Current Issue : July - September Volume : 2011 Issue Number : 3 Articles : 11 Articles
Plumbago indica (Plumbaginaceae) is an important indigenous medicinal plant commonly known as Lal Chitra. The roots contain an orange-yellow pigment plumbagin. Chemically, plumbagin is 2-methyl-5-hydroxy-1,4-napthoquinone. The roots are used as antimalarial, anticancer, antimicrobial, cardiotonic and also posses anti-fertility activity. The present work is an attempt to search for an alternative source of plumbagin. The callus was initiated from leaf and stem explants on MS medium supplemented with 2,4-D + NAA (1ppm each). The plumbagin content of callus as well as natural root were determined using HPLC and it is found that callus contain more plumbagin as compared to natural root....
Background\r\nG protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems that are cost effective. Reasons for these difficulties include inefficient transport and insertion in the plasma membrane and cytotoxicity. Additionally, GPCR purification requires detergents, which have a negative effect on receptor yields and stability.\r\nResults\r\nHere we report a detergent-free cell-free protein expression-based method to obtain pharmacologically active GPCRs in about 2 hours. Our strategy relies on the co-translational insertion of modified GPCRs into nanometer-sized planar membranes. As a model we employed an engineered �Ÿ2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (�Ÿ2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active �Ÿ2AR -T4L in cell-free systems. The binding specificity of the NLP- �Ÿ2AR-T4L complex has been determined by competitive assays. Our results demonstrate that �Ÿ2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor.\r\nConclusions\r\nAlthough the activation of �Ÿ2AR-T4L requires the insertion of the T4 lysozyme sequence and the yield of that active protein limited, our results conceptually prove that cell-free protein expression could be used as a fast approach to express these valuable and notoriously difficult-to-express proteins....
Background\r\nPlant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system.\r\nResults\r\nA LOX gene which is expressed after treatment of the viral vectors was isolated from Nicotiana benthamiana. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as Nb-9-LOX. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml-1 cell cultures) which can oxygenate linoleic acid resulting in high yields (18 �µmol ml-1 cell cultures) of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL) or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL) and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C9-aldehydes (3.3 �µmol mg-1 crude protein). The yield of C9-aldehydes from linoleic acid was 64%.\r\nConclusion\r\nThe yeast expressed Nb-9-LOX can be used to produce C9-aldehydes on a large scale in combination with a HPL gene with 9-HPL function, or to effectively produce 9-hydroxy-10(E),12(Z)-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent....
The aim of this study was to isolate and express the randomly mutated a-amylase gene from B. subtilis strain 168. BS168F: 5'-gtgtcaagaatgtttgc-3' and BS168R: 3'-gttttgttaaaagatga-5' primers were used to amplify the amylase gene using the following cycle in error-prone PCR method: 94�°C for 30?s, 40�°C for 2 min, and 72�°C for 2 min in 30 cycles that were followed with 72�°C for 2 min as a post cycle. E. coli XL1 blue was used as host for plasmid construction. Amylase enzyme activity assay was performed using continuous spectrophotometric procedures. Results of sequencing showed that sequence was cloned from the first ATG and with the correct open reading frame. Having confirmed the integrity of the insert, the gene was ligated into expression vector pET-15b and then further confirmed using digestion analysis. Amylase activity showed 3 clones with higher enzymatic activity compared with the wild type. Error-prone PCR method produced a mutated gene that provides amylase activity much higher than that of wild type. Sequencing the mutated genes should shed light on the important region of the genes that could be manipulated in future studies....
The aim of the study was to evaluate the potential antibacterial activity of Desmodium gangeticum whole plant ethanolic extract (DGEE) using in vitro antibacterial models. The in vitro antibacterial activity was performed by agar disc diffusion method .In antibacterial method DGEE was used at different concentrations 2,4,6,8,mg/ml and standard streptomycin (30µg/ml )was evaluated on bacterial strains like Bacillus subtilus ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC27853, Salmonella typhimurium ATCC 23564.The anti bacterial activity of DGEE showed concentration dependent activity against all the tested bacterium with the zone of inhibition at various concentrations. Thus the finding revealed the medicinal potential of DGEE against various infectious diseases to develop a drug. The ethanolic extract (8mg/ml) showed broad-spectrum antibacterial activity against gram positive and gram negative bacteria. The results were compared with the standard drug streptomycin (30µg/ml)....
Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (\r\n�± 0 . 5\r\n?log10 unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0?IU/mL. A high correlation (\r\n?? = 0 . 9 2 5\r\n) was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (\r\n?? = 1 4\r\n). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load....
Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics....
Live attenuated vaccines are being used to control Bluetongue in many parts of the world. Concerns regarding safety of these vaccines lead to development of inactivated vaccine. During the present study, inactivation of Bluetongue virus (BTV) using BEI at 3mM was tried and found to be effective in degrading BTV RNA, but the inactivated virus has shown residual infectivity. BTV has a tendency to form aggregates, which might have prevented inactivation by BEI, which was circumvented by addition low concentrations (0.04%) of formaldehyde....
This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8?M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution....
Abstract: Centella asiatica Linn. Commonly known as mandukparni or brahmi manduki is a traditional medical herb widely used as a brain tonic. Micro propagation offer rapid in vitro clonal multiplication of elite clones and further help in dissemination and ex situ conservation of this important yet endangered medicinal plant. In the present study, we have tried to optimize the invitro culture condition of centella asiatica for mass multiplication and callus culture study has also been initiated. Through our work, we have found that MS basal +1.0mg / l BAP+20gm/l sugar +8.0gm/l agar gives the best result for culture initiation as well as in auxiliary shoot proliferation. For rooting, MS+1.0mg/l NAA +200mg charcoal + 20 gm/l sugar +8 gm/l agar gave higher primary roots/ shoots with higher secondary roots....
Background\r\nThe ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture.\r\nResults\r\nProteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction.\r\nConclusion\r\nThese proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein....
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