Biomedical subjects

Zhimin Yin

Publications and source records attributed to Zhimin Yin.

15 recordsLinked to original sources

PubMed2006

Human glutathione S-transferase P1 suppresses MEKK1-mediated apoptosis by regulating MEKK1 kinase activity in HEK293 cells.

Glutathione S-transferase P1 (GSTP1) plays an important role in detoxification and the metabolism of xenobiotics. Here we show that GSTP1 also regulates the MEKK1-MKK7 signaling pathway. Over-expression of GSTP1 in HEK293 cells inhibited both DMEKK1- and etoposide-induced apoptosis, and inhibited pro-caspase-3 activation and PARP cleavage. MEKK1-induced apoptosis requires both its kinase activity and proteolytic cleavage. DMEKK1 activity was inhibited by over-expression of GSTP1 in vivo and MEKK1 kinase activity was also inhibited by GSTP1 in vitro when assayed with bacterially-expressed MKK7(KM) protein as substrate. GSTP1 inhibition of etoposide-induced cell apoptosis was mainly due to its ability to suppress MEKK1 kinase activity. The glutathione-conjugating activity of GSTP1 was essential for the above effects. These findings provide insight into the mechanism by which GSTP1 protects cells from genotoxin-induced apoptosis.

Antineoplastic Agents, Phytogenic↗

PubMed2006

Hsp70 inhibits lipopolysaccharide-induced NF-kappaB activation by interacting with TRAF6 and inhibiting its ubiquitination.

Inducible heat shock protein 70 (Hsp70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological and pathological processes and is essential for activating NF-kappaB signaling pathway in response to bacterial lipopolysaccharide (LPS). Here we report a novel mechanism of Hsp70 for preventing LPS-induced NF-kappaB activation in RAW264.7 macrophage-like cells. Our results show that Hsp70 can associate with TRAF6 physically in the TRAF-C domain and prevent TRAF6 ubiquitination. The stimulation of LPS dissociates the binding of Hsp70 and TRAF6 in a time-dependent manner. Hsp70 inhibits LPS-induced NF-kappaB signaling cascade activation in heat-shock treated as well as Hsp70 stable transfected RAW264.7 cells and subsequently decreases iNOS and COX-2 expression. Two Hsp70 mutants, Hsp70DeltaC(1-428aa) with N-terminal ATPase domain and Hsp70C(428-642aa) with C-terminal domain, lack the ability to influence TRAF6 ubiquitination and TRAF6-triggered NF-kappaB activation. Taken together, these findings indicate that Hsp70 inhibits LPS-induced NF-kappaB activation by binding TRAF6 and preventing its ubiquitination, and results in inhibition of inflammatory mediator production, which provides a new insight for analyzing the effects of Hsp70 on LPS-triggered inflammatory signal transduction pathways.

Animals↗

PubMed2006

Expression of SK3-type dehydrin in transporting organs is associated with cold acclimation in Solanum species.

The expression of a gene, encoding a dehydrin protein designated as DHN24 was analyzed at the protein level in two groups of Solanum species differing in cold acclimation ability. The DHN24 protein displays consensus amino acid sequences of dehydrins, termed K- and S-segments. The S-segment precedes three K-segments, classifying the protein into SK3-type dehydrins. A group of Solanum species able to cold acclimation constituted by S. sogarandinum and S. tuberosum, cv. Aster, and a second one composed of a S. sogarandinum line, that lost ability to cold acclimation, and of S. tuberosum, cv. Irga, displaying low ability to cold acclimation were studied. Under control conditions, noticeable levels of the DHN24 protein was observed in stems, tubers, and roots of Solanum species. No protein was detected in leaves. During low temperature treatment the DHN24 protein level substantially increased in tubers, in transporting organs and in apical parts, and only a small increase was observed in leaves. The increase in protein abundance was only observed in the plants able to cold acclimate and was found to parallel the acclimation capacity. Upon drought stress, the DHN24 level decreased in stems and in leaves, but increased in apical parts. These results suggest that Dhn24 expression is regulated by organ specific factors in the absence of stress and by factors related to cold acclimation processes during low temperature treatment in collaboration with organ-specific factors. A putative function of the SK3-type dehydrin proteins during plant growth and in the tolerance to low temperature is discussed.

