Effect of lower limb exercise on forearm vascular function: contribution of nitric oxide

We examined vascular function in an inactive muscle bed, the forearm, during lower limb exercise and determined the contribution of endothelium-derived nitric oxide (NO) to the hyperemic response. Eight young males were randomized to participate in two studies, each consisting of two bouts of lower limb exercise, separated by a 30-min recovery. Peak forearm blood flow (PFBF) and mean blood flow (MFBF) were continuously recorded at baseline and during exercise using continuous high-resolution vascular ultrasound and Doppler flow velocity measurement. During one session, the brachial artery was cannulated to allow continuous infusion of saline or N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthase. The alternate session was performed to control for possible effects of repeated exercise. At 60, 100, and 160 W, L-NMMA significantly decreased both PFBF and MFBF compared with the saline infusion. These results suggest that systemic production of NO occurs during exercise in resting vessel beds, which do not feed metabolically active tissue. This finding provides a plausible explanation for the antiatherogenic benefits of exercise.     

Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins

DnaJ-like proteins are defined by the presence of an approximately 73 amino acid region termed the J domain. This region bears similarity to the initial 73 amino acids of the Escherichia coli protein DnaJ. Although the structures of the J domains of E coli DnaJ and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the J domains of the various DnaJ-like proteins has yet been attempted. A multiple alignment of 223 J domain sequences was performed, and the levels of amino acid conservation at each position were established. It was found that the levels of sequence conservation were particularly high in 'true' DnaJ homologues (ie, those that share full domain conservation with DnaJ) and decreased substantially in those J domains in DnaJ-like proteins that contained no additional similarity to DnaJ outside their J domain. Residues were also identified that could be important for stabilizing the J domain and for mediating the interaction with heat shock protein 70.     

Rational Design of Holey 2D Nonlayered Transition Metal Carbide/Nitride Heterostructure Nanosheets for Highly Efficient Water Oxidation

Due to integrated advantages in electrochemical functionalities for energy conversion, 2D nonlayered heterostructure nanosheets offer new and fascinating opportunities for electrocatalysis but their fabrication is challenging when compared with the widely studied 2D layered heterostructure. Herein, a bottom‐up approach is established for facile synthesis of holey 2D transition metal carbide/nitride heterostructure nanosheets (h‐TMCN) with regulated hole sizes by controlled thermal annealing of the Mo/Zn bimetallic imidazolate frameworks (Mo/Zn BIFs). Ex situ phase and structural identifications disclose that the Mo/Zn BIFs precursor experiences interconnected three steps of transformation to produce h‐TMCN. Especially, the slow successive solid‐state diffusion of nitrogen and carbon into immediate noncrystalline molybdenum oxides allows the intergrowth of Mo₂C and Mo₂N into the 2D nonlayered heterostructure. X‐ray fine structure analysis coupled with high resolution X‐ray photoelectron spectroscopy demonstrate that Mo₂C and Mo₂N in the microdomains can chemically bond with each other, producing the abundant active N–Mo–C interfaces toward water splitting. Consequently, h‐TMCN affords low overpotentials, high turnover frequencies, rapid charge transfer, and superior long‐term stability toward electrocatalytic water oxidation. The present work demonstrates the feasibility of developing a broad range of 2D nonlayered heterostructures for high efficiency chemical energy conversion.     

Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin.

The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed both in CHO (Chinese Hamster Ovary) cells and in Pichia pastoris. The CHO and yeast expressed receptors showed identical affinity for EPO binding. Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a fourfold molar excess of receptor at high protein concentrations yielded stable EPO-EPObp complexes. Quantification of EPO and EPObp in the complex yielded a molar ratio of one EPO molecule to two receptor molecules. Residues that are responsible for EPOR glycosylation and isomerization in Pichia were identified and eliminated by site-specific mutagenesis. A thiol modification was identified and a method was developed to remove the modified species from EPObp. EPObp was complexed with erythropoietin (EPO) and purified. The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 A respectively. (Form 1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals, Inc. and Amgen, Inc. respectively.) Both contained one complex per asymmetric unit with a stoichiometry of two EPObps to one EPO.     

5-(pyridinon-1-yl)indazoles and 5-(furopyridinon-5-yl)indazoles as MCH-1 antagonists

A new series of 5-(pyridinon-1-yl)indazoles with MCH-1 antagonist activity were synthesized. Potential cardiovascular risk for these compounds was assessed based upon their interaction with the hERG potassium channel in a mini-patch clamp assay. Selected compounds were studied in a 5-day diet-induced obese mouse model to evaluate their potential use as weight loss agents. Structural modification of the 5-(pyridinon-1-yl)indazoles to give 5-(furopyridinon-5-yl)indazoles provided compounds with enhanced pharmacokinetic properties and improved efficacy.     

The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease.     

A POMC variant implicates beta-melanocyte-stimulating hormone in the control of human energy balance

The melanocortin-4 receptor (MC4R) plays a critical role in the control of energy balance. Of its two pro-opiomelanocortin (POMC)-derived ligands, alpha- and beta-MSH, the majority of attention has focused on alpha-MSH, partly reflecting the absence of beta-MSH in rodents. We screened the POMC gene in 538 patients with severe, early-onset obesity and identified five unrelated probands who were heterozygous for a rare missense variant in the region encoding beta-MSH, Tyr221Cys. This frequency was significantly increased (p < 0.001) compared to the general UK Caucasian population and the variant cosegregated with obesity/overweight in affected family members. Compared to wild-type beta-MSH, the variant peptide was impaired in its ability to bind to and activate signaling from the MC4R. Obese children carrying the Tyr221Cys variant were hyperphagic and showed increased linear growth, both of which are features of MC4R deficiency. These studies support a role for beta-MSH in the control of human energy homeostasis.