NADPH-diaphorase expression in the meibomian glands of rat palpebra in postnatal development

In the current study, we aimed at investigating the presence of nitric oxide synthase (NOS) positive nerve fibers in rat meibomian glands (MGs) at various stages of development. There is good evidence to suggest that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) is a surrogate for neuronal nitric oxide synthase (NOS). Sections of the central, upper eyelids of Wistar rats were processed histochemically for NADPH-d to investigate the presence and distribution of NOS-positive nerve fibers at the following time points: day 1 and weeks 1, 2 and 3 post partum, and in adult controls. At day 1, MG acini were lightly stained and located at a distance from the mucosal border. Vessels were accompanied by intensely stained NADPH-d positive nerve fibers. At the week 1 time point, both the vessels and the NADPH-d positive fibers were still present, but less numerous. MGs were now closer to the mucosa, so that the submucosa was thinner. The acini were mostly pale but occasionally darker. At week 3, there were fewer blood vessels in both the submucosa and within the septa. Darker acini were more common than lightly stained acini. NADPH-d positive dots were observed in the vicinity of the MGs. At the week 3 time point, MGs were adjacent to the mucosal border and stained more intensely than at earlier times; almost all acini were stained. The microscopic appearances were almost identical with those of adult palpebra. Submucosal and septal blood vessels and NADPH-d positive nerve fibers were less numerous. NADPH-d histochemical staining confirmed differences in the density of stained nerve fibers at different developmental stages. The greatest density of NADPH-d -positive nerve fibers occurred in 1-day-old rats whereas they were less numerous in adult rat eyelids. Nerves innervating MGs utilize nitric oxide (NO) as a neurotransmitter mostly in early developmental stages and this need thereafter decreases and stabilizes at 3 weeks postnatally.     

Structural basis of enantioselective inhibition of cyclooxygenase-1 by S-alpha-substituted indomethacin ethanolamides

The modification of the nonselective nonsteroidal anti-inflammatory drug, indomethacin, by amidation presents a promising strategy for designing novel cyclooxygenase (COX)-2-selective inhibitors. A series of alpha-substituted indomethacin ethanolamides, which exist as R/S-enantiomeric pairs, provides a means to study the impact of stereochemistry on COX inhibition. Comparative studies revealed that the R- and S-enantiomers of the alpha-substituted analogs inhibit COX-2 with almost equal efficacy, whereas COX-1 is selectively inhibited by the S-enantiomers. Mutagenesis studies have not been able to identify residues that manifest the enantioselectivity in COX-1. In an effort to understand the structural impact of chirality on COX-1 selectivity, the crystal structures of ovine COX-1 in complexes with an enantiomeric pair of these indomethacin ethanolamides were determined at resolutions between 2.75 and 2.85 A. These structures reveal unique, enantiomer-selective interactions within the COX-1 side pocket region that stabilize drug binding and account for the chiral selectivity observed with the (S)-alpha-substituted indomethacin ethanolamides. Kinetic analysis of binding demonstrates that both inhibitors bind quickly utilizing a two-step mechanism. However, the second binding step is readily reversible for the R-enantiomer, whereas for the S-enantiomer, it is not. These studies establish for the first time the structural and kinetic basis of high affinity binding of a neutral inhibitor to COX-1 and demonstrate that the side pocket of COX-1, previously thought to be sterically inaccessible, can serve as a binding pocket for inhibitor association.     

Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Introduction  

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.     

Effects of supplemental oxygen administration on coronary blood flow in patients undergoing cardiac catheterization

