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Articles by J Hu
Total Records ( 6 ) for J Hu
  Z Liu , Z Yu , N Liu , C Zhao , J Hu and Q. Dai
 

In our efforts for cloning novel I2-superfamily conotoxins using the signal peptide sequence, we identified a novel conotoxin Lt12.4 from Conus litteratus. This gene has a framework XII (-C-C-C-C-CC-C-C-), which is distinct from the cysteine pattern I2-superfamily conotoxin (-C-C-CC-CC-C-C-). Subsequently, we found the signal peptide sequence of Lt12.4 by 5'-RACE. Using this new sequence, we identified another five novel conotoxins with this cysteine pattern from four Conus species (Conus eburneus, Conus imperialis, Conus marmoreus, and C. litteratus). These novel conotoxins have the same cysteine pattern as the reported Gla-TxX and Gla-MII, and may contain Gla residues. Furthermore, they have the highly conserved signal peptide and hypervariable mature peptide sequences, and widely exist in Conus species. Therefore, it could be defined as a new superfamily of E-conotoxins.

  J Deng , X Liao , J Hu , X Leng , J Cheng and G. Zhao
 

In contrast to animal ferritin, relatively little information is available on phytoferritin. Black bean (Phaseolus vulgaris L.) has been consumed in many countries. In the present study, new ferritin from black bean seed was purified by two consecutive anion exchange and size exclusion chromatography. The apparent molecular mass of the native black bean seed ferritin (BSF) was found to be ~560 kDa by native PAGE analysis. N-terminal sequence, MALDI-TOF-MS and MS/MS analyses indicate that BSF and soybean seed ferritin (SSF) share very high identity in amino acid sequence. However, SDS–PAGE result indicates that BSF consists of 26.5 (H-1) and 28.0 kDa (H-2) subunits with a ratio of 2 : 1, while the ratio of these two subunits in SSF is 1 : 1. This result demonstrates that the two proteins have different subunit composition which might affect their activities in iron uptake and release. Indeed, at high iron flux, the initial rate of iron oxidative deposition in apoBSF is larger than that in apoSSF. On the contrary, the iron release from BSF is significantly slower than that from SSF. All these results indicate that phytoferritin might regulate the transit of iron into and out of the protein cavity by changing its subunit composition.

  J Hu , M Sharma , H Qin , F. P Gao and T. A. Cross
 

CorA is a constitutively expressed magnesium transporter in many bacteria. The crystal structures of Thermotoga maritima CorA provide an excellent structural framework for continuing studies. Here, the ligand binding properties of the conserved interhelical loop, the only portion of the protein exposed to the periplasmic space, are characterized by solution nuclear magnetic resonance spectroscopy. Through titration experiments performed on the isolated transmembrane domain of Mycobacterium tuberculosis CorA, it was found that two CorA substrates (Mg2+ and Co2+) and the CorA-specific inhibitor (Co(III) hexamine chloride) bind in the loop at the same binding site. This site includes the glutamic acid residue from the conserved "MPEL" motif. The relatively large dissociation constants indicate that such interactions are weak but not atypical for channels. The present data support the hypothesis that the negatively charged loop could act as an electrostatic ring, increasing local substrate concentrations before transport across the membrane.

  F Madia , M Wei , V Yuan , J Hu , C Gattazzo , P Pham , M. F Goodman and V. D. Longo
 

Oncogenes contribute to tumorigenesis by promoting growth and inhibiting apoptosis. Here we examine the function of Sch9, the Saccharomyces cerevisiae homologue of the mammalian Akt and S6 kinase, in DNA damage and genomic instability during aging in nondividing cells. Attenuation of age-dependent increases in base substitutions, small DNA insertions/deletions, and gross chromosomal rearrangements (GCRs) in sch9 mutants is associated with increased mitochondrial superoxide dismutase (MnSOD) expression, decreased DNA oxidation, reduced REV1 expression and translesion synthesis, and elevated resistance to oxidative stress-induced mutagenesis. Deletion of REV1, the lack of components of the error-prone Pol, or the overexpression of SOD1 or SOD2 is sufficient to reduce age-dependent point mutations in SCH9 overexpressors, but REV1 deficiency causes a major increase in GCRs. These results suggest that the proto-oncogene homologue Sch9 promotes the accumulation of superoxide-dependent DNA damage in nondividing cells, which induces error-prone DNA repair that generates point mutations to avoid GCRs and cell death during the first round of replication.

  F. J Moss , P.I Imoukhuede , K Scott , J Hu , J. L Jankowsky , M. W Quick and H. A. Lester
 

The mouse -aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ~30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

 
 
 
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