A universal primer set for PCR amplification of nuclear histone H4 genes from all animal species

To control the quality of genomic DNA of samples from a wide variety of animals, a heminested PCR assay specifically targeting a nuclear gene has been developed. The histone H4 gene family comprises a small number of genes considered among the most conserved genes in living organisms. Tissue samples from necropsies and from cells belonging to 43 different species were studied, eight samples from invertebrates and 35 samples from vertebrates covering all classes. Ancient DNA samples from three Siberian woolly mammoths (Mammuthus primigenius) dating between 40,000 and 49,000 years before present were also tested for PCR amplification. Performance of HIST2H4 amplification were also compared with those of previously published universal PCRs (28S rRNA, 18S rRNA, and cytochrome b). Overall, 95% of species studied yielded an amplification product, including some old samples from gorilla and chimpanzees. The data indicate that the HIST2H4 amplimers are, thus, suitable for both DNA quality testing as well as species identification in the animal kingdom.     

Correlation analyses and regression modeling between isokinetic testing and on-court performance in competitive adolescent tennis players

Tennis requires skill, physical attributes, and strategy. Ball velocity and placement are two of the most important components in winning the faster-paced modern game. Although isokinetic testing has been used to evaluate physical characteristics and injury potential in tennis players, few studies have compared isokinetics and on-court performance. Such a comparison would help establish links between speed-specific properties of functioning muscles and stroke production and could affect overall training strategy. This study compared isokinetic peak torque (PT), average power (AP), and total work (TW) during specific testing patterns correlated with ball velocity or stroke accuracy during the service, forehand, and backhand and developed predictive equations for each stroke using these variables. Thirty-five players, aged 13-18 years with at least 4 years playing experience, were evaluated using internal and external shoulder rotation, leg extension, and diagonal throwing motions. Ball velocity was measured using a radar gun. Accuracy was evaluated on the basis of shot position and depth. Significant correlations were found between ball velocity and a number of isokinetic variables, while no significant correlations were observed with shot accuracy. Significant isokinetic variables for each stroke were entered into regression models. One isokinetic speed sufficiently predicted ball velocity for each stroke, since no increase in predictive capacity was observed with the addition of other isokinetic parameters. We conclude that isokinetics at testing speeds between 1.57 and 4.71 rad.s(-1) can effectively predict ball velocity, but not accuracy, and that our results may be helpful in planning strategies for training and rehabilitation.     

Diphenylcyclohexylamine derivatives as new potent multidrug resistance (MDR) modulators

A series of compounds with a diphenylmethyl cyclohexyl skeleton, loosely related to verapamil, has been synthesized and tested as MDR modulators on anthracycline-resistant erythroleukemia K 562 cells. Their residual cardiovascular action (negative inotropic and chronotropic activity as well as vasorelaxant activity) was evaluated on guinea-pig isolated atria preparations and on guinea-pig aortic strip preparations. Most compounds of the series possess a good MDR-reverting activity together with a low cardiovascular action. Among them, compounds 3a1, 7a, and 8a are more potent than verapamil as MDR reverters and lack any cardiovascular action; they can represent useful leads for the development of new safe MDR reversing drugs.     

Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction

Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.     

Microtubule target for new antileishmanial drugs based on ethyl 3-haloacetamidobenzoates

A new family of antimicrotubule drugs named (3-haloacetamidobenzoyl) ureas and ethyl 3-haloacetamidobenzoates were found to be cytotoxic to the Leishmania parasite protozoa. While the benzoylureas were shown to strongly inhibit in vitro mammalian brain microtubule assembly, the ethyl ester derivatives were characterized as very poor inhibitors of this process. Ethyl 3-chloroacetamidobenzoate, MF29, was found to be the most efficient drug on the promastigote stage of three Leishmania species (IC50: 0.3-1.8 microM). MF29 maintained its activity against the clinical relevant intracellular stage of L. mexicana with IC50 value of 0.33 microM. It was the only compound that exhibits a high activity on all the Leishmania species tested. This compound appeared to alter parasite microtubule organisation as demonstrated by using antibodies directed against microtubule components and more precisely the class of microtubule decorated by the MAP2-like protein. It is interesting to notice that this MAP2-like protein was identified for the first time in a Leishmania parasite     

Impaired insulin sensitivity and elevated ectopic fat in healthy obese vs. nonobese prepubertal children

Insulin sensitivity is impaired and ectopic fat (accretion of lipids outside of typical adipose tissue depots) increased in obese adults and adolescents. It is unknown how early in life this occurs; thus, it is important to evaluate young children to identify potential factors leading to the development of metabolic syndrome. We examined an ethnically diverse cohort of healthy, exclusively prepubertal children (N = 123; F = 57, M = 66; age 8.04 ± 0.77 years) to examine differences in insulin sensitivity and ectopic and visceral fat deposition between obese and nonobese youth. Obesity was categorized by age- and sex-adjusted BMI z-scores (nonobese = z-score <2 (N = 94) and obese = z-score ≥2 (N = 29)). Insulin sensitivity was assessed by both a frequently sampled intravenous glucose tolerance test (S(i)) and the homeostatic model assessment of insulin resistance (HOMA(IR)). Intramyocellular lipids (IMCLs) from soleus and intrahepatic lipids (IHLs) were assessed by magnetic resonance spectroscopy, visceral adipose tissue (VAT) by magnetic resonance imaging, and total body fat by dual-energy X-ray absorptiometry. We also examined serum lipids (total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol) and blood pressure (diastolic and systolic). Obese children exhibited significantly lower S(i) (5.9 ± 5.98 vs. 13.43 ± 8.18 (mμ/l)(-1)·min(-1), P = 0.01) and HDL-C and higher HOMA(IR) (1.68 ± 1.49 vs. 0.63 ± 0.47, P < 0.0001), IMCL (0.74 ± 0.39 vs. 0.44 ± 0.21% water peak, P < 0.0001), IHL (1.49 ± 1.13 vs. 0.54 ± 0.42% water peak, P < 0.0001), VAT (20.16 ± 8.01 vs. 10.62 ± 5.44 cm(2), P < 0.0001), total cholesterol, triglycerides, low-density lipoprotein cholesterol, and systolic blood pressure relative to nonobese children. These results confirm significantly increased ectopic fat and insulin resistance in healthy obese vs. nonobese children prior to puberty. Excessive adiposity during early development appears concomitant with precursors of type 2 diabetes and the metabolic syndrome.     

Transient Inability to Manage Proteobacteria Promotes Chronic Gut Inflammation in TLR5-Deficient Mice

Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E.?coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohn's disease-associated E.?coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis.