Temporal Changes of Angiopoietins and Tie2 Expression in Rat Lungs after Monocrotaline-Induced Pulmonary Hypertension

Angiogenic factors such as vascular endothelial growth factor (VEGF) are implicated in pulmonary hypertension (PH). However, the pathway of angiogenic factor-mediated pathologic angiogenesis in PH remains unclear. In this study, we evaluated the temporal expression of angiopoietin (Ang) 1, Ang2, and their receptor (Tie2) as well as VEGF, endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and heme oxygenase 1 (HO1) in the monocrotaline-induced PH model. Histologic evaluation showed pathologic vascular remodeling in the arteries of lung sections 1 wk after monocrotaline treatment. Protein levels of Ang1, Ang2, eNOS, iNOS, HO1, and VEGF were increased 1 wk after monocrotaline treatment but Tie2 protein levels were decreased 2 wk afterward. These results suggest that Ang2 mediates vascular remodeling in PH by decreasing Tie2 expression. Therefore, the Ang–Tie2 system may play a role in the pathophysiology of PH.Abbreviations: Ang, angiopoietin; eNOS, endothelial nitric oxide synthase; HO1, heme oxygenase 1; iNOS, inducible nitric oxide synthase; PH, pulmonary hypertension; VEGF, vascular endothelial growth factorPulmonary hypertension (PH) is a disease characterized by pathologic angiogenesis caused by diffuse smooth muscle cell hyperplasia and hypertrophy of the distal pulmonary vasculature, resulting in obliteration of small pulmonary arterioles.¹³ Vascular remodeling is governed by the interaction of several angiogenic factors on endothelial and smooth muscle cells. Vascular remodeling requires complex, multistep signaling pathways and a high degree of spatial and temporal coordination among endothelial and smooth muscle cells.²⁹ However, the precise mechanisms of vascular remodeling at the cellular and molecular levels are not completely defined.The angiopoietin (Ang) family and vascular endothelial growth factor (VEGF) are 2 types of vascular regulatory molecules that have been the subject of intense investigation in both physiologic and pathologic generation of blood vessels.^2,38 Members of the Ang family have opposing effects on receptor activation, with Ang1 stimulating Tie2 and Ang2 antagonizing this stimulation.^3,8,22 In particular, Ang1 plays an important role in the assembly of newly formed vasculature and in the maintenance of vascular integrity.^7,14,36 In contrast, Ang2 antagonizes the activation of Tie2 by Ang1 and causes endothelial cell apoptosis and vascular regression.²² The functions of Ang2 appear to be more complex than those of Ang1, in that Ang2 binds to the Tie2 receptor, blocking Ang1–Tie2 signaling and acting as a vessel-destabilizing factor.²⁶ However, prolonged exposure of endothelial cells to Ang2 activates Tie2 signaling.¹⁶ Thus, the precise roles of Ang2 during the development of PH are not well understood. Tie2 is a receptor tyrosine kinase that is expressed principally on vascular endothelium and that plays a role in integrity and survival of endothelial cells.^27,30 Disrupting Tie2 function in mice results in embryonic lethality with defects in embryonic vasculature.¹¹Neither Ang1 nor Ang2 alone can trigger an angiogenic response, but both enhance angiogenesis or induce vascular remodeling, depending on the presence of VEGF or other angiogenic factors. Nitric oxide is produced by endothelial cells through the action of nitric oxide synthase (NOS). Northern blot analysis of hypoxic rat lungs showed significantly increased mRNA levels for both endothelial NOS (eNOS) and inducible NOS (iNOS).¹⁸ Increased NOS activity coincided with the beginning of the vascular remodeling process during chronic hypoxia.³⁷ Hypoxia and nitric oxide stimulate VEGF production and induce HO1 expression in vascular tissue.¹⁰ In addition, several studies have shown that VEGF works in conjunction with other angiogenic factors to produce a stable and functional microvasculature.^21,35The purpose of the present study was to demonstrate the temporal changes of several angiogenic factors during the development of PH induced by treatment of rats with monocrotaline. This research was focused on the Ang–Tie2 system and other angiogenic factors and suggested that this system plays an important role in modulating vascular remodeling during PH.     

