Activity of N-acetylmuramoyl-L-alanine amidase in phospholipidic environments

A purified preparation of N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), a murein hydrolase from Escherichia coli, was found to lose its activity during incubation in the presence of bacterial phospholipid suspensions. Whether it was co-dispersed with the phospholipids or added to sonicated phospholipid suspension, the enzyme was inhibited (or inactivated) from the first minutes of incubation at 37 degree C. As phosphatidylglycerol/cardiolipin ratio of the phospholipid suspension as increased (all other things being equal), a further decrease of amidase activity was observed. The highest losses of activity were found after co-dispersion of the enzyme and the substrate together with the phospholipids, the resulting suspension being formed of larger multilayered vesicles, as revealed by electron microscopy. In these conditions, the effect on enzyme activity was only partially accounted for by the proportion of the enzyme that was entrapped in the vesicles. The entrapment capacity of the enzyme (using a 35S-labelled enzyme preparation) and of the substrate (3H-labelled) by the multilamellar phospholipidic vesicles did not significantly change as a function of their relative content of phosphatidylglycerol and cardiolipin. The possible physiological meaning of the results is discussed is connection with our previous data and with other works related to membranous phospholipid distribution and to septum formation control in bacteria.     

Microsomal cytochromes of Candida tropicalis grown on alkanes

A comparison of methods used in isolating microsomes and in measuring microsomal cytochrome P-450 demonstrated that separation following protoplast lysis gave the best results. By this latter technique a high amount of cytochrome P-450 (0.2–0.3 nmol/mg) was recovered but cytochrome P-420, considered as the denatured form, was absent.The alkanes specifically induce cytochromes P-450 and b₅ localized on the microsomes. The denaturation in vivo of cytochrome P-450 into cytochrome P-420 even occurs during storage at 1 °C. This degradation is increased during preparation of subcellular fractions if no preventive measures are taken.     

Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes

The ionic influence and ouabain sensitivity of lymphocyte Mg²⁺-ATPase and Mg²⁺-(Na⁺ + K⁺)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5′-nucleotidase and Mg²⁺-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na⁺ + K⁺)-ATPase was located inside the membrane.Concanavalin A induced an early stimulation of Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase both on intact cells and purified plasma membranes. In contrast, 5′-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg²⁺-ATPase activity was 3–5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20 %). (Na⁺ + K⁺)-ATPase activity was undetectable in thymocytes. However, in spleen lymphocytes (Na⁺ + K⁺)-ATPase activity can be detected and was 30 % increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg²⁺-ATPase, in lymphocyte stimulation.     

Modulation of lipid synthesis,apolipoprotein biogenesis,and lipoprotein assembly by butyrate

Short-chain fatty acids (SCFAs) are potent modulators of the growth, function, and differentiation of intestinal epithelia. In addition, high-fiber diets may protect against the development of atherosclerosis because of their cholesterol-lowering effects due, in large part, to SCFA production, liver sterol metabolism, and bile acid excretion. Although the small gut plays a major role in dietary fat transport and contributes substantially to plasma cholesterol and lipoprotein homeostasis, the impact of SCFAs on intestinal lipid handling remains unknown. In the present study, the modulation of lipid synthesis, apolipoprotein biogenesis, and lipoprotein secretion by butyrate was investigated in Caco-2 cells plated on permeable polycarbonate filters, which permit separate access to the upper and lower compartments of the monolayers. Highly differentiated and polarized cells (20 days of culture) were incubated for 20 h with 20 mM butyrate in the apical medium. In the presence of [14C]oleic acid, butyrate led to a significant reduction of secreted, labeled triglycerides (27%; P < 0.01) and phospholipids (25%; P < 0.05). Similarly, butyrate significantly decreased the incorporation of [14C]acetate into exported cholesteryl ester (49%; P < 0.005). As expected from these results, with [14C]oleic acid as a precursor, butyrate significantly (P < 0.05) diminished the delivery of radiolabeled chylomicrons and very low-density lipoproteins. In parallel, [35S]methionine pulse labeling of Caco-2 cells revealed the concomitant inhibitory effect of butyrate on the synthesis of apolipoproteins B-48 (28%; P < 0.05) and A-I (32%; P < 0.01). Collectively, our data indicate that butyrate may influence lipid metabolism in Caco-2 cells, thus suggesting a potential regulation of intestinal fat absorption and circulating lipoprotein concentrations.     

