RESUMEN
Transgenic crops expressing Cry δ-endotoxins of Bacillus thuringiensis for insect resistance have been commercialized worldwide with increased crop productivity and spectacular socioeconomic gains. To attain the enhanced level of protein expression, the cry genes have to be extensively modified for RNA stability and translation efficiency in the plant systems. However, such modifications in nucleotide sequences make it difficult to express the cry genes in Escherichia coli because of the presence of E. coli rare codons. Induction of gene expression through the T7 promoter/lac operator system results in high levels of transcription but limits the availability of activated tRNA corresponding to rare codons that leads to translation stalling at ribosomes. In the present study, an Isopropyl ß-D-1-thiogalactopyranoside (IPTG)/rifampicin combination-based approach was adopted to induce transcription of cry genes through T7 promoter/lac operator while simultaneously inhibiting the transcription of host genes through rifampicin. The results show that the IPTG/rifampicin combination leads to high-level expression of four plant codon-optimized cry genes (cry2Aa, cry1F, cry1Ac, and cry1AcF). Northern blot analysis of the cry gene expressing E. coli samples showed that the RNA expression level in the IPTG-induced samples was higher as compared to that in the IPTG/rifampicin-induced samples. Diet overlay insect bioassay of IPTG/rifampicin-induced Cry toxins with Helicoverpa armigera larvae showed bioactivity (measured as LC50) similar to the previous studies. The experiment has proved that recombinant synthetic gene (plant codon-optimized gene) with the combination of Rifampicin which inhibits DNA-dependent bacterial RNA polymerase and reduces the excessive baggage of translational machinery of the bacterial cell triggers the production of synthetic protein. Purification of protein using high pH buffer increases the solubility of the protein. Further, LC50 analysis shows no reduction of protein activity leads to protein stability. Further, purified cry toxin protein can be used for crop protection against pests and a purified form of the synthetic protein can be used for antibody production and perform the immunoassay for the identification of the transgenic plant. The crystallographic structure of synthetic protein could be used for interaction study with another insect to see insecticidal activity.
Asunto(s)
Bacillus thuringiensis , Endotoxinas , Animales , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Codón , Escherichia coli/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacología , Isopropil Tiogalactósido , Larva , Rifampin/farmacologíaRESUMEN
DNA conformation and stability are critical for the normal cell functions, which control many cellular processes in life, such as replication, transcription, DNA repair, etc. The accumulation of amyloid-ß peptide (Aß) and Copper (Cu) are the etiological factors for neurodegenerative diseases and hypothesized that they can cause DNA instability. In the current investigation, we studied copper and Aß1-16 induced conformation and stability changes in CAG/CTG sequences and found alterations from B-DNA to altered B-conformation. Further, the interaction of the copper and Aß1-16 with CAG/CTG sequences was studied by molecular docking modeling and results indicated that the interaction of copper and Aß1-16 was through the hydrogen bond formation between adenine, guanine, and cytocine. This study illustrates the role of the copper and Aß1-16 in modulating the DNA conformation and stability.
