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51.
Ram Krishna Thakur Vinod Kumar Yadav Akinchan Kumar Ankita Singh Krishnendu Pal Luke Hoeppner Dhurjhoti Saha Gunjan Purohit Richa Basundra Anirban Kar Rashi Halder Pankaj Kumar Aradhita Baral MJ Mahesh Kumar Alfonso Baldi Bruno Vincenzi Laura Lorenzon Rajkumar Banerjee Praveen Kumar Viji Shridhar Debabrata Mukhopadhyay Shantanu Chowdhury 《Nucleic acids research》2014,42(18):11589-11600
52.
Marieke?Pingen Ramin?Sarrami-Forooshani Annemarie?MJ?Wensing Petra?van Ham Agata?Drewniak Charles?AB?Boucher Teunis?BH?GeijtenbeekEmail author Monique?NijhuisEmail author 《Retrovirology》2014,11(1):113
Background
Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.Results
In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5+ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5+ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.Conclusions
Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.53.
54.
Characterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases 总被引:13,自引:0,他引:13
Glycoproteins which bind concanavalin A (Con A) can be located on nitrocellulose sheets after electrophoretic transfer from slab gels, by sequential incubation of the sheets with Con A and peroxidase, and visualization of the peroxidase by an insoluble reaction product. We refer to this method as affinoblotting. Differential elution of Con A from the blots by washing the sheets with different concentrations of alpha-methylglycosides is used to demonstrate the affinity of Con A for the oligosaccharide side chains, and to differentiate between proteins with weak and those with high affinity for Con A. Concanavalin A has a high affinity for the four plant glycoproteins (phaseolin, phytohemagglutinin, jackbean alpha-mannosidase, and the glycosylated precursor of Con A) studied here. Incubation of the blots with alpha-mannosidase and endoglycosidase H (endo H) is used to demonstrate that the oligosaccharide chains can be degraded by glycosidases while the proteins are immobilized on the nitrocellulose. With this approach we show here that the four plant glycoproteins used as models in this study interact with Con A through high-mannose oligosaccharide side chains sensitive to alpha-mannosidase and endo H degradation. 相似文献
55.
56.
Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP. 总被引:24,自引:0,他引:24 下载免费PDF全文
The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
57.
Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole 总被引:7,自引:0,他引:7
To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seedspecific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope himet (Hoffman et al., 1988, Plant Mol. Biol. 11, 717–729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent. However, the protein was sufficiently destabilized to be degraded by vacuolar proteases.Abbreviations ER
endoplasmic reticulum
- BiP
binding protein
- IgG
immunoglobulin G
- Mr
relative molecular mass
The mention of vendor or product does not imply that they are endorsed or recommended by the US Department of Agriculture over vendors of similar products not mentionedThis work was supported by a grant from the National Science Foundation (Cell Biology) to M.J. Chrispeels and a fellowship from the Ministry of Education and Science, Spain-Fullbright Program to J.J. Pueyo. We thank H. Pelham for a gift of the constructs containing c-myc-SEKDEL and cmyc-FEHDEL and for a gift of anti-HDEL monoclonal antibodies. The original HiMet phaseolin construct was made by L. Hoffman and the phaseolin-HDEL or KDEL and HiMet-HDEL or KDEL constructs were made by D. Hunt as part of his doctoral research. 相似文献
58.
Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site. 总被引:20,自引:0,他引:20 下载免费PDF全文
The membranes of plant and animal cells contain aquaporins, proteins that facilitate the transport of water. In plants, aquaporins are found in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1, a mercury-sensitive cysteine residue (Cys-189) is present adjacent to a conserved Asn-Pro-Ala motif. Here, we report the molecular analysis of a new Arabidopsis aquaporin, delta-TIP (for tonoplast intrinsic protein), and show that it is located in the tonoplast. The water channel activity of delta-TIP is sensitive to mercury. However, the mercury-sensitive cysteine residue found in mammalian aquaporins is not present in delta-TIP, or in gamma-TIP, a previously characterized mercury-sensitive tonoplast aquaporin. Site-directed mutagenesis was used to identify the mercury-sensitive site in these two aquaporins as Cys-116 and Cys-118 for delta-TIP and gamma-TIP, respectively. These mutations are at a conserved position in a presumed membrane-spanning domain not previously known to have a role in aquaporin mercury sensitivity. Comparing the tissue expression patterns of delta-TIP with gamma-TIP and alpha-TIP showed that the TIPs are differentially expressed. 相似文献
59.
Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins 总被引:11,自引:5,他引:6
Garcia-Casado Gloria; Sanchez-Monge Rosa; Chrispeels Maarten J.; Armentia Alicia; Salcedo Gabriel; Gomez Luis 《Glycobiology》1996,6(4):471-477
Many plant proteins, particularly those found in foods and pollen,are known to act as sensitizing agents in humans upon repeatedexposure. Among the cereal flour proteins involved in asthmaticreactions, those members of the 相似文献
60.
Current phylogenetic tree reconstruction methods assume that there is a
single underlying tree topology for all sites along the sequence. The
presence of mosaic sequences due to recombination violates this assumption
and will cause phylogenetic methods to give misleading results due to the
imposition of a single tree topology on all sites. The detection of mosaic
sequences caused by recombination is therefore an important first step in
phylogenetic analysis. A graphical method for the detection of
recombination, based on the least squares method of phylogenetic
estimation, is presented here. This method locates putative recombination
breakpoints by moving a window along the sequence. The performance of the
method is assessed by simulation and by its application to a real data set.
相似文献