Hemodynamic Effects of the Non-Peptidic Angiotensin-(1-7) Agonist AVE0991 in Liver Cirrhosis

Background & Aims

Although in cirrhosis with portal hypertension levels of the vasoconstrictor angiotensin II are increased, this is accompanied by increased production of angiotensin (Ang)-(1–7), the endogenous ligand of the Mas receptor (MasR), which blunts hepatic fibrosis and decreases hepatic vascular resistance. Therefore, we investigated the effects of the non-peptidic Ang-(1–7) agonist, AVE0991, in experimental cirrhosis.

Methods

Cirrhosis was induced by bile duct ligation (BDL) or carbon tetrachloride (CCl₄) intoxication. The coloured microsphere technique assessed portal and systemic hemodynamic effects of AVE0991 in vivo. Hepatic expression of eNOS, p-eNOS, iNOS, JAK2, ROCK and p-Moesin were analyzed by western blots. Activities of ACE and ACE2 were investigated fluorometrically. Moreover, fibrosis was assessed in BDL rats receiving AVE0991.

Results

In vivo, AVE0991 decreased portal pressure (PP) in both rat models of cirrhosis. Importantly, systemic effects were not observed. The hepatic effects of AVE0991 were based on upregulation of vasodilating pathways involving p-eNOS and iNOS, as well as by downregulation of the vasoconstrictive pathways (ROCK, p-Moesin). Short-term treatment with AVE0991 decreased the activity of ACE2, long-term treatment did not affect hepatic fibrosis in BDL rats.

Conclusions

The non-peptidic agonist of Ang-(1–7), AVE0991, decreases portal pressure without influencing systemic pressure. Thus, although it does not inhibit fibrosis, AVE0991 may represent a promising new therapeutic strategy for lowering portal pressure.     

Structure–activity relationship and improved hydrolytic stability of pyrazole derivatives that are allosteric inhibitors of West Nile Virus NS2B-NS3 proteinase

West Nile Virus (WNV) is a potentially deadly mosquito-borne flavivirus which has spread rapidly throughout the world. Currently there is no effective vaccine against flaviviral infections. We previously reported the identification of pyrazole ester derivatives as allosteric inhibitors of WNV NS2B-NS3 proteinase. These compounds degrade rapidly in pH 8 buffer with a half life of 1–2 h. We now report the design, synthesis and in vitro evaluation of pyrazole derivatives that are inhibitors of WNV NS2B-NS3 proteinase with greatly improved stability in the assay medium.     

Comparison of Post-Mine Rehabilitated and Natural Shrubland Communities in Southwestern Australia

Following mineral sand mining near Eneabba, southwestern Australia, rehabilitation managers have the difficult task of restoring shrubland communities of exceptional plant species richness. Species diversity, composition, structure, and key functional attributes in four mined sites rehabilitated 8 (R8) to 24 (R24) years ago were compared with those of typical nearby natural areas classified on the basis of substrate type (Low and High sand Dunes, shallow sand Swales, sand over Laterite, and sand over Limestone). The rehabilitated sites (except R8) had more species (about 140) than natural sites (about 100) in 40 × 40–m plots, with 12–37% species in common with natural sites. Rehabilitated sites were more similar in composition to each other than they were to the natural sites, with two strong colonizers, the fire-killed Acacia blakelyi and the fire-tolerant Melaleuca leuropoma , universally present. Dendrograms and ordinations based on composition and cover showed that rehabilitated sites grouped with each other before they did with the Dune and Swale sites (physically closest), and last with the Laterite and Limestone sites. Plant densities for R16 and R24 were about half those of the High Dune and Limestone, and about a quarter those of the Swale and Laterite. Fire resprouters were under-represented in the rehabilitated sites. Growth form distribution in rehabilitated sites was most similar to those of the dunes, with some woody shrubs up to 2.5 m tall present. Total iron and soil hardness (penetrability) were the only soil factors consistently different (higher) in the rehabilitated sites.     

Eph/Ephrin membrane proteins: a mammalian expression vector pTIg-BOS-Fc allowing rapid protein purification

There is an urgent need for high purity, single chain, fully functional Eph/ephrin membrane proteins. This report outlines the pTIg-BOS-Fc vector and purification approach resulting in rapid increased production of fully functional single chain extracellular proteins that were isolated with high purity and used in structure-function analysis and pre-clinical studies.     