RESUMEN
The relatively short circulatory half-life (2-3 min) of staphylokinase is a major drawback in the development of SAK- (staphylokinase) based thrombolytic drug. A rapid and sensitive method, based on indirect competitive ELISA, was developed and validated for quantitative determination of SAK in rabbit plasma. The dynamic range of the assay varied between 0.41 ± 0.16 µg/L and 9.03 ± 0.38 µg/L (R(2) = 0.98) for SAK in rabbit plasma. There were no dilution linearity issues apparent with this assay. The precision (% CV) ranged from 4.6-9.7% for the intraassay and from 17.1-19.3% for interassay. This validated method was successfully employed for evaluation of various pharmacokinetic parameters of SAK in rabbit.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Fibrinolíticos/farmacocinética , Metaloendopeptidasas/sangre , Animales , Femenino , Fibrinolíticos/sangre , Masculino , Metaloendopeptidasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Conejos , Proteínas Recombinantes/sangre , Proteínas Recombinantes/metabolismoRESUMEN
Availability of adequate quantity of purified virus preparation from plant tissue is the major limitation in producing polyclonal antibodies (PAb) to begomovirus. Very few examples show successful utilization of E. coli expressed recombinant coat protein (CP) for immuno diagnosis of begomoviruses. In the present study, ~771 bp CP gene (~29.0 kDa) of Pumpkin yellow vein mosaic virus (PYVMV) was expressed as a ~71.0 kDa fusion protein with maltose binding protein (MBP) (~42.0 kDa) in E. coli. The MBP-CP was obtained in soluble state. The PAb to the purified fusion protein successfully detected PYVMV and other bipartite and monopartite begomoviruses in the field samples at 1:250 dilution in enzyme linked immunosorbent assay. Our study for the first time showed that MBP-tag fusion CP was suitable to produce diagnostic antibody to begomoviruses.
RESUMEN
In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active ß-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for ß-galactosidase production. Under optimal conditions, the production of ß-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. ß-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active ß-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active ß-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Bahías/microbiología , Análisis de Componente Principal , Rhodospirillaceae/química , beta-Galactosidasa/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Carbono/metabolismo , Frío , Medios de Cultivo/química , Fermentación , Concentración de Iones de Hidrógeno , India , Cinética , Lactosa/metabolismo , Nitrógeno/metabolismo , Rhodospirillaceae/enzimología , beta-Galactosidasa/aislamiento & purificaciónRESUMEN
In this study, we have demonstrated the conformational changes to DNA induced by abnormal interactions of copper using circular dichroism, in combination with UV-absorbance and fluorescence spectroscopy. Results confirm that binding of copper to bases of DNA in chromatin is concentration dependent. Binding efficiency of Cu2+ ions to DNA is increased in proportion to the degree of unwinding of the double helix induced by denaturation. Altered B-DNA conformation will alter the integrity of DNA which may affect the normal process of DNA replication and transcription. Copper induced DNA damage in the brain may cause neurotoxicity and the neuronal cell death and is implicated in Alzheimer's disease and other neurological disorders.
RESUMEN
The present study deals with the production of cold active polygalacturonase (PGase) by submerged fermentation using Thalassospira frigidphilosprofundus, a novel species isolated from deep waters of Bay of Bengal. Nonlinear models were applied to optimize the medium components for enhanced production of PGase. Taguchi orthogonal array design was adopted to evaluate the factors influencing the yield of PGase, followed by the central composite design (CCD) of response surface methodology (RSM) to identify the optimum concentrations of the key factors responsible for PGase production. Data obtained from the above mentioned statistical experimental design was used for final optimization study by linking the artificial neural network and genetic algorithm (ANN-GA). Using ANN-GA hybrid model, the maximum PGase activity (32.54 U/mL) was achieved at the optimized concentrations of medium components. In a comparison between the optimal output of RSM and ANN-GA hybrid, the latter favored the production of PGase. In addition, the study also focused on the determination of factors responsible for pectin hydrolysis by crude pectinase extracted from T. frigidphilosprofundus through the central composite design. Results indicated 80% degradation of pectin in banana fiber at 20 °C in 120 min, suggesting the scope of cold active PGase usage in the treatment of raw banana fibers.
