RESUMO
Investigation of cultivated fruiting bodies of Ganoderma weberianum led to the isolation of 11 previously unreported lanostane dimers, ganoweberianones C (3a), D (4a), E (5a), F (6a), G (7a), and H (8a) and isoganoweberianones A (1b), B (2b), D (4b), G (7b), and H (8b). Six new ganodermanontriol derivatives as three pairs of diastereomers (11/12, 13/14, and 15/16) and five new ganoweberianic acids (17-21) were also isolated. A method for semisynthesis of lanostane dimers by condensation of natural lanostanes was established, which was utilized in the structure elucidation and NMR data assignments of the undescribed natural lanostane dimers. Ganoweberianone D (4a) and isoganoweberianone D (4b) showed significant antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain) with IC50 values of 0.057 and 0.035 µM, respectively, whereas their cytotoxicity to Vero cells was weaker (IC50 8.1 and 19 µM, respectively).
Assuntos
Antimaláricos , Ganoderma , Triterpenos , Animais , Chlorocebus aethiops , Triterpenos/química , Antimaláricos/farmacologia , Estrutura Molecular , Células Vero , Ganoderma/química , Esteroides , Carpóforos/químicaRESUMO
The polychaete Perinereis nuntia is preferred over commercial feed pellets for boosting ovarian maturation of the female black tiger shrimp Penaeus monodon. High levels of prostaglandins in polychaetes are believed to enhance shrimp ovarian development. However, the impact of polychaete feeding on shrimp prostaglandin biosynthesis and fatty acid regulatory pathways have yet to be investigated. As polychaetes contain higher levels of arachidonic acid (ARA), eicosapentaenoic acid (EPA), prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) than feed pellets, we examined the effects of polychaete feeding alone and in combination with eyestalk ablation on shrimp hepatopancreases and ovaries. Shrimp fed with polychaetes contained higher levels of EPA, PGE2 and PGF2α in hepatopancreases than those of pellet-fed shrimp. Similarly, higher levels of ARA and higher transcription levels of cyclooxygenase (COX) and prostaglandin F synthase (PGFS) were detected in ovaries of polychaete-fed shrimp compared to those of pellet-fed shrimp. The combination of polychaete-feeding and eyestalk ablation, commonly practiced to induce ovarian development, increased levels of ARA and EPA and transcription levels of COX in hepatopancreases and ovaries of polychaete-fed shrimp compared to those of pellet-fed shrimp. In ovaries, prostaglandin biosynthesis gene transcripts were induced by polychaete feeding while transcriptional levels of fatty acid regulatory genes were regulated by shrimp feed and eyestalk ablation. Our findings not only elucidate the effects of polychaete consumption on shrimp prostaglandin biosynthesis and fatty acid regulatory pathways during larvae production, but also suggests that high levels of dietary ARA, EPA and prostaglandins are essential during P. monodon ovarian development.
Assuntos
Ração Animal/análise , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Ovário/metabolismo , Penaeidae/metabolismo , Poliquetos/fisiologia , Prostaglandinas/biossíntese , Animais , Feminino , Perfilação da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Lipogênese , Penaeidae/genética , Penaeidae/crescimento & desenvolvimentoRESUMO
Two lanostane dimers, ganoweberianones A (1) and B (2), together with seven previously undescribed lanostanes, ganoweberianic acids A-G (3-9), and three known compounds (10-12), were isolated from the artificially cultivated fruiting bodies of the basidiomycete Ganoderma weberianum. Ganoweberianone A (1) exhibited significant antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain) with an IC50 value of 0.050 µM. A method for semisynthesis of 1 by condensation of the corresponding lanostane monomers and acid-catalyzed intramolecular transesterification was demonstrated.
Assuntos
Antimaláricos/química , Carpóforos/metabolismo , Ganoderma/química , Lanosterol/análogos & derivados , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/farmacologia , Dimerização , Lanosterol/química , Ressonância Magnética Nuclear Biomolecular/métodosRESUMO
U6 RNA polymerase III promoter (PU6), which is a key element in controlling the generation of single-guide RNA (sgRNA) for gene editing through CRISPR-Cas9 system, was investigated in this work. Using bioinformatics approach, two novel U6 ribonucleic acid (U6 RNA) sequences of Aspergillus niger were identified, showing that they had conserved motifs similar to other U6 RNAs. The putative PU6 located at the upstream sequence of A. niger U6 RNA exhibited the consensus motif, CCAATYA, and the TATA box which shared highly conserved characteristics across Aspergilli, whereas the A- and B-boxes were found at the intragenic and downstream of the structural genes, respectively. Using Aspergillus oryzae as a workhorse system, the function of A. niger PU6s for controlling the transcripts of sgRNA was verified, in which the orotidine-5'-phosphate decarboxylase (pyrG) sequence was used as a target for gene disruption through CRISPR-Cas9 system. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of the selected pyrG auxotrophic strains showed the expression of sgRNA, indicating that the non-native promoters could efficiently drive sgRNA expression in A. oryzae. These identified promoters are useful genetic tools for precise engineering of metabolic pathways in the industrially important fungus through the empowered CRISPR-Cas9-associated gene-editing system.
