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61.
Jamboor K. Vishwanatha Thomas J. Tauer Solon L. Rhode III 《Molecular and cellular biochemistry》1995,146(2):121-126
A single-stranded DNA-dependent ATPase activity, consisting of two subunits of 83 kDa (p90) and 68 kDa (p70), was previously purified from HeLa cells (Vishwanatha, J.K. and Baril, E.F. (1990) Biochem 29, 8753–8759). Homology of the two subunits of single-stranded DNA-dependent ATPase with the human Ku protein (Caoet al. (1994) Biochem 33, 8548–8557) and identity of the Ku protein as the human DNA helicase II (Tutejaet al. (1994) EMBO J. 13, 4991–5001) have been reported recently. Using antisera raised against the subunits of the HDH II, we confirm that the Hela single-stranded DNA-dependent ATPase is the HDH II. Similar to the activity reported for Ku protein, ssDNA-dependent ATPase binds to double-stranded DNA and the DNA-protein complex detected by gel mobility shift assay consists of both the ATPase subunits. The p90 subunit is predominantly nuclear and is easily dissociated from chromatin. The p70 is distributed in cytosol and nucleus, and a fraction of the nuclear p70 protein is found to be associated with the nuclear matrix. Both the p90 and p70 subunits of the ATPase are present in G1 and S phase of the cell cycle and are rapidly degraded in the G2/M phase of the cell cycle.Abbreviations ssDNA
single-stranded DNA
- dsDNA
double-stranded DNA
- ATPase
adenosine triphosphatase
- HDH II
human DNA helicase II
- PGK
3-phosphoglycerate kinase 相似文献
62.
Primer recognition proteins (PRP) are cofactors of DNA polymerase alpha and may have a role in lagging strand DNA replication. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. Upon tryptic digestion, amino acid sequencing of tryptic peptides, and homology search against a protein sequence data base, we have identified PRP 2 to be the glycolytic enzyme, phosphoglycerate kinase (PGK). The activities of PRP and PGK increase coordinately in the PRP purification procedure. PRP activity is inhibited by the PGK substrate 3-phosphoglycerate and the competitive inhibitor of substrate binding, DL-alpha-glycerol 3-phosphate. 5'-p-Fluorosulfonylbenzoyl adenosine, which inactivates PGK by binding to the nucleotide binding site, also inhibits PRP. For PRP activity, the two substrate binding sites of PGK are necessary in addition to the as yet unidentified PRP 1 polypeptide. 相似文献
63.
Non‐Tuberculosis mycobacterium speciation using HPLC under Revised National TB Control Programme (RNTCP) in India
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G. Sebastian S.B. Nagaraja T. Vishwanatha M. Voderhobli N. Vijayalakshmi P. Kumar 《Journal of applied microbiology》2018,124(1):267-273
Aims
Non‐Tuberculous Mycobacteria (NTM) are ubiquitous in nature. The data on prevalence of NTM under the RNTCP is scarce. Many NTM species have clinical significance, and hence their identification and speciation are important.Methods and Results
It is a cross‐sectional study conducted at the five RNTCP accredited culture and drug susceptibility testing (CDST) laboratory. The culture isolates from AFB positive but Immunochromatographic test negative samples were taken for identification and speciation using HPLC. Of the total 266 isolates only 164 isolates had a second sample received at the laboratory. The speciation was done using HPLC for those isolates. The type of species identified are: 26·8% (44) were Mycobacterium chelonae, 12·8% (21) were Mycobacterium fortuitum, 9% (15) were Mycobacterium gordonae, 9% (15) were Mycobacterium tuberculosis complex, 6·1% (10) were Mycobacterium kansasii, 4·9% (8) were Mycobacterium simiae, 2·4% (4) were Mycobacterium thermophile, 1·2% (2) were Mycobacterium gastri, 0·6% (1) were Mycobacterium scrofulaceum, 0·6% (1) were Mycobacterium avium and 4·9% (8) isolates had chromatogram which was un‐interpretable.Conclusion
Identification and its speciation of NTM are not routinely done under TB control programme. Since HPLC could identify 95% of isolates belonging to 10 species, the speciation of NTM using HPLC should gain importance in the diagnosis of disease caused by NTM.Significance and Impact of Study
NTM are emerging as important causative agents of pulmonary and extra pulmonary disease, the ability to recognize disease caused by NTM and subsequently treat such disease has become increasingly important. The identification of NTM up to its species level should gain importance in all TB reference Laboratories. 相似文献64.
Isolated populations of drosophila pseudoobscura, separated from North
American populations by about 2,400 km, were found in Colombia in 1960. We
compared for sequences of the small ribosomal RNA (srRNA) gene on the
mitochondria between North American and Colombian D. pseudoobscura in order
to clarify the age of the Colombian isolates. The North American
populations were not genetically different from each other but were
genetically different from the Colombian populations. The Mexican strains
represent the area from which the Colombian founders might have come. The
estimated net nucleotide divergence between Mexican and Colombian D.
pseudoobscura indicates that the Colombian population is not an ancient
lineage. Phylogenies using both distance and parsimony methodologies
reinforced this conclusion. The Colombian samples group together with both
methods but, according to the bootstrap analysis, not significantly. It
appears that the populations have not been separated long enough for their
DNA sequences to show much divergence.
相似文献
65.
Vishwanatha K. Rao Gerardo Zavala Abhijit Deb Roy Richard E. Mains Betty A. Eipper 《Journal of cellular physiology》2019,234(6):8683-8697
The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar-type-H+ ATPase (V-ATPase). V-ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V-ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine α-amidating mono-oxygenase (PAM; EC 1.14.17.3), a secretory pathway membrane enzyme which shares similar topology with two V-ATPase accessory proteins (Ac45 and prorenin receptor), has a pH-sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT-20) identified distinct subcellular compartments exhibiting spatial co-occurrence of PAM and V-ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high-molecular weight complexes of PAM and V-ATPase. A PAM-1 mutant (PAM-1/H3A) with altered pH sensitivity had diminished ability to form high-molecular weight complexes. In addition, V-ATPase assembly status was altered in PAM-1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH-sensor, regulating V-ATPase action by altering its assembly status. 相似文献
66.
Megan B. Miller Kurutihalli S. Vishwanatha Richard E. Mains Betty A. Eipper 《The Journal of biological chemistry》2015,290(21):13541-13555
Previous studies revealed an essential role for the lipid-binding Sec14 domain of kalirin (KalSec14), but its mechanism of action is not well understood. Because alternative promoter usage appends unique N-terminal peptides to the KalSec14 domain, we used biophysical, biochemical, and cell biological approaches to examine the two major products, bKalSec14 and cKalSec14. Promoter B encodes a charged, unstructured peptide, whereas promoter C encodes an amphipathic helix (Kal-C-helix). Both bKalSec14 and cKalSec14 interacted with lipids in PIP strip and liposome flotation assays, with significantly greater binding by cKalSec14 in both assays. Disruption of the hydrophobic face of the Kal-C-helix in cKalSec14KKED eliminated its increased liposome binding. Although cKalSec14 showed significantly reduced binding to liposomes lacking phosphatidylinositol phosphates or cholesterol, liposome binding by bKalSec14 and cKalSec14KKED was not affected. When expressed in AtT-20 cells, bKalSec14-GFP was diffusely localized, whereas cKalSec14-GFP localized to the trans-Golgi network and secretory granules. The amphipathic C-helix was sufficient for this localization. When AtT-20 cells were treated with a cell-permeant derivative of the Kal-C-helix (Kal-C-helix-Arg9), we observed increased secretion of a product stored in mature secretory granules, with no effect on basal secretion; a cell-permeant control peptide (Kal-C-helixKKED-Arg9) did not have this effect. Through its ability to control expression of a novel, phosphoinositide-binding amphipathic helix, Kalrn promoter usage is expected to affect function. 相似文献
67.
We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids. 相似文献
68.
Lipid raft endocytosis and exosomal transport facilitate extracellular trafficking of annexin A2 总被引:1,自引:0,他引:1
Annexin A2 (AnxA2), a Ca(2+)-dependent phospholipid-binding protein, is known to associate with the plasma membrane and the endosomal system. Within the plasma membrane, AnxA2 associates in a Ca(2+) dependent manner with cholesterol-rich lipid raft microdomains. Here, we show that the association of AnxA2 with the lipid rafts is influenced not only by intracellular levels of Ca(2+) but also by N-terminal phosphorylation at tyrosine 23. Binding of AnxA2 to the lipid rafts is followed by the transport along the endocytic pathway to be associated with the intralumenal vesicles of the multivesicular endosomes. AnxA2-containing multivesicular endosomes fuse directly with the plasma membrane resulting in the release of the intralumenal vesicles into the extracellular environment, which facilitates the exogenous transfer of AnxA2 from one cell to another. Treatment with Ca(2+) ionophore triggers the association of AnxA2 with the specialized microdomains in the exosomal membrane that possess raft-like characteristics. Phosphorylation at Tyr-23 is also important for the localization of AnxA2 to the exosomal membranes. These results suggest that AnxA2 is trafficked from the plasma membrane rafts and is selectively incorporated into the lumenal membranes of the endosomes to escape the endosomal degradation pathway. The Ca(2+)-dependent exosomal transport constitutes a novel pathway of extracellular transport of AnxA2. 相似文献
69.
Background
Microsatellites are nucleotide sequences of tandem repeats occurring throughout the genome, which have been widely used in genetic linkage analysis, studies of loss of heterozygosity, determination of lineage and clonality, and the measurement of genome instability or the emergence of drug resistance reflective of mismatch repair deficiency. Such analyses may involve the parallel evaluation of many microsatellite loci, which are often limited by sample DNA, are labor intensive, and require large data processing.Results
To overcome these challenges, we developed a cost-effective high-throughput approach of microsatellite analysis, in which the amplifications of microsatellites are performed in miniaturized, multiplexed polymerase chain reaction (PCR) adaptable to 96 or 384 well plates, and accurate automated allele identification has been optimized with a collective reference dataset of 5,508 alleles using the GeneMapper software.Conclusions
In this investigation, we have documented our experience with the optimization of multiplex PCR conditions and automated allele identification, and have generated a unique body of data that provide a starting point for a cost-effective, high-throughput process of microsatellite analysis using the studied markers.70.