Catechin and epicatechin from Smilacis chinae rhizome protect cultured rat cortical neurons against amyloid beta protein (25-35)-induced neurotoxicity through inhibition of cytosolic calcium elevation

We previously reported that the Smilacis chinae rhizome inhibits amyloid beta protein (25-35) (Abeta (25-35))-induced neurotoxicity in cultured rat cortical neurons. Here, we isolated catechin and epicatechin from S. chinae rhizome and also studied their neuroprotective effects on Abeta (25-35)-induced neurotoxicity in cultured rat cortical neurons. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced neuronal cell death at a concentration of 10 microM, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Catechin and epicatechin also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC, generation of reactive oxygen species (ROS) and activation of caspase-3. These results suggest that catechin and epicatechin prevent Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity. Furthermore, these effects of catechin and epicatechin may be associated with the neuroprotective effect of the S. chinae rhizome.     

Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.)

Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6−phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Synthetic statistical approach reveals a high degree of richness of microbial eukaryotes in an anoxic water column

Molecular surveys suggest that communities of microbial eukaryotes are remarkably rich, because even large clone libraries seem to capture only a minority of species. This provides a qualitative picture of protistan richness but does not measure its real extent either locally or globally. Statistical analysis can estimate a community's richness, but the specific methods used to date are not always well grounded in statistical theory. Here we study a large protistan molecular survey from an anoxic water column in the Cariaco Basin (Caribbean Sea). We group individual 18S rRNA gene sequences into operational taxonomic units (OTUs) using different cutoff values for sequence similarity (99 to 50%) and systematically apply parametric models and nonparametric estimators to the OTU frequency data to estimate the total protistan diversity. The parametric models provided statistically sound estimates of protistan richness, with biologically meaningful standard errors, maximal data usage, and extensive model diagnostics and were preferable to the available nonparametric tools. Our clone library exceeded 700 clones but still covered only a minority of species and less than half of the larger protistan clades. Our estimates of total protistan richness portray the target community as very rich at all OTU levels, with hundreds of different populations apparently co-occurring in the small (3-liter) volume of our sample, as well as dozens of clades of the highest taxonomic order. These estimates are among the first for microbial eukaryotes that are obtained using state-of-the-art statistical methods and can serve as benchmark numbers for the local diversity of protists.     

Survivin protein expression in bovine follicular oocytes and their in vitro developmental competence

This study examined the relationship between survivin expression and the stage of development of in vitro cultured bovine oocytes and embryos; and whether survivin expression is affected by the quality of cumulus–oocyte complexes (COCS) or the quality of pre-implantation embryos. A polyclonal antibody was prepared using recombinant bovine survivin protein. Expression of survivin mRNA and protein was analyzed by real-time quantitative RT-PCR and immunocytochemistry. In the first experiment, survivin mRNA expression was examined at developmental stages from germinal vesicle (GV) oocyte to blastocyst, it was significantly decreased after fertilization of matured oocytes (P < 0.05), then increased slightly to the 8-cell stage followed by rapid increases at the morula and blastocyst stages (P < 0.05). In the second experiment, the effect of oocyte quality on survivin protein, pro-apoptotic (bax, caspase-3) and anti-apoptotic (survivin, bax inhibitor) mRNA expression was examined. Survivin protein was more strongly expressed in good quality COCS than in poor quality COCS. The expression of the anti-apoptotic genes, survivin and bax inhibitor, was significantly higher (P < 0.05) and that of the pro-apoptotic genes, bax and caspase-3, was significantly lower (P < 0.05) in good compared to poor quality COCS. The developmental competence of good quality COCS (30.4% blastocysts) was significantly better than that of poor quality COCS. In the last experiment also, we confirmed that significantly higher expression of survivin and bax inhibitor genes and significantly lower expression of bax and caspase-3 genes was resulted in good quality blastocysts than in poor quality blastocysts (P < 0.05). It was concluded that the expression of survivin was related to the quality of COCS, their developmental competence and the quality of in vitro produced blastocysts. Consequently, survivin may be a good candidate marker for embryo quality.     

Genetic characteristics of 207 microsatellite markers in the Korean population and in other Asian populations

Microsatellites, short tandem repeats, are useful markers for genetic analysis because of their high frequency of occurrence over the genome, high information content due to variable repeat lengths, and ease of typing. To establish a panel of microsatellite markers useful for genetic studies of the Korean population, the allele frequencies and heterozygosities of 207 microsatellite markers in 119 unrelated Korean, Indian and Pakistani individuals were compared. The average heterozygosity of the Korean population was 0.71, similar to that of the Indian and Pakistani populations. More than 80% of the markers showed heterozygosity of over 0.6 and were valuable as genetic markers for genome-wide screening for disease susceptibility loci in these populations. To identify the allelic distributions of the multilocus genetic data from these microsatellite markers, the population structures were assessed by clustering. These markers supported, with the most probability, three clustering groups corresponding to the three geographical populations. When we assumed only two hypothetical clusters (K), the Korean population was separate from the others, suggesting a relatively deep divergence of the Korean population. The present 207 microsatellite markers appear to reflect the historical and geographical origins of the different populations as well as displaying a similar degree of variation to that seen in previously published genetic data. Thus, these markers will be useful as a reference for human genetic studies on Asians.     