Assuntos
Aspergillus oryzae/genética , Sistemas CRISPR-Cas , Proteínas Fúngicas/genética , Edição de Genes , Regiões Promotoras Genéticas , RNA Polimerase III/genética , RNA Fúngico/genética , RNA Guia de Cinetoplastídeos/genética , Aspergillus oryzae/enzimologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Polimerase III/metabolismo , RNA Fúngico/metabolismo , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The delay in ovarian maturation in farmed black tiger shrimp Penaeus monodon has resulted in the widespread practice of feeding broodstock with the polychaete Perinereis nuntia and their unilateral eyestalk ablation. Although this practice alters fatty acid content in shrimp ovaries and hepatopancreas, its effects on fatty acid regulatory genes are yet to be systematically examined. Here, microarray analysis was performed on hepatopancreas and ovary cDNA collected from P. monodon at different ovarian maturation stages, revealing that 72 and 58 genes in fatty acid regulatory pathways were differentially expressed in hepatopancreas and ovaries respectively. Quantitative real-time PCR analysis revealed that ovarian maturation was associated with higher expression levels of acetyl-CoA acetyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase 3 and long-chain fatty acid transport protein 4 in hepatopancreas, whereas the expression levels of 15 fatty acid regulatory genes were increased in shrimp ovaries. To distinguish the effects of different treatments, transcriptional changes were examined in P. monodon with stage 1 ovaries before polychaete feeding, after 1 month of polychaete feeding and after eyestalk ablation. Polychaete feeding resulted in lower expression levels of enoyl-CoA hydratase and acyl-CoA synthetase medium-chain family member 4, while the expression level of phosphatidylinositide phosphatase SAC1 was higher in shrimp hepatopancreas and ovaries. Additionally, eyestalk ablation resulted in a higher expression level of long-chain fatty acid-CoA ligase 4 in both tissues. Together, our findings describe the dynamics of fatty acid regulatory pathways during crustacean ovarian development and provide potential target genes for alternatives to eyestalk ablation in the future.
Assuntos
Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos/genética , Ovário/metabolismo , Penaeidae/genética , Técnicas de Ablação , Ração Animal , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Ovário/crescimento & desenvolvimento , Penaeidae/crescimento & desenvolvimento , Penaeidae/metabolismo , Poliquetos , Fatores de TempoRESUMO
Dietary polyunsaturated fatty acids (PUFAs) are critical to the success of ovarian development in marine crustaceans, especially for domesticated species such as the black tiger shrimp Penaeus monodon. These fatty acids are stored in a midgut gland called the hepatopancreas and subsequently serve as an energy source or are incorporated in yolk during ovarian development. PUFAs are known precursors of hydroxy fatty acids, including hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid (HEPE), which are catalyzed by lipoxygenases (LOX). In previous studies, 8-HEPE has been shown to regulate female reproduction and adipogenesis in marine crustaceans. However, whether the biosynthesis of 8-HEPE in these species is the result of LOX activity has yet to be investigated. In this study, 8-HEPE was identified exclusively in P. monodon hepatopancreases using liquid chromatography-mass spectrometry. Treatment with nordihydroguaiaretic acid resulted in the reduction of 8-HEPE, suggesting the enzyme-dependent catalysis of 8-HEPE in hepatopancreases. Additionally, a full-length P. monodon LOX (PmLOX) was amplified from shrimp ovary cDNA. Sequence analysis revealed that the putative PmLOX contains domains and catalytic residues required for LOX catalytic function. Furthermore, PmLOX expression increased steadily as shrimp ovary maturation progressed, while PmLOX expression and the amount of 8-HEPE decreased in shrimp hepatopancreases. These findings not only suggest differential requirements for hydroxy fatty acid biosynthesis in shrimp ovaries and hepatopancreases during the P. monodon ovarian development, but also provide insights into the LOX pathway in marine crustaceans.