Acclimatization↗

PubMed2006

Tissue-culture-responsive and autotetraploidy-responsive changes in metabolic profiles of cucumber (Cucumis sativus L.).

Somaclonal variation commonly occurs during in vitro plant regeneration and may introduce unintended changes in numerous plant characters. In order to assess the range of tissue-culture-responsive changes on the biochemical level, the metabolic profiles of diploid and tetraploid cucumber R1 plants regenerated from leaf-derived callus were determined. Gas chromatography and mass spectrometry were used for monitoring of 48 metabolites and many significant changes were found in metabolic profiles of these plants as compared to a seed-derived control. Most of the changes were common to diploids and tetraploids and were effects of tissue culture. However, tetraploids showed quantitative changes in 14 metabolites, as compared to regenerated diploids. These changes include increases in serine, glucose-6P, fructose-6P, oleic acid and shikimic acid levels. Basing on this study we conclude that the variation in metabolic profiles does not correlate directly with the range of genome changes in tetraploids.

Cucumis sativus↗

PubMed2006

Cucumber (Cucumis sativus L.).

We describe two novel Agrobacterium tumefaciens-based methods of cucumber transformation. The first involves direct regeneration from leaf microexplants selected on kanamycin-containing medium. The second involves regeneration from a long-term established embryogenic suspension culture emitting green autofluorescence (GAF) and selection on medium containing hygromycin. In the latter method, GAF was used as a reporter, thereby allowing a simple and reliable identification of transgenic cells with a high regeneration capacity. (No false positives were observed.) The transformation efficiency in the leaf microexplants fluctuated from 0.8 to 6.5% of the primary explants, whereas in the embryogenic suspension-cultured cells it varied from 6.4 to 17.9% of the aggregates. In the GAF method, the step involving the elimination of the Agrobacterium cells by antibiotics could be omitted; however, this reduced the transformation efficiency to about 3%. The time required from inoculation to regenerated plant in the greenhouse was the same for both methods, but the GAF method required more preinoculation time than the leaf microexplant method.

Agrobacterium tumefaciens↗

PubMed2006

The DefH9-iaaM-containing construct efficiently induces parthenocarpy in cucumber.

Parthenocarpy (seedless fruits) is a desirable trait that has been achieved in many plant cultivars. We generated parthenocarpic cucumber fruits by introducing the chimeric DefH9-iaaM construct into the cucumber genome using an Agrobacterium tumefaciens-mediated protocol. The construct consists of the DefH9 promoter from Antirrhinum majus and the iaaM coding sequence from Pseudomonas syringae. Transgenic plants were obtained from nine independent transformation events: half of these were tetraploid and did not produce seeds following self-pollination, while the remaining half were capable of displaying parthenocarpy in the subsequent reproductive generation. Of the fruits produced by the transgenic lines, 70-90% were parthenocarpic. The segregation of the marker gene in the transgenic T(1) progeny indicated single gene inheritance. The seed set in the transgenic lines and their F(1) hybrids was lower than in the non-transgenic control plants. Some of the methodological details and the practical significance of the results are discussed.

Blotting, Southern↗

PubMed2005

Regulation of lipopolysaccharide-induced inflammatory response by glutathione S-transferase P1 in RAW264.7 cells.

Glutathione S-transferase P1(GSTP1) plays an important role in the detoxification and xenobiotics metabolism. Here, we show that GSTP1 is also involved in LPS (lipopolysaccharide)-induced inflammatory response. GSTP1 expression, determined at the transcription and translation levels, were upregulated by the LPS stimulation in RAW264.7 macrophage-like cells. GSTP1 inhibited LPS-induced mitogen-activated protein kinases MAPKs including ERK, JNK and p38 as well as NF-kappaB activation dose- and time-dependently in transient transfected and stable transfected cells. Moreover this inhibition of the signaling pathways resulted in the decrease of tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) synthesis. These data suggest that the GSTP1 prevents LPS-induced excessive production of pro-inflammatory factors and plays an anti-inflammatory role in response to LPS.

