Role of endothelin in alpha-adrenoceptor coronary vasoconstriction

It has been proposed that alpha-adrenoceptor vasoconstriction in coronary resistance vessels results not from alpha-adrenoceptors on coronary smooth muscle but from alpha-adrenoceptors on cardiac myocytes that stimulate endothelin (ET) release. The present experiments tested the hypothesis that the alpha-adrenoceptor-mediated coronary vasoconstriction that normally occurs during exercise is due to endothelin. In conscious dogs (n = 10), the endothelin ET(A)/ET(B) receptor antagonist tezosentan (1 mg/kg iv) increased coronary venous oxygen tension at rest but not during treadmill exercise. This result indicates that basal endothelin levels produce a coronary vasoconstriction at rest that is not observed during the coronary vasodilation during exercise. In contrast, the alpha-adrenoceptor antagonist phentolamine increased coronary venous oxygen tension during exercise but not at rest. The difference between the endothelin blockade and alpha-adrenoceptor blockade results indicates that alpha-adrenoceptor coronary vasoconstriction during exercise is not due to endothelin. However, in anesthetized dogs, bolus intracoronary injections of the alpha-adrenoceptor agonist phenylephrine produced reductions in coronary blood flow that were partially antagonized by endothelin receptor blockade with tezosentan. These results are best explained if alpha-adrenoceptor-induced endothelin release requires high pharmacological concentrations of catecholamines that are not reached during exercise.     

Mutation discovery in the mouse using genetically guided array capture and resequencing

Forward genetics (phenotype-driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between observable phenotype and causative genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next-generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long-range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array-based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (~200 kb) from each of five noncomplementing Kit mutants (one known allele and four unknown alleles) and have successfully identified and validated a nonsynonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover causative mutations. Importantly, these data also provide a specific methodological foundation for the development of large-scale mutation detection efforts in the laboratory mouse. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. M. D’Ascenzo and C. Meacham contributed equally to this work.     

Visualization of aggregation of the Rnq1 prion domain and cross-seeding interactions with Sup35NM

Factors triggering the de novo appearance of prions are still poorly understood. In yeast, the appearance of one prion, [PSI(+)], is enhanced by the presence of another prion, [PIN(+)]. The [PSI(+)] and [PIN(+)] prion-forming proteins are, respectively, the translational termination factor Sup35 and the yet poorly characterized Rnq1 protein that is rich in glutamines and asparagines. The prion domain of Rnq1 (RnqPD) polymerizes more readily in vitro than the full-length protein. As is typical for amyloidogenic proteins, the reaction begins with a lag phase, followed by exponential growth. Seeding with pre-formed aggregates significantly shortens the lag. A generic antibody against pre-amyloid oligomer inhibits the unseeded but not the self-seeded reaction. As revealed by electron microscopy, RnqPD polymerizes predominantly into spherical species that eventually agglomerate. We observed infrequent fiber-like structures in samples taken at 4 h of polymerization, but in overnight samples SDS treatment was required to reveal fibers among agglomerates. Polymerization reactions in which RnqPD and the prion domain of Sup35 (Sup35NM) cross-seed each other proceeded with a shortened lag that only depends weakly on the protein concentration. Cross-seeded Sup35NM fibers appear to sprout from globular RnqPD aggregates as seen by electron microscopy. RnqPD spherical aggregates appear to associate with and, later occlude, Sup35NM seed fibers. Our kinetic and morphological analyses suggest that, upon cross-seeding, the aggregate provides the surface on which oligomers of the heterologous protein nucleate their subsequent amyloid formation.     

ICAM-1 and CD11b inhibition worsen outcome in rats with E. coli pneumonia.

We investigated whether inhibiting an endothelial adhesion molecule [intracellular adhesion molecule 1 (ICAM-1)] would alter outcome and lung injury in a similar fashion to inhibition of a leukocyte adhesion molecule (integrin CD11b) in a rat model of gram-negative pneumonia. Inhibition of ICAM-1 with monoclonal antibody (MAb) 1A29 (1 mg/kg sc or 0.2 or 2 mg/kg iv, q 12 h x 3) or of CD11b with MAb 1B6 (1 mg/kg sc, q 12 h x 3) were compared against similarly administered placebo proteins in rats challenged with intrabronchial Escherichia coli. After challenge, all animals were treated with antibiotics. ICAM-1 MAb (6 mg/kg, iv, total dose) increased mortality vs. control (P = 0.03). CD11b MAb (3 mg/kg, sc, total dose) did not significantly (P = 0.16) increase mortality rates, but this was not in a range of probability to exclude a harmful effect. All other doses of MAb had no significant effect on survival rates. ICAM-1 and CD11b MAbs had significantly different effects on the time course of lung injury, circulating white cells and lymphocytes, and lung lavage white cells and neutrophils (P = 0.04-0.003). CD11b MAb decreased, whereas ICAM-1 MAb increased these measures compared with control from 6 to 12 h after E. coli. However, from 144 to 168 h after E. coli both MAbs increased these measures compared with control rats but to a greater level with CD11b MAb. Thus both ICAM-1 and CD11b appear to be necessary for survival during E. coli pneumonia. Although these adhesion molecules may participate differently in early lung injury, with CD11b increasing and ICAM-1 decreasing inflammation and injury, both are important for the resolution of later injury. During gram-negative pneumonia the protective roles of ICAM-1 and CD11b may make their therapeutic inhibition difficult.     

