Interaction of neuronal nitric oxide synthase with alpha(1)-adrenergic receptor subtypes in transfected HEK-293 cells

BACKGROUND: The C-terminal four amino acids (GEEV) of human α(1A)-adrenergic receptors (ARs) have been reported to interact with the PDZ domain of neuronal nitric oxide synthase (nNOS) in a yeast two-hybrid system. The other two α(1)-AR subtypes have no sequence homology in this region, raising the possibility of subtype-specific protein-protein interactions. RESULTS: We used co-immunoprecipitation and functional approaches with epitope-tagged α(1)-ARs to examine this interaction and the importance of the C-terminal tail. Following co-transfection of HEK-293 cells with hexahistidine/Flag (HF)-tagged α(1A)-ARs and nNOS, membranes were solubilized and immunoprecipitated with anti-FLAG affinity resin or anti-nNOS antibodies. Immunoprecipitation of HFα(1A)-ARs resulted in co-immunoprecipitation of nNOS and vice versa, confirming that these proteins interact. However, nNOS also co-immunoprecipitated with HFα(1B)- and HFα(1D)-ARs, suggesting that the interaction is not specific to the α(1A) subtype. In addition, nNOS co-immunoprecipitated with each of the three HFα(1)-AR subtypes which had been C-terminally truncated, suggesting that this interaction does not require the C-tails; and with Flag-tagged β(1)- and β(2)-ARs. Treatment of PC12 cells expressing HFα(1A)-ARs with an inhibitor of nitric oxide formation did not alter norepinephrine-mediated activation of mitogen activated protein kinases, suggesting nNOS is not involved in this response. CONCLUSIONS: These results show that nNOS does interact with full-length α(1A)-ARs, but that this interaction is not subtype-specific and does not require the C-terminal tail, raising questions about its functional significance

Optimization of Chaetoceros gracilis microalgae production for fish feeding using an airlift photobioreactor

An experimental procedure was carried out to maximize Chaetoceros gracilis growth. Chaetoceros gracilis, marine microalgae, is considered for feeding fisheries with no GMO (Genetically Modified Organisms) to avoid human health hazards. Furthermore, following United Nations Resolution on water, the microalgae is grown in photobioreactors due to its low water usage. To maximize the microalgae growth, an experimental design was carried out to analyze the effects of Light Intensity, CO2 supply per day, Sparger type, Photoperiod and Inlet airflow, pH and water temperature were monitored but not controlled. It was found that Light intensity and CO2 supply per day have statistical significance. Out of three possible scenarios, 1700 lux and 80 gr/day of CO2, leads to a cell density at day three of 310×104 cel/mL which represents 20% more of the density attained in day two under bag (standard) growing conditions. It was also found that water Ph has also a strong effect over cell density

A simple method for the quantitative analysis of tyrosol by hplc in liquid Czapek Cultures from endophytic fungi

Tyrosol is a possible quorum sensing molecule in endophytic fungi. High-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) was used for the analysis of tyrosol in liquid Czapek fungal cultures. The optimized conditions were gradient mobile phase, in linear mode, consisting initially of acetonitrile/water (1:9 v/v) and increasing up to acetonitrile (100%) in 30 minutes at a flow rate of 1 mL min-1. The column used was a ZORBAX® ODS (250 × 4.6 mm, 5 μm) at 25 ºC. Liquid-liquid extraction of 0.5 mL medium (pH 7.0) with ethyl acetate and injection of 20 μL after solvent evaporation under air flow gave good results. Some validation parameters obtained were: linearity 0.0125-5.0 μg mL-1 medium (r = 0.9967), quantification limit of 0.0125 μg mL-1 medium, %CV (precision) and %E (accuracy) bellow 15% and recovery around 80%. Therefore, the developed method presented satisfactory validation parameters and it was efficient for the analysis of tyrosol in Czapek medium.O tirosol é provavelmente uma molécula sinalizadora em fungos endofíticos. A análise do tirosol em cultura líquida Czapek de fungo endofítico foi realizada através de cromatografia líquida de alta eficiência acoplada a detector por arranjo de diodos. As análises foram obtidas em sistema de fase móvel utilizando gradiente, modo linear, iniciando em acetonitrila/água (1:9 v/v) e terminando em acetonitrila 100% em 30 minutos com vazão de 1 mL min-1. Coluna analítica ZORBAX® ODS (250 × 4,6 mm, 5 μm) à 25 ºC foi utilizada. Extração líquido-líquido de 0,5 mL do meio (pH 7,0) com acetato de etila e injeção de 20 μL após concentração do solvente sob ar comprimido originou bons resultados. Os parâmetros validados foram: linearidade 0,0125-5,0 μg mL-1 (r = 0,9967), limite de quantificação 0,0125 μg mL-1 obtidos pela média das análises; %CV (precisão) e %E (exatidão) com valores abaixo de 15% e recuperação de cerca de 80%. Além disso, o método desenvolvido apresentou valores de validação satisfatórios demonstrando eficiência na análise do tirosol em meio líquido Czapek.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPES

Study of a Brazilian family presenting non-syndromic hearing loss with mitochondrial inheritance

O presente estudo teve como objetivo descrever os achados audiológicos e genéticos de nove membros de uma família brasileira que apresenta a mutação no DNA mitocondrial. Todos os nove membros realizaram estudo genético, avaliação foniátrica e audiológica (audiometria tonal e logoaudiometria). O estudo genético revelou a presença de mutação mitocondrial A1555G no gene 12S rRNA (MT-RNR-1) do DNA mitocondrial em todos os sujeitos. Oito sujeitos apresentaram deficiência auditiva e somente um apresentou limiares auditivos normais até o término da realização do estudo. Os resultados audiológicos apontaram para perdas auditivas bilaterais, com prevalência das simétricas, de configurações e graus variados (de moderado a profundo) e pós-linguais. Progressão da perda auditiva foi observada em dois irmãos afetados. Não foi possível afirmar a época do início da perda auditiva por falta de informação dos sujeitos, no entanto, observou-se manifestação da perda em crianças e adultos. As mutações no DNA mitocondrial representam uma causa importante de perda auditiva, sendo imprescindível a realização do diagnóstico etiopatológico, a fim de retardar o início ou evitar a progressão da surdez.We hereby report on the audiological and genetic findings in individuals from a Brazilian family, with the following mitochondrial mutation A1555G in the 12SrRNA gene (MT-RNR-1). Nine individuals underwent speech, audiologic (tonal audiometry and logoaudiometry) and genetic evaluations. Eight individuals among the A1555G carriers were affected by hearing impairment and one person had normal hearing thresholds till the end of the present study. The audiologic evaluation results indicated normal hearing thresholds all the way to bilateral profound hearing loss with post-lingual onset and variable configuration. Two affected siblings presented progressive hearing loss. It was impossible to precise the time of hearing loss onset; however, the impairment was present in both children and adults. The genetic study revealed the A1555G mitochondrial mutation in the 12SrRNA gene. Given the prevalence of mitochondrial mutations as a cause of hearing loss, it is fundamental to perform the etiopathologic diagnosis in order to postpone the onset or avoid hearing impairment progression. This kind of hearing impairment represents a challenge to the professionals since there are no physical traits that indicate genetic transmission

Chaetoglobosins produced by Chaetomium globosum, endophytic fungus found in association with Viguiera robusta Gardn (Asteraceae)

Endophytes live in association with host plants during all or part of their life cycle without causing any apparent disease. They are considered outstanding and underexploited sources of novel bioactive compounds. Chaetomium globosum was isolated as an endophytic fungus from the healthy leaves of Viguiera robusta. C.globosum is a remarkable producer of chaetoglobosins, which are typically cytotoxic. In this work, chaetoglobosins B (1), D (2) and E (3) have been produced by the endophytic C. globosum strain. Chaetoglobosin B was evaluated against Jurkat (leukemia) and B16F10 (melanoma) tumoral cells and showed 89.55% and 57.10% of inhibition at 0.1 mg mL-1, respectively. Chaetoglobosin B also showed weak antibacterial activity against Staphylococcus aureus (MIC 120 µg/mL) and Escherichia coli (MIC 189 µg/mL).Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Biotransformation of a tetrahydrofuran lignan by the endophytic fungus Phomopsis Sp.

