Modulation of peroxynitrite-induced fibroblast injury by hesperetin: a role for intracellular scavenging and modulation of ERK signalling

Peroxynitrite is thought to contribute to the progression of many diseases including cardiovascular disease, cancer, and neurodegenerative disorders. We report that pre-treatment of fibroblasts with the citrus flavanone, hesperetin, prior to peroxynitrite exposure protects against peroxynitrite-mediated cytotoxicity. This protection was partially mediated by the intracellular scavenging of peroxynitrite by hesperetin as exposure of fibroblasts to peroxynitrite following hesperetin loading led to the formation of two intracellular nitro-hesperetin derivatives. In addition, protection appeared to be mediated by hesperetin-induced changes in MAP kinase signalling. Exposure of fibroblasts to hesperetin led to concentration-dependent increases in the phosphorylation of ERK1/2 and was observed to restore peroxynitrite-mediated decreases in ERK1/2 phosphorylation. We propose that the protective potential of hesperetin in fibroblasts may be mediated both by intracellular scavenging of peroxynitrite and by modulation of fibroblast signalling.     

Uterine evaluation and gestation diagnosis in owl monkey (Aotus azarai infulatus) using the B mode ultrasound

BACKGROUND: Gynecological and obstetrical ultrasonography has become an indispensable tool in the routine management, health evaluation and research on captive non-human primates. METHODS: Ultrasound was used to evaluate the uterus and estimate the gestation of owl monkeys. Twelve couples were selected, where five were primiparous and seven multiparous females from the National Primate Center reproductive colony, Ananindeua-PA, Brazil. The procedures were carried out using the GE Logiq 100 MP, equipped with a 7.5 MHz linear probe. RESULTS: The females showed a simple uterus, of elongated shape, regular outline and homogeneous echogenic texture. In the uterine measurements craniocaudal diameter, dorsoventral diameter and uterine volume (UV), significant differences were identified (P < 0.05) between ultrasound examinations of primiparous and multiparous females. The UV showed a positive correlation with the number of births. The gestational sac and the embryonic echo were visible between 28 and 38 days after mating. Between 48 and 68 days after mating, embryonic death was identified in all the gestations. CONCLUSIONS: The chemical (use of tranquilizers) and husbandry factors (capture stress) may be related to the prenatal death. The establishing methods of conditioning the female to the ultrasonographic exam may offer a solution to this problem.     

Characterization of the Melanoma miRNAome by Deep Sequencing

Background

MicroRNAs (miRNAs) are 18–23 nucleotide non-coding RNAs that regulate gene expression in a sequence specific manner. Little is known about the repertoire and function of miRNAs in melanoma or the melanocytic lineage. We therefore undertook a comprehensive analysis of the miRNAome in a diverse range of pigment cells including: melanoblasts, melanocytes, congenital nevocytes, acral, mucosal, cutaneous and uveal melanoma cells.

Methodology/Principal Findings

We sequenced 12 small RNA libraries using Illumina''s Genome Analyzer II platform. This massively parallel sequencing approach of a diverse set of melanoma and pigment cell libraries revealed a total of 539 known mature and mature-star sequences, along with the prediction of 279 novel miRNA candidates, of which 109 were common to 2 or more libraries and 3 were present in all libraries.

Conclusions/Significance

Some of the novel candidate miRNAs may be specific to the melanocytic lineage and as such could be used as biomarkers to assist in the early detection of distant metastases by measuring the circulating levels in blood. Follow up studies of the functional roles of these pigment cell miRNAs and the identification of the targets should shed further light on the development and progression of melanoma.     

Nonea pisidica (Boraginaceae-Boragineae), a new species from southwest Anatolia and its relationships inferred from karyology and cpDNA sequences

The new species Nonea pisidica (Boraginaceae-Boragineae) is described based on two collections by the authors from the region of Lake Burdur in southwest Anatolia, Turkey. It shows a combination of morphological characteristics concerning habit, flower, and fruit that distinguishes it from N. caspica and N. pallens, the two more similar taxa in Nonea sect. Nonea. Karyological analyses corroborate the separation of the three species, which have different chromosome complements and even base numbers. N. pisidica is characterised by 2n = 30, a complement previously unknown for west Asiatic species of Nonea. The dibasic haploid number x = 15 may have originated through amphidiploid hybridisation between two annual, diploid taxa with x = 7 and x = 8, such as N. lutea and N. caspica. The relationships of the new species were further analysed using trnL_UAA sequences of the chloroplast genome, which were obtained for 16 selected species of Nonea. The resulting phylogeny confirms that it is related to N. caspica and N. lutea, but not to N. pallens, in spite of morphological resemblance. Lack of relationship with the south Mediterranean N. vesicaria, the only other species of Nonea known to have 2n = 30, suggests that amphidiploidy may have played an important role in speciation processes through recurrent and polytopical occurrence.     