Multiple Shoot Regeneration from Young Shoots of Kenaf (Hibiscus cannabinus)

A method was developed to initiate multiple shoots from the young shoot of kenaf. Young shoots along with the cotyledons were excised from ten-day old aseptically germinated seeds and pre-cultured for two weeks in Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) or a combination of BA and kinetin. After two weeks in culture, elongated shoots were excised above the cotyledonary nodes and cultured on fresh medium of the same composition. Multiple shoots were initiated within eight weeks. The number of shoots varied among cultivars. The highest number of shoots (11/explant) occured in cultivar Tainung 2 (T2) cultured in MS medium supplemented with 8.8 μM BA. Concentrations of BA higher than 8.8 μM had a negative effect on the number of shoots. Furthermore, callus growth was initiated from which morphologically abnormal shoots were induced. Kinetin had a significant effect only on cultivar Everglades 41 (E41). Shoot elongation and rooting were obtained simultaneously in half strength MS basal medium with no plant growth regulators. About 98% of the rooted plants were grown to maturity under greenhouse conditions. This method was successful with all four genotypes tested. However, significant genotypic variations were observed among the genotypes.     

Tetra- and Penta-Acylated Lipid A Structures of Porphyromonas gingivalis LPS Differentially Activate TLR4-Mediated NF-κB Signal Transduction Cascade and Immuno-Inflammatory Response in Human Gingival Fibroblasts

Background

Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS), as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS_1435/1449) and penta-acylated (LPS₁₆₉₀) structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions.

Methodology/Principal Findings

This study systematically investigated the effects of P. gingivalis LPS_1435/1449 and LPS₁₆₉₀ on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs). We found that LPS_1435/1449 and LPS₁₆₉₀ differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS₁₆₉₀ but not by LPS_1435/1449. In addition, LPS₁₆₉₀ induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS₁₆₉₀ while down-regulated by LPS_1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS₁₆₉₀-induced expression of IL-6 and IL-8 in HGFs.

Conclusions/Significance

The present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in gingival tissues, thereby contributing to periodontal pathogenesis.     

Equilibration of H labeling between body water and free amino acids: Enabling studies of proteome synthesis

Protein synthesis can be estimated by measuring the incorporation of a labeled amino acid into a proteolytic peptide. Although prelabeled amino acids are typically administered, recent studies have tested ²H₂O; the assumption is that there is rapid equilibration of ²H (in body water) with the carbon-bound hydrogens of amino acids before those amino acids are incorporated into a protein(s). We have determined the temporal changes in ²H labeling of body water and amino acids which should build confidence in ²H₂O-based studies of protein synthesis when one aims to measure the ²H labeling of proteolytic peptides.     

Equilibration of (2)H labeling between body water and free amino acids: enabling studies of proteome synthesis

Protein synthesis can be estimated by measuring the incorporation of a labeled amino acid into a proteolytic peptide. Although prelabeled amino acids are typically administered, recent studies have tested (2)H(2)O; the assumption is that there is rapid equilibration of (2)H (in body water) with the carbon-bound hydrogens of amino acids before those amino acids are incorporated into a protein(s). We have determined the temporal changes in (2)H labeling of body water and amino acids which should build confidence in (2)H(2)O-based studies of protein synthesis when one aims to measure the (2)H labeling of proteolytic peptides.     

Quantification of Magnetically Induced Changes in ECM Local Apparent Stiffness

The stiffness of the extracellular matrix (ECM) is known to influence cell behavior. The ability to manipulate the stiffness of ECM has important implications in understanding how cells interact mechanically with their microenvironment. This article describes an approach to manipulating the stiffness ECM, whereby magnetic beads are embedded in the ECM through bioconjugation between the streptavidin-coated beads and the collagen fibers and then manipulated by an external magnetic field. It also reports both analytical results (obtained by formal modeling and numerical simulation) and statistically meaningful experimental results (obtained by atomic force microscopy) that demonstrate the effectiveness of this approach. These results clearly suggest the possibility of creating desired stiffness gradients in ECM in vitro to influence cell behavior.