Nav1.4 deregulation in dystrophic skeletal muscle leads to Na+ overload and enhanced cell death

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.     

Is Obesity Associated With Anemia of Chronic Disease? A Population‐based Study

Obesity is characterized by chronic, low-grade, systemic inflammation, which, in turn, has been associated with anemia of chronic disease. We hypothesized that obesity may be associated with the features of anemia of chronic disease, including low hemoglobin concentration, low serum iron and transferrin saturation (TS), and elevated serum ferritin. We compared normal-weight to overweight and obese adult participants of the third National Health and Nutrition Examination Survey with respect to hemoglobin concentration and levels of serum iron, TS, and ferritin. Measured BMI was used to categorize participants into normal weight (BMI < 25 kg/m(2), n = 6,059), overweight (BMI 25 to <30 kg/m(2), n = 5,108), mildly obese (BMI 30 to <35 kg/m(2), n = 2,366), moderately obese (BMI 35 to <40 kg/m(2), n = 850), and severely obese (BMI > or = 40 kg/m(2), n = 465). After adjustment for age, gender, menstruation, race/ethnicity, education, alcohol consumption, smoking, blood donation, and dietary iron intake, serum ferritin was progressively higher with increasing BMI category, whereas serum iron and TS were progressively lower. However, compared to normal-weight persons, those in all other higher BMI categories did not have a significant change in hemoglobin concentration after adjustment for the above-mentioned confounders. Overweight and obesity were associated with changes in serum iron, TS, and ferritin that would be expected to occur in the setting of chronic, systemic inflammation. However, overweight and obese persons were not more likely to be anemic compared with normal-weight persons.     

Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC(6')-Ib and its bifunctional, fluoroquinolone-active AAC(6')-Ib-cr variant

Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently discovered bifunctional aminoglycoside acetyltransferase (AAC(6')-Ib variant, AAC(6')-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6')-Ib-wt and its bifunctional variant AAC(6')-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6')-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 A. Observation of the direct, and optimally positioned, interaction between the 6'-NH 2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6')-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6')-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes pi-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6')-Ib-wt and the AAC(6')-Ib-cr variant involving a ternary complex.     

Proline bis-amides as potent dual orexin receptor antagonists

A series of OX(2)R/OX(1)R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered. N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed peripherally in a pharmacodynamic model of orexin activity.     

Assessment of sublethal effects of methoxyfenozide on oriental fruit Moth (Lepidoptera: Tortricidae)

Sublethal effects of the insect growth regulator methoxyfenozide were examined in oriental fruit moth, Grapholita molesta (Busck), in laboratory and field studies. In laboratory studies, oriental fruit moth larvae reared on diet amended with 0.1 ppm methoxyfenozide developed at the same rate as larvae reared on untreated diet, and paired moths reared as larvae from the same treated or untreated diets exhibited similar fecundity and fertility. Population growth differences over multiple generations were used to examine sublethal effects of methoxyfenozide on population dynamics in the field. Multiple single-tree cages were placed over apple (Malus spp.) trees treated with two applications of methoxyfenozide (70 g [AI] /ha) and nontreated trees. Cages were infested at a single time point with virgin male and female oriental fruit moth adults, and population growth was evaluated by egg counts, shoot infestation, fruit damage, and larval counts over a 12-wk period. Significantly fewer eggs, larvae, and damaged fruit were found on methoxyfenozide-treated compared with nontreated trees in 2001. Observed population differences may have been a result of direct mortality to eggs and larvae of the first generation rather than sublethal effects. In 2002, no differences were observed between treatments, but a heavy rain event shortly after the early infestation impacted the experiment. A late moth release treatment was tested in 2002 to examine the effects of residual methoxyfenozide 55 d after initial application. Significantly fewer eggs were found in the methoxyfenozide treatment compared with the control, but no differences existed among treatments in shoot infestation, percentage of damaged fruit, or larval populations. It was concluded direct mortality of eggs and larvae exposed to methoxyfenozide rather than sublethal effects were most important in reduction of subsequent generations.     

Predicting the subcellular localization of human proteins using machine learning and exploratory data analysis

Identifying the subcellular localization of proteins is particularly helpful in the functional annotation of gene products. In this study, we use Machine Learning and Exploratory Data Analysis （EDA） techniques to examine and characterize amino acid sequences of human proteins localized in nine cellular compartments. A dataset of 3,749 protein sequences representing human proteins was extracted from the SWISS-PROT database. Feature vectors were created to capture specific amino acid sequence characteristics. Relative to a Support Vector Machine, a Multi-layer Perceptron, and a Naive Bayes classifier, the C4.5 Decision Tree algorithm was the most consistent performer across all nine compartments in reliably predicting the subcellular localization of proteins based on their amino acid sequences （average Precision=0.88; average Sensitivity=0.86）. Furthermore, EDA graphics characterized essential features of proteins in each compartment. As examples, proteins localized to the plasma membrane had higher proportions of hydrophobic amino acids; cytoplasmic proteins had higher proportions of neutral amino acids; and mitochondrial proteins had higher proportions of neutral amino acids and lower proportions of polar amino acids. These data showed that the C4.5 classifier and EDA tools can be effective for characterizing and predicting the subcellular localization of human proteins based on their amino acid sequences.