Influenza and Other Respiratory Viruses Detected by Influenza-Like Illness Surveillance in Leyte Island,the Philippines, 2010–2013

This study aimed to determine the role of influenza-like illness (ILI) surveillance conducted on Leyte Island, the Philippines, including involvement of other respiratory viruses, from 2010 to 2013. ILI surveillance was conducted from January 2010 to March 2013 with 3 sentinel sites located in Tacloban city, Palo and Tanauan of Leyte Island. ILI was defined as fever ≥38°C or feverish feeling and either cough or running nose in a patient of any age. Influenza virus and other 5 respiratory viruses were searched. A total of 5,550 ILI cases visited the 3 sites and specimens were collected from 2,031 (36.6%) cases. Among the cases sampled, 1,637 (75.6%) were children aged <5 years. 874 (43.0%) cases were positive for at least one of the respiratory viruses tested. Influenza virus and respiratory syncytial virus (RSV) were predominantly detected (both were 25.7%) followed by human rhinovirus (HRV) (17.5%). The age distributions were significantly different between those who were positive for influenza, HRV, and RSV. ILI cases were reported throughout the year and influenza virus was co-detected with those viruses on approximately half of the weeks of study period (RSV in 60.5% and HRV 47.4%). In terms of clinical manifestations, only the rates of headache and sore throat were significantly higher in influenza positive cases than cases positive to other viruses. In conclusion, syndromic ILI surveillance in this area is difficult to detect the start of influenza epidemic without laboratory confirmation which requires huge resources. Age was an important factor that affected positive rates of influenza and other respiratory viruses. Involvement of older age children may be useful to detect influenza more effectively.     

Predicting enzyme class from protein structure using Bayesian classification

Predicting enzyme class from protein structure parameters is a challenging problem in protein analysis. We developed a method to predict enzyme class that combines the strengths of statistical and data-mining methods. This method has a strong mathematical foundation and is simple to implement, achieving an accuracy of 45%. A comparison with the methods found in the literature designed to predict enzyme class showed that our method outperforms the existing methods.     

Association between different growth curve definitions of overweight and obesity and cardiometabolic risk in children

Background:

Overweight and obesity in young people are assessed by comparing body mass index (BMI) with a reference population. However, two widely used reference standards, the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) growth curves, have different definitions of overweight and obesity, thus affecting estimates of prevalence. We compared the associations between overweight and obesity as defined by each of these curves and the presence of cardiometabolic risk factors.

Methods:

We obtained data from a population-representative study involving 2466 boys and girls aged 9, 13 and 16 years in Quebec, Canada. We calculated BMI percentiles using the CDC and WHO growth curves and compared their abilities to detect unfavourable levels of fasting lipids, glucose and insulin, and systolic and diastolic blood pressure using receiver operating characteristic curves, sensitivity, specificity and kappa coefficients.

Results:

The z scores for BMI using the WHO growth curves were higher than those using the CDC growth curves (0.35–0.43 v. 0.12–0.28, p < 0.001 for all comparisons). The WHO and CDC growth curves generated virtually identical receiver operating characteristic curves for individual or combined cardiometabolic risk factors. The definitions of overweight and obesity had low sensitivities but adequate specificities for cardiometabolic risk. Obesity as defined by the WHO or CDC growth curves discriminated cardiometabolic risk similarly, but overweight as defined by the WHO curves had marginally higher sensitivities (by 0.6%–8.6%) and lower specificities (by 2.6%–4.2%) than the CDC curves.

Interpretation:

The WHO growth curves show no significant discriminatory advantage over the CDC growth curves in detecting cardiometabolic abnormalities in children aged 9–16 years.Pediatric obesity is associated with dyslipidemia, insulin resistance and elevated blood pressure.^1–6 Thus, accurately identifying children with obesity is crucial for clinical management and public health surveillance.Lipid screening is recommended for young people who are overweight,^7,8 but studies show that estimates of the prevalence of overweight and obesity are 1%–7% lower using the growth curves of the Centers for Disease Control and Prevention (CDC) versus those of the World Health Organization (WHO).^9–11 Although the CDC and WHO definitions of overweight and obesity both use approximations of overweight and obese values of body mass index (BMI) when children reach 19 years of age, the CDC growth curves use data from more recent samples of young people.^12,13 Given the recent rise in the prevalence of obesity among young people, using a heavier reference population may lead to fewer children being identified as overweight and obese, and an identical BMI value may not trigger a clinical investigation.⁷ The Canadian Paediatric Society, in collaboration with the College of Family Physicians of Canada, Dietitians of Canada and Community Health Nurses of Canada, recently recommended that physicians switch from the CDC to the WHO growth curves for monitoring growth for Canadian children aged 5–19 years.¹⁴ This is a major change for health providers caring for the estimated 8 million children in Canada.¹⁵Understanding how using the different growth curves affects the identification of adverse cardiometabolic risk profiles is essential for the appropriate management of overweight and obesity among young people. Thus, our objectives were to assess whether the association between BMI percentiles and cardiometabolic risk differs between the definitions of overweight and obesity based on the WHO and CDC growth curves, and to compare the sensitivity and specificity of these definitions in detecting cardiometabolic risk.