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.     

Synthesis, pharmacological evaluation and electrochemical studies of novel 6-nitro-3,4-methylenedioxyphenyl-N-acylhydrazone derivatives: Discovery of LASSBio-881, a new ligand of cannabinoid receptors

We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inflammatory, and in vitro antiproliferative and antioxidant compound, with a cannabinoid ligand profile. We observed that LASSBio-881 (3c) was able to bind to CB1 receptors (71% at 100microM) and also to inhibit T-cell proliferation (66% at 10microM) probably by binding to CB2 receptors, in a non-proapoptotic manner, different from anandamide (1). It was also demonstrated that LASSBio-881 (3c) had an important antioxidant profile toward free radicals (DPPH and hydroxyl), probably due to its particular redox behavior, which reflects the presence of both nitro and 3,5-di-tert-butyl-4-hydroxyphenyl sub-units, as demonstrated by cyclic voltammetry studies. In addition, we showed that these structural sub-units are essential for the observed pharmacological activity.     

Purification and characterization of 4-N-trimethylamino-1-butanol dehydrogenase of Pseudomonas sp. 13CM

A new enzyme, NAD+-dependent 4-N-trimethylamino-1-butanol dehydrogenase from Pseudomonas sp. 13CM, was purified 526-fold to apparent homogeneity in 5 chromatographic steps. The enzyme had a molecular mass of 45 kDa and appeared to be a monomer enzyme. The isoeletric point was found to be 4.8. The optimum temperature was 50 degrees C, and the optimum pHs for the oxidation and reduction reactions were 9.5 and 6.0 respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and amino acid terminal sequence. The Km values for trimethylamino-1-butanol and NAD+ were 0.54 mM and 0.22 mM respectively. In the reduction reaction, the apparent Km values for trimethylaminobutylaldehyde and NADH were 0.67 mM and 0.04 mM, respectively. The enzyme was inhibited by SH reagents, chelating reagents, and heavy metal ions. The N-terminal 12 amino acid residues were sequenced.     

PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.     

Role of humic acid fraction with higher aromaticity in enhancing the activity of a biomimetic catalyst, tetra(p-sulfonatophenyl)porphineiron(III)

To elucidate the structural features of humic acids (HAs) that potentially contribute to enhancing the activity of a tetra(p-sulfonatophenyl)porphineiron(III) (Fe(III)-TPPS) catalyst, the effects of the chemical properties of molecular weight fractionated HAs on the catalytic activity of Fe(III)-TPPS were investigated. Three fractions were obtained as the following order of molecular size: F3 < F2 < F1. The deactivation of Fe(III)-TPPS, which can be attributed to the self-degradation of Fe(III)-TPPS, was retarded in the presence of HAs, and the pseudo-first-order rate constant in the presence of F3 was the smallest of the three fractions. In addition, the highest catalytic activity, determined as the percent degradation of an organic substrate, was observed in the presence of F3. The enhanced catalytic activity of Fe(III)-TPPS was due to the formation of supramolecular complexes with HAs, and the formation constant for F3 was the largest. Thus, the F3 fraction was the most effective fraction. Solid-state CPMS 13C NMR spectra indicated that the aromaticity of F3 was the highest of all of the fractions. Thus, it can be concluded that aromatic moieties in HAs play an important role in the formation of supramolecular complexes with Fe(III)-TPPS, leading to an enhancement in catalytic activity.     

Neutralising antibodies for Mayaro virus in Pantanal, Brazil

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.     

Specific calpain activity evaluation in Plasmodium parasites

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.     

Inactivation of GSK3β and activation of NF-κB pathway via Axl represents an important mediator of tumorigenesis in esophageal squamous cell carcinoma

The receptor tyrosine kinase Axl has been described as an oncogene, and its deregulation has been implicated in the progression of several human cancers. While the role of Axl in esophageal adenocarcinoma has been addressed, there is no information about its role in esophageal squamous cell carcinoma (OSCC). In the current report, we identified, for the first time, deregulation of Axl expression in OSCC. Axl is consistently overexpressed in OSCC cell lines and human tumor samples, mainly in advanced stages of the disease. Blockage of Axl gene expression by small interfering RNA inhibits cell survival, proliferation, migration, and invasion in vitro and esophageal tumor growth in vivo. Additionally, repression of Axl expression results in Akt-dependent inhibition of pivotal genes involved in the nuclear factor-kappaB (NF-κB) pathway and in the induction of glycogen synthase kinase 3β (GSK3β) activity, resulting in loss of mesenchymal markers and induction of epithelial markers. Furthermore, treatment of esophageal cancer cells with the Akt inhibitor wortmannin inhibits NF-κB signaling, induces GSK3β activity, and blocks OSCC cell proliferation in an Axl-dependent manner. Taken together, our results establish a clear role for Axl in OSCC tumorigenesis with potential therapeutic implications.     

Characterization of Mesorhizobium loti L-rhamnose isomerase and its application to L-talose production

The L-rhamnose isomerase gene (rhi) of Mesorhizobium loti was cloned and expressed in Escherichia coli, and then characterized. The enzyme exhibited activity with respect to various aldoses, including D-allose and L-talose. Application of it in L-talose production from galactitol was achieved by a two-step reaction, indicating that it can be utilized in the large-scale production of L-talose.