Asunto(s)
Fermentación , Pectinas/biosíntesis , Poligalacturonasa/biosíntesis , Medios de Cultivo , Hidrólisis , Rhodospirillaceae/enzimología , Rhodospirillaceae/crecimiento & desarrolloRESUMEN
Archaea are ubiquitous in their presence and abundant not only in extreme environments, but also in soil, oceans and freshwater, where they may fulfil a key role in the biogeochemical cycles of the earth. The identification of archaeal genomic signatures elucidates us a measure of distinctiveness of Archaea as a coherent group, although these signatures can differ according to the degree of stringency. The 16S rRNA and the Rad A genes are highly conserved in living organisms and are very useful for the phylogenetic analysis. Phylogenetic trees are constructed using the molecular evolutionary genetics analysis (MEGA) tool by neighbour joining (NJ) method and repeated bootstrapping for 5000 times was performed. The two trees were then compared using the Compare2trees and statistically analysed using the MEGA tool. The two phylogenetic trees show a similarity of 54.9%. In both the trees, the taxon Thaumarchaeota shows a high level of variance. The species Cenarchaeum symbiosum A shows a high level of similarity with the sequences of higher organisms (Euryarcheota-eukaryota), which shows that it has branched away to higher organisms from a closely related Protozoa, Eubacteria ancestor.
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Archaea/genética , Evolución Molecular , Genómica/métodos , Filogenia , ARN de Archaea/genética , ARN Ribosómico 16S/genética , GenomaRESUMEN
The cold active ß-galactosidase from psychrophilic bacteria accelerate the possibility of outperforming the current commercial ß-galactosidase production from mesophilic sources. The present study is carried out to screen and isolate a cold active ß-galactosidase producing bacterium from profound marine waters of Bay-of-Bengal and to optimize the factors for lactose hydrolysis in milk. Isolated bacterium 3SC-21 was characterized as marine psychrotolerant, halophile, gram negative, rod shaped strain producing an intracellular cold active ß-galactosidase enzyme. Further, based upon the 16S rRNA gene sequence, bacterium 3SC-21 was identified as Thalassospira sp. The isolated strain Thalassospira sp. 3SC-21 had shown the enzyme activity between 4 and 20 °C at pH of 6.5 and the enzyme was completely inactivated at 45 °C. The statistical method, central composite rotatable design of response surface methodology was employed to optimize the hydrolysis of lactose and to reveal the interactions between various factors behind this hydrolysis. It was found that maximum of 80.18 % of lactose in 8 ml of raw milk was hydrolysed at pH of 6.5 at 20 °C in comparison to 40 % of lactose hydrolysis at 40 °C, suggesting that the cold active ß-galactosidase from Thalassospira sp. 3SC-21 would be best suited for manufacturing the lactose free dairy products at low temperature.
Asunto(s)
Lactosa/metabolismo , Leche/química , Rhodospirillaceae/enzimología , Rhodospirillaceae/aislamiento & purificación , beta-Galactosidasa/genética , Animales , Bahías/microbiología , Frío , Concentración de Iones de Hidrógeno , Hidrólisis , India , ARN Ribosómico 16S/genética , Rhodospirillaceae/clasificación , Rhodospirillaceae/genética , Agua de Mar/microbiología , Cloruro de Sodio/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Laccase- and peroxidase-free tyrosinase has commercial importance in the production of L-3, 4-dihydroxyphenylalanine (L-DOPA), which is mainly used in the treatment of Parkinson's disease. In the present study, isolation of an actinomycetes microbial strain capable of producing only tyrosinase is reported. Among all soil isolates, three individual colonies revealed black color around the colony in the presence of tyrosine. Further screening for laccase and peroxidase activities using syringaldazine denoted that one of the isolates, designated as RSP-T1, is laccase and peroxidase negative and produces only tyrosinase. The microbe was authenticated as Streptomyces antibioticus based on 16S ribotyping. Effective growth of this isolate was noticed with the use of medium (pH 5.5) containing casein acid hydrolysate (10.0 g/l), K(2)HPO(4) (5.0 g/l), MgSO(4) (0.25 g/l), L-tyrosine (1.0 g/l), and agar (15 g/l). The scanning electron micrograph depicted that the microbe is highly branched and filamentous in nature. The enzyme production was positively regulated in the presence of copper sulfate. The impact of different fermentation parameters on tyrosinase production depicted that the maximized enzyme titer values were observed when this isolate was grown at 6.5 pH and at 30 degrees C temperature under agitated conditions (220 rpm). Among all the studied physiological parameters, agitation played a significant role on tyrosinase production. Upon optimization of the parameters, the yield of tyrosinase was improved more than 100% compared with the initial yield.
