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31.
A simple and fairly rapid procedure is described for separation of a mixture of five 3′, 5′-cyclic ribonucleotides. The separation was achieved by high pressure liquid chromatography on a Vydac pellicular anion exchange column using a linear gradient of 0.5 to 1.0 m acetic acid as the eluting solvent. 相似文献
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Vibrio cholerae hemolysin (HlyA) is a 65?kDa pore-forming toxin which causes lysis of target eukaryotic cells by forming heptameric channels in the plasma membrane. Deletion of the 15?kDa C-terminus β-prism carbohydrate-binding domain generates a 50?kDa truncated variant (HlyA50) with 1000-fold-reduced pore-forming activity. Previously, we showed by cryo-electron microscopy that the two toxin oligomers have central channels, but the 65?kDa toxin oligomer is a seven-fold symmetric structure with bowl-, ring-, and arm-like domains, whereas the 50?kDa oligomer is an asymmetric jar-like heptamer. In the present study, we determined three-dimensional(3D) structures of HlyA and HlyA50 in presence of erythrocyte stroma and observed that interaction of the 65?kDa toxin with the stroma induced a significant decrease in the height of the β-barrel oligomer with a change in conformation of the ring- and arm-like domains of HlyA. These features were absent in interaction of HlyA50 with stroma. We propose that this conformational transition is critical for membrane-insertion of the toxin. 相似文献
35.
Nitesh Kumar Somlata Mohit Mazumder Priyanka Dutta Sankar Maiti Samudrala Gourinath 《PLoS pathogens》2014,10(9)
Entamoeba histolytica is a protist parasite that is the causative agent of amoebiasis, and is a highly motile organism. The motility is essential for its survival and pathogenesis, and a dynamic actin cytoskeleton is required for this process. EhCoactosin, an actin-binding protein of the ADF/cofilin family, participates in actin dynamics, and here we report our studies of this protein using both structural and functional approaches. The X-ray crystal structure of EhCoactosin resembles that of human coactosin-like protein, with major differences in the distribution of surface charges and the orientation of terminal regions. According to in vitro binding assays, full-length EhCoactosin binds both F- and G-actin. Instead of acting to depolymerize or severe F-actin, EhCoactosin directly stabilizes the polymer. When EhCoactosin was visualized in E. histolytica cells using either confocal imaging or total internal reflectance microscopy, it was found to colocalize with F-actin at phagocytic cups. Over-expression of this protein stabilized F-actin and inhibited the phagocytic process. EhCoactosin appears to be an unusual type of coactosin involved in E. histolytica actin dynamics. 相似文献
36.
Mammalian Na+/H+ exchanger isoform one (NHE1) is a plasma membrane protein responsible for pH regulation in mammalian cells. Excess activity of the protein promotes heart disease and is a trigger of metastasis in cancer. Inhibitors of the protein exist but problems in specificity have delayed their clinical application. Here we examined amino acids involved in two modeled inhibitor binding sites (A, B) in human NHE1. Twelve mutations (Asp159, Phe348, Ser351, Tyr381, Phe413, Leu465, Gly466, Tyr467, Leu468, His473, Met476, Leu481) were made and characterized. Mutants S351A, F413A, Y467A, L468A, M476A and L481A had 40–70% of wild type expression levels, while G466A and H473A expressed 22% ~ 30% of the wild type levels. Most mutants, were targeted to the cell surface at levels similar to wild type NHE1, approximately 50–70%, except for F413A and G466A, which had very low surface targeting. Most of the mutants had measurable activity except for D159A, F413A and G466A. Resistance to inhibition by EMD87580 was elevated in mutants F438A, L465A and L468A and reduced in mutants S351A, Y381A, H473A, M476A and L481A. All mutants with large alterations in inhibitory properties showed reduced Na+ affinity. The greatest changes in activity and inhibitor sensitivity were in mutants present in binding site B which is more closely associated with TM4 and C terminal of extracellular loop 5, and is situated between the putative scaffolding domain and transport domain. The results help define the inhibitor binding domain of the NHE1 protein and identify new amino acids involved in inhibitor binding. 相似文献
37.
