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991.
The objective of the present study was to optimise and evaluate a procedure for detection of Mycobacterium avium subsp. paratuberculosis in archived formalin-fixed, paraffin embedded tissue sections from cases of naturally occurring paratuberculosis in goats. A pilot study assessed 3 procedures for extraction of DNA for detection by PCR. The procedure that gave the most consistent results involved removal of paraffin by treatment with xylene and ethanol, disruption of tissue pellets by beating with zirconium/silica beads, extraction of DNA using a DNeasy kit (Qiagen) with overnight proteinase K digestion and final ethanol precipitation. This procedure was used to analyse 82 paraffin embedded tissues (44 small intestine, 38 mesenteric lymph node) with various grades of histological lesions of paratuberculosis and acid-fast bacilli (AFB) loads. The overall sensitivity of the PCR was about 72% of all samples including both paucibacillary and multibacillary lesions. The sensitivity of the assay was 87.5% (42/48) in all paraffin sections having clearly and easily demonstrable AFB. Fifty percent of the tissue sections with rarely detectable AFB were positive by PCR. There was no significant difference (<0.05) between the sensitivity of the PCR analyses carried out on intestinal and mesenteric lymph node tissues. The results of this study suggest that IS900 PCR on formalin-fixed, paraffin embedded histological sections is a practical and important tool for confirming diagnosis of paratuberculosis in goats, where fresh tissues for bacterial culture or PCR are not available due to problem of maintaining a cold chain system. 相似文献
992.
Namukwaya B Tripathi L Tripathi JN Arinaitwe G Mukasa SB Tushemereirwe WK 《Transgenic research》2012,21(4):855-865
Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, is one of the most important diseases of banana (Musa sp.) and currently considered as the biggest threat to banana production in Great Lakes region of East and Central Africa. The pathogen is highly contagious and its spread has endangered the livelihood of millions of farmers who rely on banana for food and income. The development of disease resistant banana cultivars remains a high priority since farmers are reluctant to employ labor-intensive disease control measures and there is no host plant resistance among banana cultivars. In this study, we demonstrate that BXW can be efficiently controlled using transgenic technology. Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of banana. These transgenic lines were characterized by molecular analysis. After challenge with X. campestris pv. musacearum transgenic lines showed high resistance. About 67% of transgenic lines evaluated were completely resistant to BXW. These transgenic lines did not show any disease symptoms after artificial inoculation of in vitro plants under laboratory conditions as well as potted plants in the screen-house, whereas non-transgenic control plants showed severe symptoms resulting in complete wilting. This study confirms that expression of the Pflp gene in banana results in enhanced resistance to BXW. This transgenic technology can provide a timely solution to the BXW pandemic. 相似文献
993.
Siddharth M Datta SK Bansal S Mustafa M Banerjee BD Kalra OP Tripathi AK 《Journal of biochemical and molecular toxicology》2012,26(6):241-247
Nephrotoxicity of organochlorine pesticides (OCPs) has been established in experimental animal models. This study was designed to evaluate the relationship of the blood OCPs level with the estimated glomerular filtration rate (eGFR) and oxidative stress (OS) in chronic kidney disease (CKD) patients. Patients in different stages of CKD (n = 150) and age, sex matched healthy controls (n = 96) were recruited. The blood OCPs level were analyzed by gas chromatography, and plasma levels of several OS parameters such as malondialdehyde (MDA), protein carbonyl, advanced oxidation protein products (AOPP), and total thiols were quantified by standard spectrophotometric methods. We observed significantly higher levels of hexachlorocyclohexane (α, γ), endosulfan, aldrin, p,p'-dichlorodiphenyldichloroethylene (DDE), and total pesticides in CKD patients. Negative correlation was also observed for aldrin, p,p'-DDE and total pesticides (p < 0.05) with eGFR. Plasma levels of MDA and AOPP showed significant positive association with the total pesticides level, indicating augmentation of OS with increased accumulation of OCPs in CKD patients. 相似文献
994.