Measurement and meaning of markers of reactive species of oxygen, nitrogen and sulfur in healthy human subjects and patients with inflammatory joint disease

Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO?, another cell-signalling gas produced in the inflamed joint is H2S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H2S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H2S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.     

Comparative Assessment of the Artificial Neural Network and Response Surface Modelling Efficiencies for Biohydrogen Production on Sugar Cane Molasses

This study comparatively evaluates the modelling efficiency of the Response Surface Methodology (RSM) and the Artificial Neural Network (ANN). Twenty-nine biohydrogen fermentation batches were carried out to generate the experimental data. The input parameters consisted of a concentration of molasses (50–150 g/l), pH (4–8), temperature (35–40 °C) and inoculum concentration (10–50 %). The obtained data were used to develop the RSM and ANN models. The ANN model was a committee of networks with a topology of 4-(6-10)-1 structured on multilayer perceptrons. RSM and ANN models gave R ² values of 0.75 and 0.91, respectively, with predicted optimum conditions of 150 g/l, 8 and 35 °C for molasses, pH and temperature, respectively, with differences in inoculum concentrations (10.11 and 15 %) for RSM and ANN, respectively. Upon validation, 15.12 and 119.08 % prediction errors on hydrogen volume were found for ANN and RSM, respectively. These findings suggest that ANN has greater accuracy in modelling the relationships between the considered process inputs for fermentative biohydrogen production and thus, is more reliable to navigate the optimization space.     

Engagement of ubiquitination and de-ubiquitination at rostral ventrolateral medulla in experimental brain death

Background

Whereas brain death is a vitally important clinical phenomenon, our contemporary understanding on its underlying cellular mechanisms remains elusive. This study evaluated whether the ubiquitin-proteasome system (UPS) in the rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is engaged in this fatal process.

Methods

We performed proteomics, Western Blot, real-time PCR, ELISA and pharmacological experiments in conjunction with a clinically relevant experimental endotoxemia model of brain death based on intravenous administration of Escherichia coli lipopolysaccharide in adult male Sprague–Dawley rats.

Results

Proteomics, Western blot and enzyme activity analyses demonstrated that polyubiquitination was preserved and de-ubiquitination by ubiquitin C-terminal hydrolase isozyme-L1 (UCH-L1) was sustained, alongside increased monoubiquitin availability or proteasome activity in RVLM over the course of experimental endotoxemia. However, real-time PCR revealed no significant alteration in proteasome subunit alpha type-1, ubiquitin or UCH-L1 at mRNA level. Functionally, whereas microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) potentiated survival, an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or an UCH-L1 inhibitor exacerbated mortality.

Conclusions

We proposed previously that the progression towards brain death entails a tug-of-war between pro-death and pro-life programs in RVLM. It is conceivable that ubiquitination or de-ubiquitination in RVLM participate in brain death by regulating the degradation of the proteins involved in those programs.     

Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Hydrogen sulfide (H(2)S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H(2)S in response to pro-inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H(2)S. HACs and MPCs were exposed to IL-1β, IL-6, TNF-α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H(2)S synthesizing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) were determined by Western blot and zinc-trap spectrophotometry, respectively. Cellular oxidative stress was induced by H(2)O(2), the peroxynitrite donor SIN-1 and 4-hydroxynonenal (4-HNE). Cell death was assessed by 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H(2) S synthesis was inhibited by siRNA-mediated knockdown of CSE and CBS and pharmacological inhibitors D,L-propargylglycine and aminoxyacetate, respectively. Exogenous H(2)S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H(2)S in a CBS-dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL-1β, TNF-α, IL-6 or LPS. Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment. These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs. H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.     

Loss of 3-chlorotyrosine by inflammatory oxidants: implications for the use of 3-chlorotyrosine as a bio-marker in vivo

Activated neutrophils generate the potent oxidant hypochlorous acid (HOCl) from the enzyme myeloperoxidase (MPO). A proposed bio-marker for MPO-derived HOCl in vivo is 3-chlorotyrosine, elevated levels of which have been measured in several human inflammatory pathologies. However, it is unlikely that HOCl is produced as the sole oxidant at sites of chronic inflammation as other reactive species are also produced during the inflammatory response. The work presented shows that free and protein bound 3-chlorotyrosine is lost upon addition of the pro-inflammatory oxidants, HOCl, peroxynitrite, and acidified nitrite. Furthermore, incubation of 3-chlorotyrosine with activated RAW264.7 macrophages or neutrophil-like HL-60 cells resulted in significant loss of 3-chlorotyrosine. Therefore, at sites of chronic inflammation where there is concomitant ONOO⁻ and HOCl formation, it is possible measurement of 3-chlorotyrosine may represent an underestimate of the true extent of tyrosine chlorination. This finding could account for some of the discrepancies reported between 3-chlorotyrosine levels in tissues in the literature.