Ashok Das Soumajit Dutta Moumita Sen Abha Saxena Jitendra Kumar Lopamudra Giri David W. Murhammer Jayanta Chakraborty 《Biotechnology and bioengineering》2021,118(1):238-252
Baculoviruses have enormous potential for use as biopesticides to control insect pest populations without the adverse environmental effects posed by the widespread use of chemical pesticides. However, continuous baculovirus production is susceptible to DNA mutation and the subsequent production of defective interfering particles (DIPs). The amount of DIPs produced and their genome length distribution are of great interest not only for baculoviruses but for many other DNA and RNA viruses. In this study, we elucidate this aspect of virus replication using baculovirus as an example system and both experimental and modeling studies. The existing mathematical models for the virus replication process consider DIPs as a lumped quantity and do not consider the genome length distribution of the DIPs. In this study, a detailed population balance model for the cell‐virus culture is presented, which predicts the genome length distribution of the DIP population along with their relative proportion. The model is simulated using the kinetic Monte Carlo algorithm, and the results agree well with the experimental results. Using this model, a practical strategy to maintain the DIP fraction to near to its maximum and minimum limits has been demonstrated. 相似文献
38.
The results of pervaporation-coupled esterification of various carboxylic acids with ethanol catalyzed by Porcine pancreatic lipase are reported. The effect of lipase and substrate concentrations has been studied and the advantage of pervaporation on the equilibrium conversion has been deduced. The kinetics of reaction were analyzed with a three-parameter model which coupled the effect of pervaporation. The intrinsic kinetic constants for all the reactions were estimated and correlated with the carbon number, an indicator of hydrophobicity of the acids. It was found that the rate constant increases with decrease in carbon number. The experimental concentration profiles were simulated from the model for all the reactions and the model prediction was found to be reasonably good. The water permeability was also correlated well with acid hydrophobicity. The pervaporation coupled reaction efficiency, as represented by the reaction time for equilibrium conversion, was found to bear a profound relation to membrane surface area per unit volume of the reaction mixture (A/V). The time for equilibrium conversion was found to decrease with an increase in A/V value, reaching a minimum and then increasing with a further increase of A/V. A probable explanation has been postulated for such an observation. 相似文献
39.
Shib P. Dutta Ralph J. Bernacki Alexander Bloch Girish B. Chheda 《Nucleosides, nucleotides & nucleic acids》2013,32(2):151-162
Abstract N 6 ?(1-hydroxyguanidino)purine IIa, and its 9-β-D-ribonucleoside derivative IIb were prepared by reacting at room temperature 6-hydroxyadenine Ia and 6-hydroxyadenosine Ib, with 1-guanyl-3,5-dimethylpyrazole nitrate in DMF. Refluxing IIa and IIb in 95% ethanol gave N6?(1-hydroxyureido)purine and its ribonucleoside derivative respectively; the latter compound was also obtained by refluxing Ib with 1-guanyl-3,5-dimethylpyrazole nitrate in ethanol. The two base analogs were inactive against L1210 cells in vitro, but the nucleoside derivatives inhibited the growth of these cells by 50% at 5 × 10 -6 and 6 × 10?7 M respectively. Compound IIb, at 200 mg/kg/day × 5, increased the life span of L1210-bearing DBA/2N mice by 57%. Cytofluorometric determinations showed that IIb inhibited cell growth in the G2 phase of the cell cycle. also found to inhibit adenosine deaminase activity with a Ki = 3.47 μM. 相似文献
40.
Minakshi Mazumdar Arghya Adhikary Samik Chakraborty Shravanti Mukherjee Argha Manna Shilpi Saha Suchismita Mohanty Amrita Dutta Pushpak Bhattacharjee Pallab Ray Sreya Chattopadhyay Shuvomoy Banerjee Juni Chakraborty Arun K. Ray Gaurisankar Sa Tanya Das 《Apoptosis : an international journal on programmed cell death》2013,18(5):589-604
Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy. 相似文献