High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro 总被引:1,自引:0,他引:1
Tripathi A Chaube SK 《Apoptosis : an international journal on programmed cell death》2012,17(5):439-448
The present study was aimed to find out whether an increase of cytosolic free calcium level induces egg apoptosis through
mitochondria-caspase mediated pathway. To increase cytosolic free calcium level and morphological apoptotic changes, ovulated
eggs were cultured in Ca2+/Mg2+ free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 μM) for 3 h in vitro. The morphological
apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H2O2) concentration, catalase activity, cytochrome c concentration, caspase-9 and caspase-3 activities and DNA fragmentation were
analyzed. Calcium ionophore induced morphological apoptotic features in a concentration-dependent manner followed by degeneration
at higher concentrations (3 and 4 μM). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen
peroxide (H2O2) and inhibited catalase activity in treated eggs. The increased H2O2 concentration was associated with increased cytochrome c concentration, caspase-9 and caspase-3 activities that resulted
in the induction of morphological features characteristic of egg apoptosis. The increased caspase-3 activity finally induced
DNA fragmentation as evidenced by TUNEL positive staining in calcium ionophore-treated eggs. These findings suggest that high
cytosolic free calcium level induces generation of H2O2 that leads to egg apoptosis through mitochondria-caspase mediated pathway. 相似文献
995.
Nucleation temperature‐controlled synthesis and in vitro toxicity evaluation of l‐cysteine‐capped Mn:ZnS quantum dots for intracellular imaging 下载免费PDF全文
Vivek Pandey Gajanan Pandey Vinay Kumar Tripathi Sapna Yadav Mohana Krishna Reddy Mudiam 《Luminescence》2016,31(2):341-347
Quantum dots (QDs), one of the fastest developing and most exciting fluorescent materials, have attracted increasing interest in bioimaging and biomedical applications. The long‐term stability and emission in the visible region of QDs have proved their applicability as a significant fluorophore in cell labelling. In this study, an attempt has been made to explore the efficacy of l ‐cysteine as a capping agent for Mn‐doped ZnS QD for intracellular imaging. A room temperature nucleation strategy was adopted to prepare non‐toxic, water‐dispersible and biocompatible Mn:ZnS QDs. Aqueous and room temperature QDs with l ‐cysteine as a capping agent were found to be non‐toxic even at a concentration of 1500 µg/mL and have wide applications in intracellular imaging. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
996.
Generally, pellets obtained from extrusion/spheronization, containing microcrystalline cellulose (MCC), do not disintegrate. An attempt has been made to develop melt-in-mouth pellets of taste-masked atomoxetine hydrochloride, using extrusion-spheronization, for pediatric patients. Melt-in-mouth pellets were prepared using extrusion-spheronization method and optimized using 33 FFD. MCC (X1, %), mannitol (X2, %) and Indion 414: Pharmaburst 500 ratio (X3, ratio) were the factors (independent variables) studied, whereas responses studied (dependent variables) were friability (Y1, %), yield (Y2, %) shape (Y3, roundness) in vitro disintegration time (Y4, seconds). The optimized formulation obtained from FFD was characterized for friability, shape and morphology, in vitro disintegration time, porosity, moisture uptake, in vitro release study and in vivo taste and disintegration time in healthy human volunteers. Randomized, two-treatment, two-sequence, two-period, single dose, crossover sensory evaluation study of taste-masked melt-in-mouth pellet was carried out in 10 healthy human subjects. A statistically significant polynomial mathematical relationship was generated between the factors and responses to obtain an optimized formulation. The optimized formulation was characterized (in vitro and in vivo) and exhibited a rapid drug release in vitro attributed to fast disintegration of pellets and high solubility of drug in 0.1 N HCl and buffer (pH 6.8). In vivo, 40% of volunteers ranked taste-masked optimized formulation as slightly bitter while 60% ranked it as no taste. The optimized pellets were conveniently administered in volunteers and exhibited rapid in-vivo disintegration in the oral cavity. Melt-in-mouth pellets can be a used as a platform technology for administering drugs to paediatric patients accurately and conveniently resulting in patient compliance. 相似文献
997.
The effective and robust separation of biomolecules of interest from patient samples is an essential step in diagnostic applications. We present a platform for the fast extraction of nucleic acids from clinical specimens utilizing paramagnetic PMPs, an oil-water interface, a small permanent magnet and a microfluidic channel to separate and purify captured nucleic acids from lysate in less than one minute, circumventing the need for multiple washing steps and greatly simplifying and expediting the purification procedure. Our device was able to isolate influenza RNA from clinical nasopharyngeal swab samples with high efficiency when compared to the Ambion® MagMAXTM Viral RNA Isolation Kit, sufficiently separating nucleic acid analytes from PCR-inhibiting contaminants within the lysate while also critically maintaining high integrity of the viral genome. We find that this design has great potential for rapid, efficient and sensitive nucleic acid separation from patient sample. 相似文献
998.
