Elafin-overexpressing mice have improved cardiac function after myocardial infarction

Elevated serine elastase activity after myocardial infarction can contribute to remodeling associated with left ventricular dilatation and dysfunction. We therefore assessed the effects of overexpressing the selective serine elastase inhibitor elafin in transgenic mice in which a myocardial infarction was caused by ligation of the left anterior descending coronary artery (LAD). Elevated serine elastase activity was observed in nontransgenic littermates as early as 6 h after LAD ligation and persisted at 4 and 7 days but not in sham-operated or elafin-overexpressing transgenic mice. Myeloperoxidase activity (index of inflammatory cells) and matrix metalloproteinase 2 were also increased but only at 4 and 7 days and only in nontransgenic mice (P < 0.05 for both comparisons), and this increase correlated with inflammatory cell infiltration. Echocardiographic study at 4 days revealed indexes of diastolic dysfunction in nontransgenic versus elafin-overexpressing mice (P < 0.05). Morphometric and biochemical analyses at 28 days indicated impairment in cardiac performance, with greater scar thinning and infarct expansion in nontransgenic versus elafin transgenic littermates (P < 0.05 for all comparisons). Thus serine elastase inhibition appears to suppress inflammation, cardiac dilatation, and dysfunction after myocardial infarct.     

Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence

We present a novel method for mapping proximity within proteins. The method exploits the quenching of the fluorescent label bimane by nearby Trp residues. In studies of T4 lysozyme we show that this effect appears to be distance dependent and orientation specific. Specifically, we show that a proximal Trp residue can reduce bimane fluorescence intensity by up to 500% and induce complicated fluorescence decay kinetics. Replacing the neighboring Trp residue with phenylalanine removes these spectral perturbations. The advantages of using the Trp quenching of bimane fluorescence for protein structural studies include the low amount of protein required and the substantial simplification of labeling strategies. We anticipate this method will prove suitable for a wide array of high-throughput protein studies such as protein folding, the detection of protein-protein interactions, and, most importantly, the dynamic monitoring of conformational changes.     

Differential regulation of gonadotropin-releasing hormone secretion and gene expression by androgen: membrane versus nuclear receptor activation

Steroid hormones induce rapid membrane receptor-mediated effects that appear to be separate from long-term genomic events. The membrane receptor-mediated effects of androgens on GT1-7 GnRH-secreting neurons were examined. We observed androgen binding activity with a cell-impermeable BSA-conjugated testosterone [testosterone 3-(O-carboxymethyl)oxime (T-3-BSA)] and were able to detect a 110-kDa protein recognized by the androgen receptor (AR) monoclonal MA1-150 antibody in the plasma membrane fraction of the GT1-7 cells by Western analysis. Further, a transfected green fluorescent protein-tagged AR translocates and colocalizes to the plasma membrane of the GT1-7 neuron. Treatment with 10 nM 5alpha-dihydrotestosterone (DHT) inhibits forskolin-stimulated accumulation of cAMP, through a pertussis toxin-sensitive G protein, but has no effect on basal cAMP levels. The inhibition of forskolin-stimulated cAMP accumulation by DHT was blocked by hydroxyflutamide, a specific inhibitor of the nuclear AR. DHT, testosterone (T), and T-3-BSA, all caused significant elevations in intracellular calcium concentrations ([Ca(2+)](i)). T-3-BSA stimulates GnRH secretion 2-fold in the GT1-7 neuron, as did DHT or T. Interestingly GnRH mRNA levels were down-regulated by DHT and T as has been reported, but not by treatment with T-3-BSA or testosterone 17beta-hemisuccinate BSA. These studies indicate that androgen can differentially regulate GnRH secretion and gene expression through specific membrane-mediated or nuclear mechanisms.     

Structural analysis of chick ephrin-A2 by function-blocking and non-blocking monoclonal antibodies

Ephrins, ligands for the Eph family of receptor tyrosine kinases, play key roles in diverse biological processes. In this study, we determined the epitopes and kinetic parameters of function-blocking (B3) and non-blocking (IV) monoclonal antibodies (mAbs) recognizing chick ephrin-A2. We show that the epitope for the non-blocking mAb is the residue Asp(105) of chick ephrin-A2. However, the binding of the function-blocking mAb depends mostly on residue Ser(108) and its epitope may reside within residues 105-132, which appear crucial for the receptor interaction site. Kinetic studies suggest a possible mechanism why mAb IV, despite recognizing a region very close to the mAb B3 epitope, fails to block the ligand-receptor interaction.     