Assuntos
Hepatopâncreas/embriologia , Hepatopâncreas/enzimologia , Lipoxigenase/metabolismo , Ovário/embriologia , Ovário/enzimologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Masculino , Especificidade de Órgãos/fisiologia , Organogênese/fisiologia , Penaeidae/embriologia , Penaeidae/enzimologia , Transdução de Sinais/fisiologiaRESUMO
In a continuation of our research into antitubercular lanostane triterpenoids from submerged cultures of Ganoderma species, three strains, Ganoderma orbiforme BCC 22325, Ganoderma sp. BCC 60695, and Ganoderma australe BCC 22314, have been investigated. Fourteen new lanostane triterpenoids, together with 35 known compounds, were isolated. Antitubercular activities of these mycelium-associated Ganoderma lanostanoids against Mycobacterium tuberculosis H37Ra were evaluated. Taken together with the assay data of previously isolated compounds, structure-activity relationships of the antitubercular activity are proposed. Most importantly, 3ß- and 15α-acetoxy groups were shown to be critical for antimycobacterial activity. The most potent compound was (24E)-3ß,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid (35).
Assuntos
Antituberculosos/isolamento & purificação , Antituberculosos/farmacologia , Carpóforos/química , Ganoderma/isolamento & purificação , Lanosterol/análogos & derivados , Micélio/química , Mycobacterium tuberculosis/química , Antituberculosos/química , Ganoderma/química , Lanosterol/química , Lanosterol/isolamento & purificação , Lanosterol/farmacologia , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The isolation of lanostane triterpenoids possessing significant anti-tuberculosis (anti-TB) activity from mycelial cultures of the basidiomycete Ganoderma australe strain TBRC-BCC 22314 was previously reported. To demonstrate the potential of the dried mycelial powder for utilization in anti-TB medicinal products, its authentic chemical analysis was performed. Considering the possibility of the changes in the lanostane compositions and anti-TB activity by sterilization, both autoclave treated and non-autoclaved mycelial powder materials were chemically investigated. The study led to the identification of the lanostanes responsible for the activity of the mycelial extract against Mycobacterium tuberculosis H37Ra. The anti-TB activity of the extracts from autoclaved and non-autoclaved mycelial powders were the same (MIC 3.13 µg/mL). However, the analytical results revealed several unique chemical conversions of the lanostanes under the sterilization conditions. The most potent major lanostane, ganodermic acid S (1), was shown to be significantly active also against the extensively drug-resistant (XDR) strains of M. tuberculosis.
Assuntos
Ganoderma , Mycobacterium tuberculosis , Pós , Estrutura Molecular , Testes de Sensibilidade Microbiana , Antituberculosos/farmacologia , Ganoderma/químicaRESUMO
In a laboratory assay, it was shown that B. bassiana BCC48145, BCC2660, and P. lilacinum TBRC10638 were the three strains that exhibited the highest insecticidal activity against chili thrips, causing 92.5% and 91.86% and 92.3% corrected mortality, respectively. The fungi B. bassiana BCC48145 and P. lilacinum TBRC10638 were selected for greenhouse spraying. Cytotoxicity test of the extracts from both fungi evaluated against 4 animal cell lines: KB; human oral cavity carcinoma, MCF7; human breast adenocarcinoma, NCI-H187; human small cell lung carcinoma and GFP-expressing Vero cells, showed none-cytotoxic to all cell lines. An efficacy validation in the greenhouse showed that P. lilacinum TBRC 10638 was more effective than B. bassiana BCC48145 and could control the thrips up to 80% when using the fungus at 108 spores/mL. The LC50 values of P. lilacinum TBRC 10638 against chili thrips based on total thrips count from two experiments were 1.42 × 108 and 1.12 × 107 spores/mL when the fungal spores were sprayed once a week. The optimal concentration of P. lilacinum TBRC 10638 spores for effective control of chili thrips was determined at 1.41 × 109 spores/mL. The average efficacy of P. lilacinum TBRC 10638 for thrips control from 3 field trials was 30.08%, 14.39%, and 29.92%. This result was not significantly different from that of the chemical insecticide treatment group, which showed efficacy at 19.27%, 14.92%, and 19.97%. Furthermore, there was no difference in productivity among the different treatment groups. Our results demonstrated that P. lilacinum TBRC 10638 is a promising biocontrol agent that could be used as an alternative to chemical insecticide for controlling chili thrips.
