Current Issue : October - December Volume : 2012 Issue Number : 4 Articles : 8 Articles
Background: To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a\r\ncost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of\r\nthe N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The\r\nexpression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombininlike\r\npeptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs\r\nwere purified and evaluated for antimicrobial activity.\r\nResults: Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene\r\nwas then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then\r\nexpressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in\r\nthe cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in\r\nvitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving\r\nAMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All\r\nrecombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.\r\nConclusions: The method described in this report allows the fast synthesis of genes that are optimized for overexpression\r\nin E. coli and for the production of sufficiently large amounts of peptides for functional and structural\r\ncharacterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is\r\na low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic\r\nanalyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/\r\nHis-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning....
Background: Bone physiology is increasingly appreciated as an important contributor to metabolic disorders such\r\nas type 2 diabetes. However, progress in understanding the role of bone in determining metabolic health is\r\nhampered by the well-described difficulty of obtaining high quality RNA from bone for gene expression analysis\r\nusing the currently available approaches.\r\nResults: We developed a simple approach to isolate bone RNA that combines pulverizing the bone and the\r\nphenol-guanidinium based RNA extraction in a single step while maintaining near-freezing temperatures. This\r\nsingle step method increases the yield of high quality RNA by eight-fold, with RNA integrity numbers ranging from\r\n6.7 to 9.2.\r\nConclusions: Our streamlined approach substantially increases the yield of high-quality RNA from bone tissue\r\nwhile facilitating safe and efficient processing of multiple samples using readily available platforms. The RNA\r\nobtained from this method is suitable for use in gene expression analysis in real-time quantitative PCR, microarray,\r\nand next generation sequencing applications...
The organic solvent extracts of Chloroform, Methanol, Ethanol and isolated marine cyanobacteria strains were tested for antibacterial activity against Bacterial pathogens, Anti bacterial activity was based on the disc diffusion method. The results indicated that the Chloroform extract of cyanobacteria Synechococcus elongatus was maximum inhibition and showed tested microorganisms maximum inhibition was Bacillus subtilis (18mm) and minimum in Staphylococcus aureus (10mm), and followed ethanol extract of cyanobacteria Synechococcus elongatus maximum inhibition was showed in Bacillus subtilis (13mm), and minimum in proteus vulgaris (6mm), and minimum in methanol extract of cyanobacteria Synechococcus elongatus and inhibition of tested microorganism was showed maximum in proteus vulgaris (12 mm) and minimum Staphylococcus aureus (4mm). Therefore, suggested that further works performed on the isolation and characterization of the compound....
The biomass production, chlorophyll a, protein contents was varied with temperature, pH and salinity for all the species tested in the present study. There was an increase in biomass production, chlorophyll a, protein contents with increasing temperature. In most of the species, the maximum biomass was obtained at 30 - 400C and minimum at 10- 200C and 500C. The maximum biomass, chlorophyll a and protein content was obtained at pH 9 and 10, while the pH of 7, 8 and 11 showed minimum production of biomass, chlorophyll a and protein contents in almost all the species. Regarding salinity the maximum biomass was obtained at 30 – 40 ppt and minimum in 10, 20 and 50 ppt....
Epigenetic changes to our genes, assisted by non-coding DNA are just as important as genetic changes in coding DNA which determines when and where genes are switched on and off to produce a person. It refers to changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence or Changes in gene expression affect us throughout our lives, not just during fertilization and development. The main epigenetic regulatory mechanisms are histone modifications and DNA methylation. Recently, studies have demonstrated that these processes are acting together in concert to regulate gene Expression like DNA methylation and Histone are sites of reversible post-translational modifications such as acetylation, methylation and phosphorylation in future cancer therapy. It is chemical modifications of genes that are heritable from one cell generation to the next and that affects gene expression but do not alter the DNA sequence. Human Epigenome project aims to identify and catalogue all of the DNA methylation patterns and histone modification in chromatin across the human genome and identify which ones are associated with disease and to imagine the possibilities that it could bring for the future of medicine in cancer....
Background: Mucosal immune surveillance is thought to be largely achieved through uptake by specialized\r\nepithelial M cells. We recently identified Claudin 4 as an M cell target receptor and developed a Claudin 4\r\ntargeting peptide (CPE) that can mediate uptake of nanoparticles through Nasal Associated Lymphoid Tissue\r\n(NALT) M cells.\r\nMethods: Recombinant influenza hemagglutinin (HA) and a version with the CPE peptide at the C-terminal end\r\nwas used to immunize mice by the intranasal route along with a single dose of cholera toxin as an adjuvant.\r\nSerum and mucosal IgG and IgA responses were tested for reactivity to HA.\r\nResults: We found that the recombinant HA was immunogenic on intranasal administration, and inclusion of the\r\nCPE targeting peptide induced higher mucosal IgA responses. This mucosal administration also induced systemic\r\nserum IgG responses with Th2 skewing, but targeting did not enhance IgG responses, suggesting that the IgG\r\nresponse to mucosal immunization is independent of the effects of CPE M cell targeting.\r\nConclusions: M cell targeting mediated by a Claudin 4-specific targeting peptide can enhance mucosal IgA\r\nresponses above the response to non-targeted mucosal antigen. Since Claudin 4 has also been found to be\r\nregulated in human Peyer�s patch M cells, the CPE targeting peptide could be a reasonable platform delivery\r\ntechnology for mucosal vaccination...
Background: During a global influenza pandemic, the vaccine requirements of developing countries can surpass\r\ntheir supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not\r\nbe available in time. There is thus a need for developing countries in general to produce their own pandemic and\r\npossibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing\r\ninfluenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and\r\npotentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this\r\nstudy, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype\r\nH5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA)\r\nsurface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene\r\n(H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of\r\nAgrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell\r\ncompartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic\r\ntobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy.\r\nResults: For both transient and transgenic expression systems the highest accumulation of full-length H5 protein\r\noccurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The\r\nH5 proteins were produced at relatively high concentrations in both systems. Following partial purification,\r\nhaemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced\r\nHA variants was correct and the proteins were functional. The immunisation of chickens and mice with the\r\ncandidate vaccines elicited HA-specific antibody responses.\r\nConclusions: We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic\r\nexpression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine\r\npotential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic\r\nresponse strategy for South Africa and other developing countries....
Background: The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination\r\nbetween its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has\r\ncatalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and\r\nmammalian cells.\r\nResults: In this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis\r\nthaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1\r\nrecombinase successfully excised the target sequence in a conservative manner and the resulting recombination\r\nevent was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In\r\naddition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target\r\ntransgenic plants to generate excised progeny.\r\nConclusion: The Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana\r\ngerminal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1\r\nin planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without\r\nselectable marker transgenes or other undesirable exogenous sequences....
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