In Young Park Pratim Chowdhury Durga Nand Tripathi Reid T. Powell Ruhee Dere Esteban A. Terzo 《MABS-AUSTIN》2016,8(8):1590-1597
Posttranslational modifications (PTMs) on microtubules differentiate these cytoskeletal elements for a variety of cellular functions. We recently identified SETD2 as a dual-function histone and microtubule methyltransferase, and methylation as a new microtubule PTM that occurs on lysine 40 of α-tubulin, which is trimethylated (α-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (α-TubK40me3 pAb) and monoclonal (α-TubK40me3 mAb) antibodies to a methylated α-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di- or tri-methylated lysine residues they recognize. While both the pAb and mAb antibodies recognized lysines methylated by SETD2 on microtubules and histones, the clone 18 mAb was more specific for methylated microtubules, with little cross-reactivity for methylated histones. The clone 18 mAb recognized specific subsets of microtubules during mitosis and cytokinesis, and lacked the chromatin staining seen by immunocytochemistry with the pAb. Western blot analysis using these antibodies revealed that methylated α-tubulin migrated faster than unmethylated α-tubulin, suggesting methylation may be a signal for additional processing of α-tubulin and/or microtubules. As the first reagents that specifically recognize methylated α-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules. 相似文献
999.
Binu M. Tripathi David P. Edwards Lucas William Mendes Mincheol Kim Ke Dong Hyoki Kim Jonathan M. Adams 《Molecular ecology》2016,25(10):2244-2257
Selective logging and forest conversion to oil palm agriculture are rapidly altering tropical forests. However, functional responses of the soil microbiome to these land‐use changes are poorly understood. Using 16S rRNA gene and shotgun metagenomic sequencing, we compared composition and functional attributes of soil biota between unlogged, once‐logged and twice‐logged rainforest, and areas converted to oil palm plantations in Sabah, Borneo. Although there was no significant effect of logging history, we found a significant difference between the taxonomic and functional composition of both primary and logged forests and oil palm. Oil palm had greater abundances of genes associated with DNA, RNA, protein metabolism and other core metabolic functions, but conversely, lower abundance of genes associated with secondary metabolism and cell–cell interactions, indicating less importance of antagonism or mutualism in the more oligotrophic oil palm environment. Overall, these results show a striking difference in taxonomic composition and functional gene diversity of soil microorganisms between oil palm and forest, but no significant difference between primary forest and forest areas with differing logging history. This reinforces the view that logged forest retains most features and functions of the original soil community. However, networks based on strong correlations between taxonomy and functions showed that network complexity is unexpectedly increased due to both logging and oil palm agriculture, which suggests a pervasive effect of both land‐use changes on the interaction of soil microbes. 相似文献
1000.
Uridine 5′-diphospho-glucuronosyltransferase-1A9 (UGT1A9) expressed in the liver, shows good sequence identity with UGT1A10, expressed in the intestine. Both uridine 5′-diphospho-glucuronosyltransferase (UGT) isoforms show comprehensive overlapping substrate selectivity but there are differences in stereoselectivity, regiospecificity and rate of glucuronidation of the substrates. Multiple sequence alignment analyses of UGT1A9 and UGT1A10 showed that 13% of the residues in N-terminal domain (NTD) are non-identical between them. Herein, authors attempted homology modelling of UGT1A9 and UGT1A10 and validation using software tools and reported mutagenic studies. A molecular docking study of the known substrates is performed on UGT1A9 and UGT1A10 homology models. The non-identical N-terminal residues ranging from 111 to 117 in UGT1A9 and UGT1A10 were identified to play a central role in the substrate selectivity. However, substrate binding is performed by Ser111, Gly115 and Leu117 in UGT1A10 and Gly111, Asp115 and Phe117 in UGT1A9. This study reports new residues in NTD, showing interaction with uridine 5′-diphospho-glucuronic acid which binds with C-terminal domain. Further, molecular dynamics simulations were carried out to study the role of non-identical residues in substrate identification. The study demonstrates the folding of the UGT enzyme, particularly, helix-loop-helix transition and movement of Nα3-2 helix, in response to substrate and co-substrate binding. 相似文献