Universal primers for the PCR-mediated amplification of DNA β

DNA β is an approx 1350 nucleotide, single-stranded DNA molecule which has been shown to be associated with some monopartite geminiviruses of the genus Begomovirus. This component requires the helper begomovirus for replication in the cells of host plants and for insect transmission, possibly by trans-encapsidation. Sequence comparisons of the two available DNA β sequences has identified a highly conserved region upstream of a predicted hairpin structure. Abutting primers designed to this conserved region allows PCR-mediated amplification of the full-length DNA β component from total nucleic acid extracts isolated from infected plants originating from a variety of geographically distinct sources and host plants.     

A 5-day intensive curriculum for interns utilizing simulation and active-learning techniques: addressing domains important across internal medicine practice

Objective

Simulation-based learning strategies have demonstrated improved procedural competency, teamwork skills, and acute patient management skills in learners. “Boot camp” curricula have shown immediate and delayed performance in surgical and medical residents. We created a 5-day intensive, simulation and active learning-based curriculum for internal medicine interns to address perceived gaps in cognitive, affective and psychomotor domains. Intern confidence and self-perceived competence was assessed via survey before and after the curriculum, along with qualitative data.

Results

A total of 33 interns completed the curriculum in 2014, 32 in 2015. Interns had a significant increase in confidence and self-perceived competence in procedural, cognitive and affective domains (all p values?<?.05).

     

Beating cold by being tough: impact of elevation on leaf characteristics in <Emphasis Type="Italic">Phleum himalaicum</Emphasis> Mez. endemic to Himalaya

Effect of altitude on leaf responses in Phleum himalaicum populations was evaluated at three different elevation levels, viz. (Low 1200 m.a.s.l.), middle (1600 m a.s.l.) and high (1900 m a.s.l.) in western part of Himalaya. We hypothesized that physico-chemical properties of soil varied along elevation and Phleum populations located at high elevation would adapt more distinct morphological and physiological traits than those originating from middle and low elevation sites. Our study revealed that soil pH, Ec Mg, Ca, and P decreased at high elevation however, significant increase was recorded in soil K, organic matter, and total nitrogen along the elevation gradient. A significant correlation between leaf characteristics and elevation sites was recorded along the gradient. The outcomes of this study showed that highland population had better adjustments under low temperature and exhibited adaptive traits. These were, decreased number of leaves and leaf area, increased leaf blade thickness, intensive sclerification, and greater stomatal and trichome density. Apart from these, high elevation population had more physiological adjustment in terms of low stomatal conductance, low transpiration rate, high water use efficiency, and synthesis of more osmolytes in leaf. We argued that certain level of sugar and protein must be attained by high population to dodge the aggressive climatic forces in order to grow successfully at the highest elevation. Furthermore, altitude between 1600 and 1900 m was more likely an optimum zone for vigorous growth of P. himalaicum at the highest level of elevation.     

Changes in insulin receptor,hexokinase and NADPH producing enzymes in Choroid plexus during experimental diabetes

The activities of insulin receptor and the enzymes hexokinase (EC 2.7.1.1) and NADP-dependent malic enzyme (EC1.1.1.40), glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in rat choroid plexus in alloxan induced diabetes. A significant decrease was observed in the activities of all the enzymes except isocitrate dehydrogenase and also the choroid plexus insulin receptor activity was decreased. A reversal of the efect was observed with insulin administration to diabetic rats. It may be concluded that the enzymes of choroid plexus together with insulin receptor are directly controlled by-the concentration of insulin.     

Synthesis of anionic poly(ethylene glycol) derivative for chitosan surface modification in blood-contacting applications

To improve blood compatibility, chitosan surface was modified by the complexa-tion-interpenetration method using an anionic derivative of poly(ethylene glycol) (PEG). Methoxypoly(ethylene glycol) sulfonate (MPEG sulfonate)-modified chitosan was prepared by allowing the base polymer to swell in an acidic medium, followed by polyelectrolyte complexation and interpenetration of MPEG sulfonate with the chitosan matrix. Addition of a strong base collapsed the base polymer to permanently immobilize the modifying agent on the surface. Electron spectroscopy for chemical analysis (ESCA) confirmed the presence of MPEG sulfonate on chitosan and the high resolution Cls peak showed an increase in -C—O- which is indicative of the ethylene oxide residues. The number of adherent platelets and the extent of platelet activation was significantly reduced on MPEG sulfonate-modified chitosan. Compared to an average of more than 66 fully activated platelets on unmodified chitosan surface, only 3.0 contact-adherent platelets were present on MPEG sulfonate-modified chitosan. Plasma recalcification time, a measure of the intrinsic coagulation reaction, was about 11.5 min in contact with modified chitosan. The results of this study show that chitosan surface can be modified by the complexation-interpenetration method with anionic PEG derivative. Surface-immobilized MPEG sulfonate was effective in preventing plasma protein adsorption and platelet adhesion and activation by the steric repulsion mechanism.