RESUMO
Eicosanoids, which are oxygenated derivatives of polyunsaturated fatty acids (PUFAs), serve as signaling molecules that regulate spermatogenesis in mammals. However, their roles in crustacean sperm development remain unknown. In this study, the testis and vas deferens of the black tiger shrimp Penaeus monodon were analyzed using ultra-high performance liquid chromatography coupled with Orbitrap high resolution mass spectrometry. This led to the identification of three PUFAs and ten eicosanoids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and (±)15-hydroxyeicosapentaenoic acid ((±)15-HEPE), both of which have not previously been reported in crustaceans. The comparison between wild-caught and domesticated shrimp revealed that wild-caught shrimp had higher sperm counts, higher levels of (±)8-HEPE in testes, and higher levels of prostaglandin E2 (PGE2) and prostaglandin F2α in vas deferens than domesticated shrimp. In contrast, domesticated shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and eicosapentaenoic acid (EPA) in testes and higher levels of 15d-PGJ2, (±)12-HEPE, EPA, arachidonic acid (ARA), and docosahexaenoic acid (DHA) in vas deferens than wild-caught shrimp. To improve total sperm counts in domesticated shrimp, these broodstocks were fed with polychaetes, which contained higher levels of PUFAs than commercial feed pellets. Polychaete-fed shrimp produced higher total sperm counts and higher levels of PGE2 in vas deferens than pellet-fed shrimp. In contrast, pellet-fed shrimp contained higher levels of (±)12-HEPE, (±)18-HEPE, and EPA in testes and higher levels of (±)12-HEPE in vas deferens than polychaete-fed shrimp. These data suggest a positive correlation between high levels of PGE2 in vas deferens and high total sperm counts as well as a negative correlation between (±)12-HEPE in both shrimp testis and vas deferens and total sperm counts. Our analysis not only confirms the presence of PUFAs and eicosanoids in crustacean male reproductive organs, but also suggests that the eicosanoid biosynthesis pathway may serve as a potential target to improve sperm production in shrimp.
Assuntos
Penaeidae , Animais , Ácido Araquidônico , Dinoprosta , Dinoprostona/metabolismo , Ácidos Docosa-Hexaenoicos , Eicosanoides , Ácido Eicosapentaenoico , Ácidos Graxos Insaturados , Masculino , Mamíferos/metabolismo , Prostaglandinas E , Sêmen/metabolismo , Contagem de Espermatozoides , Espermatozoides/metabolismoRESUMO
Antitubercular lanostane triterpenoids isolated from mycelial cultures of the basidiomycete Ganoderma australe were structurally modified by semisynthesis. One of the synthetic compounds, named GA003 (9), showed more potent activity against Mycobacterium tuberculosis H37Ra than the lead natural lanostane (1). GA003 was also significantly active against the virulent strain (H37Rv) as well as extensively drug-resistant tuberculosis strains.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Ganoderma/química , Triterpenos/química , Animais , Antituberculosos/toxicidade , Chlorocebus aethiops , Ganoderma/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , Triterpenos/síntese química , Triterpenos/farmacologia , Células VeroRESUMO
Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.
Assuntos
Penaeidae/enzimologia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Sequência de Bases , Inibidores de Ciclo-Oxigenase/farmacologia , Análise Mutacional de DNA/métodos , DNA Complementar/genética , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Feminino , Glicosilação/efeitos dos fármacos , Células HEK293 , Hemolinfa/metabolismo , Humanos , Ibuprofeno/farmacologia , Peso Molecular , Ovário/metabolismo , Prostaglandina-Endoperóxido Sintases/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção , Tunicamicina/farmacologiaRESUMO
In this paper we investigated the possible involvement of prostaglandin E synthases (PGESs) in compensatory mechanism. Our findings showed that microsomal (m)PGES-1 expression was significantly up-regulated in COX knock-out (K/O) cells whereas the expression of cytosolic PGES was not changed indicating that the induction of mPGES-1 may, at least in part, contribute to the substantial increase of PGE(2) production in COX K/O cell lines. The selective up-regulation of mPGES-1 in COX-2 K/O cells suggests that mPGES-1 may be metabolically coupled with COX-1 for PGE(2) formation. Addition of arachidonic acid caused significant induction of mPGES-1 and COX-2 in WT cells, whereas COX-1 and cPGES were not affected. Our earlier and the current studies demonstrate the coregulation of cPLA(2), COX, and mPGES-1, in PGE(2) synthesis pathway, and that these enzymes contribute to the elevation of PGE(2) level when one COX isoform is absent.