Asunto(s)
Monofenol Monooxigenasa/metabolismo , Streptomyces antibioticus/enzimología , Streptomyces antibioticus/aislamiento & purificación , Análisis por Conglomerados , Sulfato de Cobre/metabolismo , Medios de Cultivo/química , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Activadores de Enzimas/metabolismo , Hidrazonas/metabolismo , Concentración de Iones de Hidrógeno , Lacasa/metabolismo , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Peroxidasa/metabolismo , Filogenia , ARN Ribosómico 16S/genética , Ribotipificación , Análisis de Secuencia de ADN , Microbiología del Suelo , Streptomyces antibioticus/citología , Streptomyces antibioticus/genética , Temperatura , Tirosina/metabolismoRESUMEN
Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.
Asunto(s)
Antígenos Virales/inmunología , Glicoproteínas/inmunología , Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/aislamiento & purificación , Baculoviridae , Tampones (Química) , Línea Celular , Clonación Molecular , Detergentes , Expresión Génica , Vectores Genéticos , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Insectos , Ratones , Rabia/inmunología , Rabia/prevención & control , Vacunas Antirrábicas/administración & dosificación , Vacunas Antirrábicas/química , Vacunas Antirrábicas/aislamiento & purificación , Virus de la Rabia/genética , Solubilidad , Spodoptera , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
Permanent loss of cardiomyocytes and scar tissue formation after myocardial infarction (MI) results in an irreversible damage to the cardiac function. Cardiac repair (replacement, restoration, and regeneration) is, therefore, essential to restore function of the heart following MI. Existing therapies lower early mortality rates, prevent additional damage to the heart muscle, and reduce the risk of further heart attacks. However, there is need for treatment to improve the infarcted area by replacing the damaged cells after MI. Thus, the cardiac tissue regeneration with the application of stem cells may be an effective therapeutic option. Recently, interest is more inclined toward myocardial regeneration with the application of stem cells. However, the potential benefits and the ability to improve cardiac function with the stem cell-based therapy need to be further addressed. In this review, we focus on the clinical applications of stem cells in the cardiac repair.
RESUMEN
Beauveria bassiana is a biocontrol agent which shows entomopathogenecity on insect pests especially the Lepidopterons invading the agriculturally important crops. The mode of infection is through the cuticle by degrading the chitin present on it which is enabled by the exochitinase enzyme of Bbchit1 gene. A good quality genomic DNA was isolated from Beauveria bassiana NCIM 1216 and amplified with specific primers to isolate the gene corresponding to Bbchit1 which codes for the exochitinase enzyme that is responsible for pathogenesis. The Bbchit1 gene of B. bassiana was transformed with the binary plasmid pBANF-bar-pAN-Bbchit1, in which the Bbchit1 gene was placed downstream of the constitutive gpd promoter, which was mediated by A. tumefaciens, and transformants were selected on the basis of herbicide resistance. Fifty herbicide resistant colonies were obtained and analyzed. The exochitinase produced by these transformants was observed maximum on the 7th day of inoculation in both which was 0.09 µmol/ml/min for the purified fraction and 0.06 µmol/ml/min for the crude extract. The chitinolytic activity was observed maximum at pH 5 and at temperature of 40°C. The genetically modified pure form can be used in the production of transgenic plants and in bringing out commercial formulation for the control of Lepidopteran pests.
RESUMEN
Exposure of fish to a sublethal concentration of malathion showed a significant inhibition of acetylcholinesterase (AChE) activity. The levels of protease were markedly elevated with a consequent increase in most of the free amino acids. However, the levels of glutamic acid and valine, phenylalanine and methionine complex remained unchanged, while aspartic acid showed a marked drop. These changes are discussed in relation to the sublethal stress induced by malathion.