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61.
Use of short-lived green fluorescent protein for the detection of proteasome inhibition 总被引:1,自引:0,他引:1
Andreatta C Nahreini P Hovland AR Kumar B Edwards-Prasad J Prasad KN 《BioTechniques》2001,30(3):656-660
Human embryonic kidney (HEK293) cells were stably transduced with a retroviral vector containing an expression cassette for a short-lived green fluorescent protein (d2EGFP) and the neomycin resistance gene (Neor). When Neor HEK293 clones were treated with proteasome inhibitors, lactacystin or MG132, an increase in the constitutive levels of d2EGFP expression was observed. Based on flow cytometry, proteasome inhibitors induced a 5- to 10-fold increase in the fluorescent intensity of d2EGFP in HEK293 cell clones. However, in the presence of proteasome inhibitors, HEK293 clones showed a 4- to 6.5-fold increase in d2EGFP concentration as determined by western blot analysis. Our data suggest that d2EGFP is a useful indicator of proteasome inhibition. Therefore, stable expression of d2EGFP in mammalian cells is potentially useful for high-throughput screening of cDNAs or pharmaceutical drugs that repress proteasome functions in vivo. 相似文献
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Russell Vassell Yong He Prasad Vennakalanti Antu K. Dey Min Zhuang Wei Wang Yide Sun Zohar Biron-Sorek Indresh K. Srivastava Celia C. LaBranche David C. Montefiori Susan W. Barnett Carol D. Weiss 《PloS one》2015,10(6)
The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols. 相似文献
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Pre-incubation of cells of Saccharomyces cerevisiae with 2 M-ethanol led to decreased rates of L-alanine uptake, H+ efflux and fermentation rate. However, these responses were modified in yeast cells with altered phospholipid composition. Using L-alanine transport and H+ efflux as indices of ethanol tolerance, it was observed that cells enriched with phosphatidylserine had greater tolerance to ethanol. This resulted from altered charge of membrane phospholipids rather than changes in membrane fluidity. It is suggested that the anion:zwitterion ratio of phospholipids may be one of the important determinants of ethanol tolerance in S. cerevisiae. 相似文献
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Arvind Gulati Natasha Sharma Pratibha Vyas Swati Sood Praveen Rahi Vijaylata Pathania Ramdeen Prasad 《Archives of microbiology》2010,192(11):975-983
An efficient phosphate-solubilizing plant growth–promoting Acinetobacter rhizosphaerae strain BIHB 723 exhibited significantly higher solubilization of tricalcium phosphate (TCP) than Udaipur rock phosphate (URP),
Mussoorie rock phosphate (MRP) and North Carolina rock phosphate (NCRP). Qualitative and quantitative differences were discerned
in the gluconic, oxalic, 2-keto gluconic, lactic, malic and formic acids during the solubilization of various inorganic phosphates
by the strain. Gluconic acid was the main organic acid produced during phosphate solubilization. Formic acid production was
restricted to TCP solubilization and oxalic acid production to the solubilization of MRP, URP and NCRP. A significant increase
in plant height, shoot fresh weight, shoot dry weight, root length, root dry weight, and root, shoot and soil phosphorus (P)
contents was recorded with the inoculated treatments over the uninoculated NP0K or NPTCPK treatments. Plant growth promotion as a function of phosphate solubilization suggested that the use of bacterial strain
would be a beneficial addition to the agriculture practices in TCP-rich soils in reducing the application of phosphatic fertilizers. 相似文献
68.
Prasad Kaparaju María Serrano Anne Belinda Thomsen Prawit Kongjan Irini Angelidaki 《Bioresource technology》2009,100(9):2562-2568
The production of bioethanol, biohydrogen and biogas from wheat straw was investigated within a biorefinery framework. Initially, wheat straw was hydrothermally liberated to a cellulose rich fiber fraction and a hemicellulose rich liquid fraction (hydrolysate). Enzymatic hydrolysis and subsequent fermentation of cellulose yielded 0.41 g-ethanol/g-glucose, while dark fermentation of hydrolysate produced 178.0 ml-H2/g-sugars. The effluents from both bioethanol and biohydrogen processes were further used to produce methane with the yields of 0.324 and 0.381 m3/kg volatile solids (VS)added, respectively. Additionally, evaluation of six different wheat straw-to-biofuel production scenaria showed that either use of wheat straw for biogas production or multi-fuel production were the energetically most efficient processes compared to production of mono-fuel such as bioethanol when fermenting C6 sugars alone. Thus, multiple biofuels production from wheat straw can increase the efficiency for material and energy and can presumably be more economical process for biomass utilization. 相似文献
69.