Preparation of sodium deoxycholate (DOC) conjugated heparin derivatives for inhibition of angiogenesis and cancer cell growth

We describe new DOC (sodium deoxycholate)-heparin nanoparticles for in vivo tumor targeting and inhibition of angiogenesis based on chemical conjugation and the enhanced permeability and retention (EPR) effect. Heparin has been used as a potent anticoagulant agent for 70 years, and has recently been found to inhibit the activity of growth factors which stimulate the smooth muscle cells around tumor. From the results, DOC and heparin were conjugated by bonding carboxyl groups of heparin with amine groups of aminated sodium deoxycholate. Larger antitumor effects of the DOC-heparin VI (8.5 mol of DOC coupled with 1.0 mol heparin) were achieved in animal studies, compared to heparin alone. We confirmed that the conjugated heparin retained its ability to inhibit binding with angiogenic factor, showing a significant decrease in endothelial tubular formation. These results provide new insights into the nontoxic anticancer drug carrier as well as the design of multifunctional bioconjugates for targeted drug delivery.     

Host discrimination and interspecific competition of Trissolcus nigripedius and Telenomus gifuensis (Hymenoptera: Scelionidae), sympatric parasitoids of Dolycoris baccarum (Heteroptera: Pentatomidae)

Trissolcus nigripedius Nakagawa and Telenomus gifuensis Ashmead (Hymenoptera: Scelionidae) are solitary egg parasitoids of Dolycoris baccarum L. (Heteroptera: Pentatomidae), a polyphagous seed-sucking bug of agricultural crops. Field examinations revealed that the two parasitoids are the most common species, but only a single species emerge from a host egg mass. To explain this observation we tested two hypotheses of interspecific host discrimination and asymmetry in competitive interaction between Tr. nigripedius and Te. gifuensis. Trissolcus nigripedius and Te. gifuensis could discriminate host egg pre-parasitized by either self or conspecific like other scelionid parasitoids that use external mark on host eggs. When provided with host eggs pre-parasitized by each other, both Tr. nigripedius and Te. gifuensis multiparasitized 94% and 100% of the host eggs without interspecific host discrimination, respectively. Interestingly, from the multiparasitized eggs irrespective of oviposition sequence, progeny of Te. gifuensis always survive better than Tr. nigripedius. Telenomus gifuensis is superior in immature competition probably due to shorter egg incubation period after oviposition, hence progeny of Te. gifuensis become first instar faster than that of Tr. nigripedius. However, adult Tr. nigripedius is always superior competitor in possessing and guarding the host eggs even after oviposition against Te. gifuensis. Therefore, the asymmetry in competitive interaction between Tr. nigripedius and Te. gifuensis may explain the emergence of a single species from a host egg mass in the field in spite of no interspecific host discrimination.     

A plant growth promoting rhizobacterium, Paenibacillus polymyxa strain GBR-1, suppresses root-knot nematode

Exposure of root-knot nematode, Meloidogyne incognita to various concentrations (5-100%) of culture filtrate of Paenibacillus polymyxa GBR-1 under in vitro conditions significantly reduced egg hatch and caused substantial mortality of its juveniles. The increase in the exposure durations of juveniles to culture filtrate and its concentrations increased the mortality rate. Similarly, higher concentrations increased its inhibitory effect on egg hatch. In higher concentrations (25-100%) egg hatch was inhibited by 84-91% after 2 days of exposures as compared to control in sterile distilled water. Application of various concentrations of culture filtrate extract or bacterial suspension of P. polymyxa GBR-1 into potting soil infested with 2000 J2 of M. incognita, reduced the root galling and nematode populations and increased tomato plant growth and root-mass production compared with untreated control (P< or = 0.05). The beneficial effect of P. polymyxa GBR-1 into potted soil increased exponentially with the increase in dose concentrations. Root gall index was reduced from 4.8 to 1.4 and 1.8 when potting soil was treated with 10% concentrations of culture filtrate extract and bacterial suspension, respectively, compared with untreated control. Application of bacterial suspension of P. polymyxa GBR-1 into potted soil at 3 day pre-inoculation of nematode was the most effective followed by simultaneously and at 2 days post-inoculation; as root galling was reduced by 62.5%, 58.3% and 50.0%, respectively, compared with untreated control.