Patients with heart disease are frequently treated with supplemental oxygen. Although oxygen can exhibit vasoactive properties in many vascular beds, its effects on the coronary circulation have not been fully characterized. To examine whether supplemental oxygen administration affects coronary blood flow (CBF) in a clinical setting, we measured in 18 patients with stable coronary heart disease the effects of breathing 100% oxygen by face mask for 15 min on CBF (via coronary Doppler flow wire), conduit coronary diameter, CBF response to intracoronary infusion of the endothelium-dependent dilator ACh and to the endothelium-independent dilator adenosine, as well as arterial and coronary venous concentrations of the nitric oxide (NO) metabolites nitrotyrosine, NO(2)(-), and NO(3)(-). Relative to breathing room air, breathing of 100% oxygen increased coronary resistance by approximately 40%, decreased CBF by approximately 30%, increased the appearance of nitrotyrosine in coronary venous plasma, and significantly blunted the CBF response to ACh. Oxygen breathing elicited these changes without affecting the diameter of large-conduit coronary arteries, coronary venous concentrations of NO(2)(-) and NO(3)(-), or the coronary vasodilator response to adenosine. Administering supplemental oxygen to patients undergoing cardiac catheterization substantially increases coronary vascular resistance by a mechanism that may involve oxidative quenching of NO within the coronary microcirculation.     

Phylogenetic analysis of ecomorphological divergence, community structure, and diversification rates in dusky salamanders (Plethodontidae: Desmognathus)

An important dimension of adaptive radiation is the degree to which diversification rates fluctuate or remain constant through time. Focusing on plethodontid salamanders of the genus Desmognathus, we present a novel synthetic analysis of phylogeographic history, rates of ecomorphological evolution and species accumulation, and community assembly in an adaptive radiation. Dusky salamanders are highly variable in life history, body size, and ecology, with many endemic lineages in the southern Appalachian Highlands of eastern North America. Our results show that life-history evolution had important consequences for the buildup of plethodontid-salamander species richness and phenotypic disparity in eastern North America, a global hot spot of salamander biodiversity. The origin of Desmognathus species with aquatic larvae was followed by a high rate of lineage accumulation, which then gradually decreased toward the present time. The peak period of lineage accumulation in the group coincides with evolutionary partitioning of lineages with aquatic larvae into seepage, stream-edge, and stream microhabitats. Phylogenetic simulations demonstrate a strong correlation between morphology and microhabitat ecology independent of phylogenetic effects and suggest that ecomorphological changes are concentrated early in the radiation of Desmognathus. Deep phylogeographic fragmentation within many codistributed ecomorph clades suggests long-term persistence of ecomorphological features and stability of endemic lineages and communities through multiple climatic cycles. Phylogenetic analyses of community structure show that ecomorphological divergence promotes the coexistence of lineages and that repeated, independent evolution of microhabitat-associated ecomorphs has a limited role in the evolutionary assembly of Desmognathus communities. Comparing and contrasting our results to other adaptive radiations having different biogeographic histories, our results suggest that rates of diversification during adaptive radiation are intimately linked to the degree to which community structure persists over evolutionary time.     

STIM1, an essential and conserved component of store-operated Ca2+ channel function

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.     

DOES NICHE CONSERVATISM PROMOTE SPECIATION? A CASE STUDY IN NORTH AMERICAN SALAMANDERS

Recent speciation research has generally focused on how lineages that originate in allopatry evolve intrinsic reproductive isolation, or how ecological divergence promotes nonallopatric speciation. However, the ecological basis of allopatric isolation, which underlies the most common geographic mode of speciation, remains poorly understood and largely unstudied. Here, we explore the ecological and evolutionary factors that promote speciation in Desmognathus and Plethodon salamanders from temperate eastern North America. Based on published molecular phylogenetic estimates and the degree of geographic range overlap among extant species, we find strong evidence for a role for geographic isolation in speciation. We then examine the relationship between climatic variation and speciation in 16 sister-taxon pairs using geographic information system maps of climatic variables, new methods for modeling species' potential geographic distributions, and data on geographic patterns of genetic variation. In contrast to recent studies in tropical montane regions, we found no evidence for parapatric speciation along climatic gradients. Instead, many montane sister taxa in the Appalachian Highlands inhabit similar climatic niches and seemingly are allopatric because they are unable to tolerate the climatic conditions in the intervening lowlands. This temporal and spatial-ecological pattern suggests that niche conservatism, rather than niche divergence, plays the primary role in promoting allopatric speciation and montane endemism in this species-rich group of vertebrates. Our results demonstrate that even the relatively subtle climatic differences between montane and lowland habitats in eastern North America may play a key role in the origin of new species.