Regional differences in neurovirulence have been documented among subtype/clade-C HIV-1 isolates in India and Southern Africa. We previously demonstrated that a C31S substitution in Clade-C Tat dicysteine motif reduces monocyte recruitment, cytokine induction and direct neurotoxicity. Therefore, this polymorphism is considered to be a causative factor for these differences in neurovirulence. We previously reported on the genotypic differences in Tat protein between clade-C and rest of the clades showing that approximately 90% of clade-C HIV-1 Tat sequences worldwide contained this C31S polymorphism, while 99% of non-clade C isolates lacked this Tat polymorphism at C31 residue (Ranga et al. (2004) J Virol 78∶2586–2590). Subsequently, we documented intra-clade-C differences in the frequency of Tat dicysteine variants between India and Southern Africa, as the basis for differential disease severity and showed the importance of the Tat dicysteine motif for neuropathogenesis using small animal models. We have now examined if determinants of neurovirulence besides Tat are different between the clade-C HIV-1 isolates from Southern Africa and India. Envelope glycoprotein gp120 is a well-documented contributor to neurotoxicity. We found that gp120 sequences of HIV-1 isolates from these two regions are genetically distinct. In order to delineate the contribution of gp120 to neurovirulence, we compared direct in vitro neurotoxicity of HIV-infected supernatants of a representative neurovirulent US clade-B isolate with two isolates each from Southern Africa and India using primary human neurons and SH-SY5Y neuroblastoma cells. Immunodepletion of gp120 of both US clade B and the Southern African clade C isolates revealed robust decreases in neurotoxicity, while that of the Indian isolates showed minimal effect on neurotoxicity. The gp120 as a cause of differential neurotoxicity was further confirmed using purified recombinant gp120 from HIV isolates from these regions. We conclude that gp120 is one of the key factors responsible for the decreased neurovirulence of Indian clade C HIV-1 isolates when compared to South African clade C HIV-1. 相似文献
70.
Major zinc binding protein purified from renal brush border membrane (BBM) (R. Kumar, R. Prasad, Biochim. Biophys. Acta 1419 (1999) 23) was reconstituted into liposomes and its functional characteristics were investigated. Physical incorporation of the major zinc binding protein into the proteoliposomes was checked by SDS-PAGE, which showed a single band on silver staining. The structural integrity of the proteoliposomes was assessed by phase contrast microscopy, which revealed the proteoliposomes as globular structures and intact boundaries. Further structural integrity/leakiness of the proteoliposomes was checked by monitoring efflux of Zn(2+) from the pre-loaded proteoliposomes in the presence of either 2 mM Ca(2+) or Cd(2+) or Zn(2+). It was observed that even after 2 h of the initiation of efflux, 85-95% of Zn(2+) was retained in the proteoliposomes, thereby indicating that proteoliposomes were not leaky and maintained structural integrity during the uptake study. Zinc uptake into the proteoliposomes followed Michaelis-Menten kinetics with affinity constant (K(m)) of 1.03 mM and maximal velocity (V(max)) of 1333 nmol/mg protein per min. The uptake process followed first-order kinetics with a rate constant (k) of 1. 09x10(-3) s(-1). The specificity of zinc transport system was determined by studying the interaction of divalent cations viz. Ca(2+) and Cd(2+) with the zinc uptake. It was observed that Cd(2+) competitively inhibited the zinc uptake process with inhibitory concentration (K(i)) of 2.9 mM. Kinetic analysis of inhibitory effect of Cd(2+) on zinc uptake revealed an increase in K(m) to 1.74 mM without influencing V(max). Zn(2+) uptake into the proteoliposomes was found to be temperature sensitive and Arrhenius plot showed a breakpoint at 27 degrees C. The apparent energies of activation (E(a)) were found to be 7.09 and 2.74 kcal/mol below and above the breakpoint, respectively. The initial velocity of Zn(2+) uptake increased with the increase in outwardly directed proton gradient ([H](i) greater than [H](o)). The Zn(2+) uptake was inhibited by DCCD, thereby suggesting the involvement of -COOH groups in the translocation of Zn(2+) across the lipid bilayer. The ratio of acidic to basic amino acids (1.26) strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding to zinc. The findings of the present study confirms that 40 kDa major zinc binding glycoprotein purified from renal BBM is a zinc transporter involved in the influx of Zn(2+) into the epithelial cells of the renal tubular system. 相似文献