Control of coronary blood flow during exercise

Under normal physiological conditions, coronary blood flow is closely matched with the rate of myocardial oxygen consumption. This matching of flow and metabolism is physiologically important due to the limited oxygen extraction reserve of the heart. Thus, when myocardial oxygen consumption is increased, as during exercise, coronary vasodilation and increased oxygen delivery are critical to preventing myocardial underperfusion and ischemia. Exercise coronary vasodilation is thought to be mediated primarily by the production of local metabolic vasodilators released from cardiomyocytes secondary to an increase in myocardial oxygen consumption. However, despite various investigations into this mechanism, the mediator(s) of metabolic coronary vasodilation remain unknown. As will be seen in this review, the adenosine, K(+)(ATP) channel and nitric oxide hypotheses have been found to be inadequate, either alone or in combination as multiple redundant compensatory mechanisms. Prostaglandins and potassium are also not important in steady-state coronary flow regulation. Other factors such as ATP and endothelium-derived hyperpolarizing factors have been proposed as potential local metabolic factors, but have not been examined during exercise coronary vasodilation. In contrast, norepinephrine released from sympathetic nerve endings mediates a feed-forward betaadrenoceptor coronary vasodilation that accounts for approximately 25% of coronary vasodilation observed during exercise. There is also a feed-forward alpha-adrenoceptor-mediated vasoconstriction that helps maintain blood flow to the vulnerable subendocardium when heart rate, myocardial contractility, and oxygen consumption are elevated during exercise. Control of coronary blood flow during pathophysiological conditions such as hypertension, diabetes mellitus, and heart failure is also addressed.     

Neurexin in Embryonic Drosophila Neuromuscular Junctions

Background

Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described.

Methodology/Principal Findings

We use a variety of techniques, including immunohistochemistry, electron microscopy, in situ hybridization, and electrophysiology, to characterize neurexin expression and phenotypes in embryonic Drosophila neuromuscular junctions (NMJs). Our results surprisingly suggest that neurexin in embryos is present both pre and postsynaptically. Presynaptic neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic neurexin, also suppresses formation of ectopic glutamate receptor clusters. Interestingly, we find that loss of neurexin only affects receptors containing the subunit GluRIIA.

Conclusions/Significance

Our study extends previous results and provides important detail regarding the role of neurexin in Drosophila glutamate receptor abundance. The possibility that neurexin is present postsynaptically raises new hypotheses regarding neurexin function in synapses, and our results provide new insights into the role of neurexin in synapse development.     

A finite element analysis of masticatory stress hypotheses

Understanding how the skull transmits and dissipates forces during feeding provides insights into the selective pressures that may have driven the evolution of primate skull morphology. Traditionally, researchers have interpreted masticatory biomechanics in terms of simple global loading regimes applied to simple shapes (i.e., bending in sagittal and frontal planes, dorsoventral shear, and torsion of beams and cylinders). This study uses finite element analysis to examine the extent to which these geometric models provide accurate strain predictions in the face and evaluate whether simple global loading regimes predict strains that approximate the craniofacial deformation pattern observed during mastication. Loading regimes, including those simulating peak loads during molar chewing and those approximating the global loading regimes, were applied to a previously validated finite element model (FEM) of a macaque (Macaca fascicularis) skull, and the resulting strain patterns were compared. When simple global loading regimes are applied to the FEM, the resulting strains do not match those predicted by simple geometric models, suggesting that these models fail to generate accurate predictions of facial strain. Of the four loading regimes tested, bending in the frontal plane most closely approximates strain patterns in the circumorbital region and lateral face, apparently due to masseter muscle forces acting on the zygomatic arches. However, these results indicate that no single simple global loading regime satisfactorily accounts for the strain pattern found in the validated FEM. Instead, we propose that FE models replace simple cranial models when interpreting bone strain data and formulating hypotheses about craniofacial biomechanics.     

Evolutionarily conserved thermal biology across continents: The North American lizard Plestiodon gilberti (Scincidae) compared to Asian Plestiodon

1.

The lizard genus Plestiodon (Scincidae; formerly Eumeces) is widespread in North America and Asia, but the thermal biology of only a few species has been studied.