Sulfated homologues of heparin inhibit hepatitis C virus entry into mammalian cells

The mechanism of entry of hepatitis C virus (HCV) through interactions between the envelope glycoproteins and specific cell surface receptors remains unclear at this time. We have previously shown with the vesicular stomatitis virus (VSV)/HCV pseudotype model that the hypervariable region 1 of the HCV E2 envelope glycoprotein helps in binding with glycosaminoglycans present on the cell surface. In this study, we have examined the binding of HCV envelope glycoproteins with chemically modified derivatives of heparin. Furthermore, we have determined the functional relevance of the interaction of heparin derivatives with HCV envelope glycoproteins for infectivity by using a human immunodeficiency virus (HIV)/HCV pseudotype, a VSV/HCV pseudotype, and cell culture-grown HCV genotype 1a. Taken together, our results suggest that the HCV envelope glycoproteins rely upon O-sulfated esters of a heparin homologue to facilitate entry into mammalian cells.     

Exercise-induced activation of cardiac sympathetic nerve triggers cardioprotection via redox-sensitive activation of eNOS and upregulation of iNOS

We investigated the mechanism of exercise-induced late cardioprotection against ischemia-reperfusion (I/R) injury. C57BL/6 mice received treadmill exercise (60 min/day) for 7 days at a work rate of 60-70% maximal oxygen uptake. Exercise transiently increased oxidative stress and activated endothelial isoform of nitric oxide synthase (eNOS) during exercise and increased expression of inducible isoform of NOS (iNOS) in the heart after 7 days of exercise. The mice were subjected to regional ischemia by 30 min of occlusion of the left coronary artery, followed by 2 h of reperfusion. Infarct size was significantly smaller in the exercised mice. Ablation of cardiac sympathetic nerve by topical application of phenol abolished oxidative stress, activation of eNOS, upregulation of iNOS, and cardioprotection mediated by exercise. Treatment with the antioxidant N-(2-mercaptopropionyl)-glycine during exercise also inhibited activation of eNOS, upregulation of iNOS, and cardioprotection. In eNOS(-/-) mice, exercise-induced oxidative stress was conserved, but upregulation of iNOS and cardioprotection was lost. Exercise did not confer cardioprotection when the iNOS selective inhibitor 1400W was administered just before coronary artery occlusion or when iNOS(-/-) mice were employed. These results suggest that exercise stimulates cardiac sympathetic nerves that provoke redox-sensitive activation of eNOS, leading to upregulation of iNOS, which acts as a mediator of late cardioprotection against I/R injury.     

Impaired NO-mediated vasodilation with increased superoxide but robust EDHF function in right ventricular arterial microvessels of pulmonary hypertensive rats

Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload, eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n=126) to induce PH or with saline as controls (n=114). After 3 wk, coronary arterioles (diameter = 30-100 microm) and small arteries (diameter = 100-200 microm) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats, ACh-induced vasodilation was significantly attenuated in both arterioles and small arteries, especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity, whereas the EDHF-mediated vasodilation was relatively robust. The suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels.     

Large-scale, lineage-specific expansion of a bric-a-brac/tramtrack/broad complex ubiquitin-ligase gene family in rice

Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain-encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host.     

Neonatal lung side population cells demonstrate endothelial potential and are altered in response to hyperoxia-induced lung simplification

Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45(pos)/Flk-1(pos) and CD45(neg)/Flk-1(pos) bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.     

The nonsynonymous/synonymous substitution rate ratio versus the radical/conservative replacement rate ratio in the evolution of mammalian genes

There are 2 ways to infer selection pressures in the evolution of protein-coding genes, the nonsynonymous and synonymous substitution rate ratio (K(A)/K(S)) and the radical and conservative amino acid replacement rate ratio (K(R)/K(C)). Because the K(R)/K(C) ratio depends on the definition of radical and conservative changes in the classification of amino acids, we develop an amino acid classification that maximizes the correlation between K(A)/K(S) and K(R)/K(C). An analysis of 3,375 orthologous gene groups among 5 mammalian species shows that our classification gives a significantly higher correlation coefficient between the 2 ratios than those of existing classifications. However, there are many orthologous gene groups with a low K(A)/K(S) but a high K(R)/K(C) ratio. Examining the functions of these genes, we found an overrepresentation of functional categories related to development. To determine if the overrepresentation is stage specific, we examined the expression patterns of these genes at different developmental stages of the mouse. Interestingly, these genes are highly expressed in the early middle stage of development (blastocyst to amnion). It is commonly thought that developmental genes tend to be conservative in evolution, but some molecular changes in developmental stages should have contributed to morphological divergence in adult mammals. Therefore, we propose that the relaxed pressures indicated by the K(R)/K(C) ratio but not by K(A)/K(S) in the early middle stage of development may be important for the morphological divergence of mammals at the adult stage, whereas purifying selection detected by K(A)/K(S) occurs in the early middle developmental stage.     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.