent. The films prepared in the presence of the enzyme showed low solubility at a wide range of pH, a lower degree of swelling, and good biodegradability following protease treatments. The presence of transglutaminase induced also an enhancement in film mechanical resistance and a reduction in their deformability. Finally, the barrier efficiency toward oxygen and carbon dioxide was found to be markedly improved in the cross-linked films which showed also a lower permeability to water vapor. Some potential practical applications of transglutaminase-treated chitosan-whey protein films are suggested.