Non-peptide alpha(v)beta(3) antagonists. Part 3: identification of potent RGD mimetics incorporating novel beta-amino acids as aspartic acid replacements.

Potent non-peptidic alpha(v)beta(3) antagonists have been prepared incorporating various beta-amino acids as aspartic acid mimetics. Modification of the beta-alanine 3-substituents alters the potency and physicochemical properties of these receptor antagonists and in some cases provides orally bioavailable alpha(v)beta(3) inhibitors.     

Locus-specific Changes in Cell Wall Composition Characteristic of Osmotic Mutants of Neurospora crassa

The osmotic phenotype of Neurospora crassa is characterized by inhibition of growth at high osmolalities of growth medium. Mutations at six osmotic loci of linkage group I were examined to assess the biochemical and physiological effects of these mutants. Isolated cell walls from 23 osmotic strains were compared with the wild type with regard to quantitative levels of the following components: percentage of total dry weight, total glucose, alkali-soluble glucose, nonglucose carbohydrates, amino acids, glucosamine, galactosamine, and a compound tentatively identified as quinovosamine. The last component has not previously been observed in N. crassa cell walls. Although the cell wall dry weight content of osmotic mutants was not altered, walls isolated from all of the osmotic strains had less alkali-insoluble glucose than those from the wild type. In addition, all of the loci except cut exhibited other consistent differences from the wild type. The os-1, os-3, and os-5 mutants had low levels of alkali-soluble glucose. The os-3 and os-5 mutants had high levels of nonglucose carbohydrates, and flm-2 had a low level of nonglucose carbohydrates. The os-4 mutants had low levels of galactosamine and amino acids and high levels alkali-soluble glucose. An os-1 mutant, B135, produced less of the whole alkali-soluble fraction of the cell wall.     

Functional anatomy of the female reproductive systems of two spider crabs (Decapoda,Majoidea)

To better understand the mating systems of majoid crabs, we studied the functional anatomy of the female reproductive systems of the spider crabs Leurocyclus tuberculosus and Libinia spinosa, comparing them with those of other Majoidea. Adult females were measured and dissected, and their reproductive systems described macroscopically and histologically. In females of both species, the seminal receptacles are paired globular structures of ecto‐mesodermal origin. The mesoderm‐derived region is lined by a stratified epithelium. The anchoring, proliferative, and secretory strata are clearly recognizable . The ectoderm‐derived region is lined by a simple cylindrical epithelium underlying a cuticle that increases in thickness toward the vagina. The transition between the ectoderm and mesoderm‐derived regions is abrupt, with differences between the studied species: Li. spinosa has a “velum,” whereas Le. tuberculosus presents prominent “folds.” In both species, the position in which the oviduct is connected to the seminal receptacles is intermediate between the dorsal and ventral types previously described in other eubrachyurans. The seminal receptacles of the studied species show four different conditions, which can be distinguished macroscopically based on their shape and amount of sperm stored. We compare our data with those from other Majoidea in an attempt to determine whether the morphology of the seminal receptacles is related to different mating strategies or behaviors.     

Myristoylation exerts direct and allosteric effects on Gα conformation and dynamics in solution

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen-deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.     

Unconventional Chemiosmotic Coupling of NHA2, a Mammalian Na+/H+ Antiporter,to a Plasma Membrane H+ Gradient

Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na⁺-Li⁺ counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H⁺ gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na⁺ electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H⁺-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na⁺ efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H⁺ gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H⁺-coupled transport is more widespread. The coexistence of Na⁺ and H⁺-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney.     

Complete Genome Sequence of Hepatitis E Virus from Rabbits in the United States

Hepatitis E virus (HEV) is a single-strand positive-sense RNA virus in the family Hepeviridae. The disease caused by HEV, hepatitis E, is an important public health problem in developing countries of Asia and Africa and is also endemic in many industrialized countries, including the United States. HEV has been identified from several other animal species in addition to humans, including the pig, chicken, mongoose, deer, rabbit, ferret, bat, and fish. Here we report the complete genome sequence of the first strain of HEV from rabbits in the United States. Sequence and phylogenetic analyses revealed that the U.S. rabbit HEV is a distant member of the zoonotic genotype 3 HEV, thus raising a concern for potential zoonotic human infection. A unique 90-nucleotide insertion within the X domain of the ORF1 was identified in the rabbit HEV, and this insertion may play a role in the species tropism of HEV.     

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0285-7) contains supplementary material, which is available to authorized users.     

Functional Feeds Reduce Heart Inflammation and Pathology in Atlantic Salmon (Salmo salar L.) following Experimental Challenge with Atlantic Salmon Reovirus (ASRV)

Heart and Skeletal Muscle Inflammation (HSMI), recently associated with a novel Atlantic salmon reovirus (ASRV), is currently one of the most prevalent inflammatory diseases in commercial Atlantic salmon farms in Norway. Mortality varies from low to 20%, but morbidity can be very high, reducing growth performance and causing considerable financial impact. Clinical symptoms, including myocarditis, myocardial and red skeletal muscle necrosis, correlate with the intensity of the inflammatory response. In the present study, the effects of two functional feeds (FF1 and FF2) were compared to a standard commercial reference feed (ST) in Atlantic salmon subjected to an ASRV challenge. The functional feeds had reduced levels of total lipid and digestible energy, and different levels and proportions of long-chain polyunsaturated fatty acids (LC-PUFA). The objective was to determine whether these feeds could provide effective protection by decreasing the inflammatory response associated with HSMI. Histopathology, viral load, fatty acid composition and gene expression of heart tissue were assessed over a period of 16 weeks post-infection with ASRV. The viral load and histopathology scores in heart tissue in response to ASRV infection were reduced in fish fed both functional feeds, with FF1 showing the greatest effect. Microarray hierarchical cluster analysis showed that the functional feeds greatly affected expression of inflammation/immune related genes over the course of the ASRV infection. Viral load correlated with up-regulation of pro-inflammatory genes at the early-mid stages of infection in fish fed the ST diet. Expression of inflammatory genes 16-weeks after ASRV challenge reflected the difference in efficacy between the functional feeds, with fish fed FF1 showing lower expression. Thus, severity of the lesions in heart tissue correlated with the intensity of the innate immune response and was associated with tissue fatty acid compositions. The present study demonstrated that dietary modulation through clinical nutrition had major influences on the development and severity of the response to ASRV infection in salmon. Thus, HSMI was reduced in fish fed the functional feeds, particularly FF1. The modulation of gene expression between fish fed the different feeds provided further insight into the molecular mechanisms and progression of the inflammatory and immune responses to ASRV infection in salmon.     

Framing the Salmonidae Family Phylogenetic Portrait: A More Complete Picture from Increased Taxon Sampling

Considerable research efforts have focused on elucidating the systematic relationships among salmonid fishes; an understanding of these patterns of relatedness will inform conservation- and fisheries-related issues, as well as provide a framework for investigating evolutionary mechanisms in the group. However, uncertainties persist in current Salmonidae phylogenies due to biological and methodological factors, and a comprehensive phylogeny including most representatives of the family could provide insight into the causes of these difficulties. Here we increase taxon sampling by including nearly all described salmonid species (n = 63) to present a time-calibrated and more complete portrait of Salmonidae using a combination of molecular markers and analytical techniques. This strategy improved resolution by increasing the signal-to-noise ratio and helped discriminate methodological and systematic errors from sources of difficulty associated with biological processes. Our results highlight novel aspects of salmonid evolution. First, we call into question the widely-accepted evolutionary relationships among sub-families and suggest that Thymallinae, rather than Coregoninae, is the sister group to the remainder of Salmonidae. Second, we find that some groups in Salmonidae are older than previously thought and that the mitochondrial rate of molecular divergence varies markedly among genes and clades. We estimate the age of the family to be 59.1 MY (CI: 63.2-58.1 MY) old, which likely corresponds to the timing of whole genome duplication in salmonids. The average, albeit highly variable, mitochondrial rate of molecular divergence was estimated as ∼0.31%/MY (CI: 0.27–0.36%/MY). Finally, we suggest that some species require taxonomic revision, including two monotypic genera, Stenodus and Salvethymus. In addition, we resolve some relationships that have been notoriously difficult to discern and present a clearer picture of the evolution of the group. Our findings represent an important contribution to the systematics of Salmonidae, and provide a useful tool for addressing questions related to fundamental and applied evolutionary issues.     

C. elegans BLOC-1 Functions in Trafficking to Lysosome-Related Gut Granules

The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that C. elegans glo-2 and snpn-1, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in glo-2 and snpn-1,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for glo-2 function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in C. elegans. To address the function of C. elegans BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(-) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(-) and BLOC-1(-) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in C. elegans.