Assuntos
Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Oxirredutases Intramoleculares/metabolismo , Regulação para Cima , Animais , Ácido Araquidônico/farmacologia , Western Blotting , Linhagem Celular , Oxirredutases Intramoleculares/genética , Camundongos , Camundongos Knockout , Prostaglandina-E Sintases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/efeitos dos fármacosRESUMO
Malaria continues to be a major infectious disease of the developing world and the problem is compounded not only by the emergence of drug resistant strains but also from a lack of a vaccine. The situation for tuberculosis (TB) infection is equally problematic. Once considered a "treatable" disease for which eradication was predicted, TB has re-emerged as highly lethal, multi-drug resistant strains after the outbreak of AIDS. Worldwide, the disease causes millions of deaths annually. Similarly, treatments for chronic inflammatory diseases such as arthritis have been impeded due to the potentially lethal side effects of the new and widely prescribed non-steroidal anti-inflammatory compounds. Thais have utilized bioresources from plants and some microorganisms for medicine for thousands of years. Because of the need for new drugs to fight malaria and TB, with radically different chemical structures and mode of actions other than existing drugs, efforts have been directed towards searching for new drugs from bioresources. This is also true for anti-inflammatories. Although Thailand is considered species-rich, only a small number of potential bioresources has been investigated. This article briefly describes the pathogenesis of 2 infectious diseases, malaria and TB, and modern medicines employed in chemotherapy. Diversities of Thai flora and fungi and their chemical constituents with antagonistic properties against these 2 diseases are described in detail. Similarly, anti-inflammatory compounds, mostly cyclooxygenase (COX) inhibitors, are also described herein to demonstrate the potential of Thai bioresources to provide a wide array of compounds for treatment of diseases of a different nature.
Assuntos
Fitoterapia , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Animais , Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Antituberculosos/isolamento & purificação , Antituberculosos/farmacologia , Inibidores de Ciclo-Oxigenase/isolamento & purificação , Inibidores de Ciclo-Oxigenase/farmacologia , Fungos/química , Humanos , Malária/tratamento farmacológico , Medicina Tradicional do Leste Asiático , Mycobacterium tuberculosis/efeitos dos fármacos , Extratos Vegetais/química , Tailândia , Tuberculose/tratamento farmacológicoRESUMO
The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon ovaries along with the previously identified PUFAs and prostaglandin E2 (PGE2) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF2α biosynthesis in shrimp ovarian development.
Assuntos
Proteínas de Artrópodes/genética , Vias Biossintéticas/genética , Enzimas/genética , Ovário/metabolismo , Penaeidae/metabolismo , Prostaglandinas/biossíntese , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Enzimas/classificação , Enzimas/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Oxirredutases Intramoleculares/classificação , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Ovário/crescimento & desenvolvimento , Penaeidae/crescimento & desenvolvimento , Filogenia , Prostaglandina-E Sintases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.
Assuntos
Contagem de Células/métodos , Colorimetria/métodos , Rodaminas/metabolismo , Testes de Toxicidade/métodos , Técnicas de Cultura de Células/métodos , Corantes Fluorescentes , Sensibilidade e EspecificidadeRESUMO
The first naturally occurring atropisomeric flavans, myristinins B (2), C (2a), E (4), and F (4a), together with their corresponding trans-isomers, myristinins A (1) and D (3), were isolated from the CH(2)Cl(2) extract of Myristica cinnamomea fruits. Compounds 1, the mixture of 2 and 2a, and the mixture of 4 and 4a, exhibited antifungal activity against Candida albicans with IC(50) values ranging from 5.9 to 8.8 microg/mL, and they selectively inhibited the enzyme cyclooxygenase-2 (COX-2).
Assuntos
Antifúngicos/síntese química , Inibidores de Ciclo-Oxigenase/síntese química , Flavonoides/síntese química , Isoenzimas/metabolismo , Myristicaceae/química , Prostaglandina-Endoperóxido Sintases/metabolismo , Antifúngicos/química , Antifúngicos/farmacologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/farmacologia , Desenho de Fármacos , Flavonoides/química , Flavonoides/farmacologia , Indicadores e Reagentes , Isomerismo , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A novel Theta class glutathione transferase (GST) isoenzyme from mouse termed mGSTT3 has been identified by analysis of the expressed sequence tag database. The gene encoding mGSTT3 is clustered with the mGSTT1 and mGSTT2 genes on chromosome 10 and has an exon/intron structure that is similar to that of the other Theta class genes. mGSTT3 is expressed strongly in the liver and to a decreasing extent in the kidney and testis. Recombinant mGSTT3-3 expressed in Escherichia coli had a substrate-specificity profile that differed significantly from that of GSTT1-1 and GSTT2-2 isoenzymes. A molecular model of mGSTT3 suggested that, in comparison with GSTT2, a decrease in volume of the hydrophobic substrate-binding site and the loss of the sulphate-binding pocket prevents its use of the GSTT2 substrate 1-menaphthyl sulphate.