The biotrasformation of the tetrahydrofuran lignan, (-)-grandisin, by the endophitic fungus Phomopsis sp, obtained from Viguiera arenaria, led to the formation of a new compound determined as 3,4-dimethyl-2-(4'-hydroxy-3',5'-dimethoxyphenyl)-5-methoxy-tetrahydrofuran. The metabolite was evaluated against the parasite Trypanosoma cruzi, the causative agent of Chagas's disease, and showed a trypanocidal activity (IC50 9.8 μmol L-1) similar to the natural precursor (IC50 3.7 μmol L-1).A biotransformação da lignana tetraidrofurânica, (-)-grandisina, pelo fungo endofítico Phomopsis sp, obtido de Viguiera arenaria, conduziu à formação de um novo metabólito caracterizado como 3,4-dimetil-2-(4'-hidróxi-3',5'-dimetóxifenil)-5-metóxi-tetraidrofurano. O metabólito foi analisado contra o parasita Trypanosoma cruzi, o agente causador da doença de Chagas, e mostrou uma atividade tripanocida (IC50 9,8 μmol L-1) similar ao precursor natural (IC50 3,7 μmol L-1).Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Fast HPLC analysis of omeprazole, 5-hydroxyomeprazole and omeprazole sulfone in liquid culture medium using a monolithic column for application in biotransformation studies with fungi

A fast liquid chromatography method was developed and validated for the simultaneous determination of omeprazole (OMZ), 5-hydroxyomeprazole (5-HOMZ) and omeprazole sulphone (OMZ SUL) in liquid culture medium for application in biotransformation studies employing phytopathogenic and endophytic fungi. The separation was achieved using a monolithic Chromolith Fast gradient RP 18 endcapped column, using a mobile phase consisting of 0.15% (v/v) trifluoroacetid acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B), under linear gradient of 5 to 90% of B in 1 min, flow rate of 1.0 mL min-1, temperature at 30 ºC and detection at 220 nm. Sample preparation was performed by liquid-liquid extraction, with recoveries in the range of 62.3 to 76.6% for all analytes. The method was linear in the range of 0.2 to 10.0 µg mL-1 (r &#8805; 0.995). The values for intra- and inter-day precision (% coefficient of variation) and accuracy (% relative error) were < 15% for all analytes. The validated method was used to evaluate OMZ biotransformation to their mammalian metabolites by selected fungi. In general, the phytopathogenic fungi studied were more efficient to biotransform OMZ. The sulfonation reaction was more prevalent for all studied fungi.Um método rápido por cromatografia líquida foi desenvolvido para a determinação simultânea de omeprazol (OMZ), 5-hidroxiomeprazol (5-HOMZ) e omeprazol sulfona (OMZ SUL) em meio de cultura líquido, para aplicação em estudos de biotransformação empregando fungos fitopatogênicos e endofíticos. A separação foi realizada empregando uma coluna monolítica Chromolith Fast gradient RP 18 com a fase móvel constituída por ácido trifluoroacético (TFA) 0,15% (v/v) em água (solvente A) e TFA 0,15% (v/v) em acetonitrila (solvente B). Foi empregado um gradiente linear de 5 a 90% de B em 1 minuto, vazão de 1,0 mL min-1, temperatura de 30 ºC e detecção em 220 nm. A extração líquido-líquido foi empregada na preparação das amostras, com recuperações na faixa de 62,3-76,6% para todos os analitos. O método foi linear na faixa de 0,2-10,0 µg mL-1 (r &#8805; 0,995). Os valores de precisão e exatidão intra- e inter-dias (coeficiente de variação e erro relativo) foram inferiores a 15% para todos os analitos. O método validado foi utilizado para avaliar a biotransformação do OMZ em seus principais metabólitos humanos pelos fungos selecionados. Em geral, os fungos fitopatogênicos foram mais eficientes para biotransformar o OMZ. A reação de sulfonação foi mais prevalente em todos os fungos estudadosFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES