RESUMO
An aggregation-induced emission (AIE) active Schiff base L was obtained by reacting pyridoxal and 2-hydroxy-1-naphthaldehyde with p-phenylenediamine in two simple steps. The colorimetric, UV/VIS and fluorescence studies of L revealed that the yellow emissive L (λem =540â nm, λex =450â nm) in pure DMSO turned to a red-emissive L, when the poor solvent fraction (HEPES buffer, 10â mM, pHâ 7.4) was increased above 50 % in DMSO. The SEM and DLS results indicated the formation of self-aggregates of L that restricted the intramolecular motion and promoted the excited state intramolecular proton transfer (ESIPT) process. The cations sensing ability of the AIEgen L was explored in HEPES buffer (5 % DMSO, 10â mM, pHâ 7.4), where Cu2+ selectively quenched the fluorescence at 608â nm due to the chelation-enhanced fluorescence quenching (CHEQ) effect with an estimated sensitivity limit of 0.9â µM. Subsequently, the inâ situ formed AIEgen L-Cu2+ complex was applied for the cascade detection of glutathione (GSH), cysteine (Cys) and homocysteine (Hcy). The decomplexation of Cu2+ from the AIEgen L-Cu2+ by GSH, Cys and Hcy restored the quenched fluorescence emission of AIEgen L at 608â nm. With this Cu2+ displacement approach, the concentration of Cys, Hcy and GSH can be detected down to 2.8â µM, 3.12â µM and 2.0â µM, respectively. The practical utility of AIEgen L and AIEgen L-Cu2+ was examined by monitoring the selective analytes in real environmental and biological samples, and also applied successfully for the cell imaging applications.
Assuntos
Cobre , Cisteína , Cobre/química , Dimetil Sulfóxido , Corantes Fluorescentes/química , Glutationa , HEPES , Homocisteína , Prótons , Piridoxal , Bases de Schiff , Solventes , Espectrometria de FluorescênciaRESUMO
Prophenoloxidase (proPO) is very important to protect the invertebrates from microbial infections. Our previous studies revealed that proPO was up-regulated in WSSV-injected Macrobrachium rosenbergii and is responsible for protecting M. rosenbergii from WSSV. In order to prove this mechanism, an attempt was made in the present study to silence the proPO gene in freshwater prawn by injection of dsRNA-proPO followed by WSSV challenge. Two partial fragments of proPO with the size of 251 and 331 bp were used to synthesize dsRNA using LITMUS38i vector and E. coli. The bacterially synthesized dsRNA-proPO was used to silence proPO gene to determine its involvement in developing resistance in prawn against WSSV. In proPO gene-silenced prawn, 100% mortality was observed after WSSV challenge whereas no mortality was observed in prawn injected with WSSV alone. The WSSV infection in gene-silenced prawn was confirmed by PCR, and its propagation was quantified by ELISA and real-time PCR at different time intervals. Real-time PCR assay revealed a significant reduction in the expression of proPO gene in WSSV-challenged proPO-silenced prawn when compared to normal prawn. Level of proPO was reduced significantly in the haemolymph of proPO-silenced prawn when compared to prawn injected with PBS.
Assuntos
Proteínas de Artrópodes/genética , Catecol Oxidase/genética , Precursores Enzimáticos/genética , Inativação Gênica , Palaemonidae/virologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Palaemonidae/enzimologia , Palaemonidae/genéticaRESUMO
White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6-histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD-infected post-larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection. The antiserum raised against r-MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti-rMCP43 and pure r-MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT-PCR to test the efficiency of antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV-positive coded samples as detected by RT-PCR.
Assuntos
Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Imunoensaio/métodos , Nodaviridae/isolamento & purificação , Nodaviridae/metabolismo , Palaemonidae/virologia , Animais , Regulação Viral da Expressão Gênica/fisiologia , Interações Hospedeiro-Patógeno , Larva/virologia , Estágios do Ciclo de VidaRESUMO
The present study was designed to isolate a potential compound from the extracts of Elytraria acaulis (E. acaulis) for ovarian cancer. n-Hexane, ethyl acetate, chloroform, acetone and methanol extract were taken using the Soxhlet method. Thin layer, column chromatography, NMR and MASS studies were done for the isolation and structural characterization of the compound. Finally, the novel compound (Z)-3-(2-methyl-3-oxoprop-1-en-1-yl) phenyl heptanoate was identified. MTT assay, cell morphology and cell cycle analysis were done to evaluate the anticancer property of the compound. In the MTT assay, the percentage of the cell viability treated with the isolated compound was decreased while increasing the concentration of the compound. Cancer cells treated with the isolated compound showed distinct morphological changes when compared to the control untreated cells. In the cell cycle analysis, the isolated compound induced a significant increase in the percentage of cells in G0/G1 phase and a decrease in the percentage of cells in the S phase and G2-M phase of the PA 1 cell lines. The cell cycle arrest induced by the isolated compound may account for its antiproliferative capacity. Hence, the novel compound isolated from E. acaulis can be a potent candidate in the designing of anticancer drugs.
Assuntos
Apoptose , Neoplasias Ovarianas , Humanos , Feminino , Proliferação de Células , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Folhas de Planta/metabolismo , Sobrevivência Celular , Extratos Vegetais/químicaRESUMO
Shrimp farming is an important socioeconomic activity worldwide. Infectious myonecrosis virus (IMNV) is an important shrimp virus responsible for significant mortality (up to 70%) in Litopenaeus vannamei. We produced recombinant capsid protein (r-IMNV31) and obtained a highly specific antibody, anti-r-IMNV31, which was used in WOAH-approved ELISA and Western blot to detect IMNV. Further, anti-r-IMNV31 was employed in an indigenously developed lateral flow immunoassay (LFA) with gold nanoparticles as a visual label. Using LFA, IMNV could be detected rapidly (20 min) from tissue homogenate with high specificity, reproducibility, and sensitivity (LOD = 103 viral particles). LFA was validated with "gold standard" qRT-PCR using 60 samples with high sensitivity (100%), specificity (86%). A Cohen's kappa coefficient of 0.86 suggested "good agreement" between LFA and qRT-PCR. With a shelf-life of ~ 1 year at ambient temperature, the use of LFA in the on-site detection of IMNV by shrimp farmers will be a reality.
Assuntos
Nanopartículas Metálicas , Penaeidae , Animais , Reprodutibilidade dos Testes , Ouro , ImunoensaioRESUMO
A new Lu3+ selective fluorescent probe L was synthesized and characterized. The optical properties of L were investigated by using absorption and fluorescence spectral studies in 7 : 3 (v/v) aqueous dimethyl sulphoxide. Upon addition of Lu3+ in a pH 4 (acetate buffer) solution of L, the weakly fluorescent probe L became highly fluorescent. The fluorescence intensity increased five-fold at 490 nm with excitation at 437 nm. The formation of 2 : 1 complexation between L and Lu3+ was confirmed by Job's plot. The binding constant (Ka, 1.43 × 104 M-1) was determined by the Benesi-Hildebrand (BH) method. The limit of detection (LOD) of Lu3+ using L was found to be 23 nM. The binding mechanism of L with Lu3+ was studied by 1H-NMR, ESI mass spectroscopy, and theoretical studies. Further, the probe L was successfully used to bioimage Lu3+ in a zebrafish gill cell line (DrG) and in the yolk, papillae of the eyes, and head of zebrafish embryos (Danio rerio), therefore providing a powerful live imaging approach for investigating chemical signaling in complex multicellular systems.
Assuntos
Corantes Fluorescentes , Peixe-Zebra , Animais , Corantes Fluorescentes/análise , Células HeLa , Humanos , Espectrometria de Fluorescência , ÁguaRESUMO
The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1â¯mM, 1â¯mM, 10â¯mM, 100â¯mM and 500â¯mM) and chlorpromazine hydrochloride (1⯵M, 10⯵M, 100⯵M, 200⯵M and 500⯵M) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1â¯mM, 1â¯mM and 10â¯mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1⯵M, 10⯵M and 100⯵M were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1â¯mM and 10â¯mM, and chlorpromazine hydrochloride of 10⯵M and 100⯵M could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.
Assuntos
Cloreto de Amônio/farmacologia , Antivirais/farmacologia , Clorpromazina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Nodaviridae/efeitos dos fármacos , Cloreto de Amônio/toxicidade , Animais , Antivirais/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Clorpromazina/toxicidade , Efeito Citopatogênico Viral , Ensaio de Imunoadsorção Enzimática , Peixes , Testes de Sensibilidade Microbiana , Nodaviridae/crescimento & desenvolvimento , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/análise , Replicação Viral/efeitos dos fármacosRESUMO
The VP28 gene of white spot syndrome virus was amplified by PCR using gene specific primer set and cloned into pRSET B vector to produce recombinant VP28 (r-VP28) in E. coli GJ1158. The chitosan tripolyphosphate nanoparticles (CS/TPP) were prepared by ionic gelation process and characterized. The purified r-VP28 protein was encapsulated by CS/TPP nanoparticles. The encapsulation efficiency of CS/TPP nanoparticles was found to be 84.8% for r-VP28 protein binding with CS/TPP nanoparticles. The in vitro release profile of encapsulated r-VP28 was determined after treating with protease and chitosanase. The different types of feed were formulated and named as normal feed with PBS, Feed A coated with crude r-VP28, Feed B with purified r-VP28 and Feed C with CS/TPP encapsulated r-VP28 (Purified). Tissue distribution and clearance of r-VP28 at different time intervals were examined in shrimp fed with different types of feed by ELISA and the results showed the presence of r-VP28 protein in different organs. Various immunological parameters were assessed in experimental shrimp. The mRNA expression of five immune-related genes was analysed by qPCR in order to investigate their response to all types of feed in shrimp. A cumulative percentage mortality was also recorded in treated shrimp challenged with WSSV.
Assuntos
Quitosana/química , Nanopartículas/química , Proteínas do Envelope Viral/genética , Vírus da Síndrome da Mancha Branca 1/genética , Animais , Quitosana/farmacologia , Escherichia coli/genética , Géis/química , Penaeidae/genética , Penaeidae/virologia , Proteínas Recombinantes/genética , Proteínas do Envelope Viral/química , Vírus da Síndrome da Mancha Branca 1/patogenicidadeRESUMO
A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect the venom of Indian cobra (Naja naja naja) in various tissues (brain, heart, lungs, liver, spleen, blood, kidneys, and tissue at the site of injection) of mice after cobra venom injected at different time intervals (0, 2, 4, 6, 8, and 12 h intervals up to 24 h). Whole venom antiserum or individual venom protein antiserum (14, 29, 65, 72, and 99 kDa) could recognize N. n. naja venom by Western blotting and ELISA, and antibody titer was also assayed by ELISA. Antiserum raised against cobra venom in rabbit significantly neutralized the toxicity of venom-injected mice at different time intervals after treatment. The assay could detect N. n. naja venom levels up to 2.5 ng/ml of tissue homogenate, and the venom was detected up to 24 h after venom injection. Venom was detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area, and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart, and lungs. Development of a simple, rapid, and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.
Assuntos
Anticorpos Neutralizantes/farmacologia , Antivenenos/farmacologia , Venenos Elapídicos/toxicidade , Ensaio de Imunoadsorção Enzimática/métodos , Mordeduras de Serpentes/diagnóstico , Mordeduras de Serpentes/tratamento farmacológico , Animais , Western Blotting , Modelos Animais de Doenças , Venenos Elapídicos/sangue , Venenos Elapídicos/imunologia , Eletroforese em Gel de Poliacrilamida , Dose Letal Mediana , Masculino , Camundongos , Valor Preditivo dos Testes , Coelhos , Mordeduras de Serpentes/sangue , Mordeduras de Serpentes/imunologia , Fatores de TempoRESUMO
The cytotoxicity, genotoxicity and oxidative stress of malachite green (MG) was investigated using the fish Channa striata kidney (CSK) and Channa striata gill (CSG) cell lines. Five concentrations ranging from 0.001 to 10 µg mL(-1) were tested in three independent experiments. Cytotoxicity was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Rhodamine 123 and Alamar Blue. The mitochondrial changes and apoptosis of MG-exposed cells were observed by Rhodamine 123 and acridine orange/ethidium bromide (AO/EB) staining, respectively. In vitro potential DNA damaging effect of MG was tested using comet assay. Mitochondrial damage, apoptosis and DNA fragmentation increased in a concentration-dependent manner. Additionally, DNA electrophoretic mobility experiments were carried out to study the binding effect of MG to double-stranded DNA (dsDNA) of cells. DNA shift mobility experiments showed that MG is capable of strongly binding to linear dsDNA causing its degradation. Biochemical parameters such as lipid peroxidation (MDA), catalase (CAT) activity and reduced glutathione (GSH) levels were evaluated after exposure to MG. In CSK and CSG cell lines exposed to MG for 48 h, a significant increase in lipid peroxidation, which might be associated with decreased levels of reduced glutathione and catalase activity in these cell lines (p < 0.001), was observed.
Assuntos
Brânquias/efeitos dos fármacos , Rim/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Perciformes , Corantes de Rosanilina/toxicidade , Animais , Linhagem Celular , Ensaio Cometa , Dano ao DNA , Peixes/metabolismo , Água Doce , Brânquias/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Oxirredução , Perciformes/metabolismoRESUMO
The isolated and identified triterpenoid, 1-hydroxytetratriacontane-4-one (C34H68O2), obtained from the methanolic leaf extract of Leucas aspera Linn. was explored for the first time for antisnake venom activity. The plant (L. aspera Linn.) extract significantly antagonized the spectacled cobra (Naja naja naja) venom induced lethal activity in a mouse model. It was compared with commercial antiserum obtained from King Institute of Preventive Medicine (Chennai, Tamil Nadu, India). N. naja naja venom induced a significant decrease in antioxidant superoxide dismutase, glutathione (GSH) peroxidase, catalase, reduced GSH and glutathione-S-transferase activities and increased lipid peroxidase (LPO) activity in different organs such as heart, liver, kidney and lungs. The histological changes following the antivenom treatment were also evaluated in all these organs. There were significant alterations in the histology. Triterpenoid from methanol extract of L. aspera Linn. at a dose level of 75 mg per mouse significantly attenuated (neutralized) the venom-induced antioxidant status and also the LPO activity in different organs.
Assuntos
Antioxidantes/farmacologia , Venenos Elapídicos/toxicidade , Triterpenos/farmacologia , Animais , Catalase/metabolismo , Venenos Elapídicos/antagonistas & inibidores , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Dose Letal Mediana , Camundongos , Extratos Vegetais/farmacologia , Superóxido Dismutase/metabolismoRESUMO
Cell lines of Etroplus suratensis established in our laboratory were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent. The cytotoxic effect of tannery effluent in three cell lines derived from eye, kidney and gill tissue of E. suratensis was assessed using multiple endpoints such as Neutral Red (NR) assay, Coomassie Blue (CB) protein assay and Alamar Blue (AB) assay. Acute toxicity tests on fish were conducted by exposing E. suratensis for 96 h to tannery effluent under static conditions. The toxic effect of tannery effluent on the survival of fish was found to be concentration and time dependent. The tannery effluent at the concentration of 15% caused 100% mortality at 96 h whereas the lower concentration (0.5%) caused 13.33% mortality. The cytotoxicity of tannery effluent was found to be similar in the three cell lines tested, independent of the toxic endpoints employed. EC(50) values, the effective concentration of tannery effluent resulting in 50% inhibition of cytotoxicity parameters after 48 h exposure to tannery effluent were calculated for eye, kidney and gill cell lines using NR uptake, AB and cell protein assays. Statistical analysis revealed good correlation with r(2)=0.95-0.99 for all combinations between endpoints employed. Linear correlations between each in vitro EC(50) and the in vivo LC(50) data, were highly significant p<0.001 with r(2)=0.977, 0.968 and 0.906 for AB(50), NR(50), and CB(50), respectively.