Animals↗

PubMed2005

Human glutamylcysteine synthetase protects HEK293 cells against UV-induced cell death through inhibition of c-Jun NH2-terminal kinase.

Human glutamylcysteine ligase catalytic subunit (GCLC) is the rate-limiting enzyme for glutathione synthesis. The heavy subunit possesses all the catalytic activities. UV irradiation (UV-C, 30 J/m(2)) induced apoptosis in HEK293 cells, but the morphological changes were inhibited significantly by expression of GCLC. MTS assay and flow cytometry results also indicated that GCLC and JNK1(APF) expression enhanced cellular resistance to UV irradiation. Western blotting showed that irradiation strongly activated the c-Jun NH(2)-terminal kinases (JNKs) and caspase-3 as well as p38 in HEK293 cells. Interestingly, existing data show that GCLC blocks JNK1 phosphorylation but does not affect p38 phosphorylation. Therefore, overexpression of GCLC protected HEK293 cells against UV irradiation-induced cell death by inhibiting the phosphorylation and activation of JNK1, concomitantly with the inhibition of caspase-3 activation and p21(WAF1)-luciferase activity downstream of JNK.

Apoptosis↗

PubMed2005

The metabolic profiles of transgenic cucumber lines vary with different chromosomal locations of the transgene.

The metabolic profiles of five transgenic cucumber lines were compared taking into consideration their transgene integration sites. The plants analyzed were homozygous and contained transgenes integrated in a single locus on chromosomes I, II, III or IV. The transgenes were preferentially located in the euchromatic regions. Each of these locations possessed a specific metabolic profile. The number of altered compounds in the transgenic lines varied between 9 and 23 of the 47 metabolites identified. These alterations seem to be specific for each independent transgene integration. However, some changes are common: a decrease in the levels of phenylalanine, aspartate, ethanolamine and pipecolate, and an increase in the level of benzoic acid. The observed effects of transgene introduction are discussed in this paper.

Chromosome Banding↗

PubMed2004

Tobacco PR-2d promoter is induced in transgenic cucumber in response to biotic and abiotic stimuli.

The PR-2d promoter/uidA (GUS) gene construct was introduced into the cucumber (Cucumis sativus L.) genome and several transgenic lines were produced. Activation of the PR-2d promoter was investigated in these plants in response to inoculation with fungal pathogens and after salicylic acid (SA) or cold treatments. Treatment with exogenous SA increased GUS activity 2 to 11 fold over that of the control. Endogenous SA and its conjugate salicylic acid glucoside (SAG) rose in parallel after inoculation with the fungal pathogen Pseudoperonospora cubensis, with SAG becoming the predominant form. The free SA levels increased 15 fold above the basal level at 5 dpi and preceded the induction of the PR-2d promoter by five days, which occurred at 10 dpi with a 12 fold increase over the control. Inoculation with another fungal pathogen, Erysiphe polyphage, increased GUS activity 4 to 44 fold over that of the control. During normal development of flowers in the cucumber, the PR-2d/uidA gene expressed in the floral organs was similar to that of the primary host. In addition, we present the first evidence that the PR-2d promoter was induced (624 fold) under cold stress. We demonstrate that in the heterologous state the gene construct was expressed according to the signalling pattern of the native species and was stably transmitted to progeny over four generations.

Cold Temperature↗

PubMed2004

Transgene inheritance in plants.

The patterns of transgene inheritance in plants and the possible explanations for non-Mendelian transmission are reviewed. The non-Mendelian inheritance of a transgene has been recorded with a frequency between 10% and 50% in transgenic plants produced either by Agrobacterium-mediated transformation or through particle bombardment. Different effects such as deletion, duplication, rearrangement, repeated sequence recombination as well as gene interaction have been observed for transgenic loci. The nature of the recipient genome, nature of the transgene and the interactions between them seem to contribute to the non-Mendelian segregation of transgenes.

Gene Deletion↗

PubMed2004

Differences in the inheritance stability of kanamycin resistance between transgenic cucumbers (Cucumis sativus L.) containing two constructs.

A phenotypic segregation of kanamycin resistance conferred by nosnptII was investigated in two groups of transgenic cucumber lines one containing the PR-2duidA-nosnptII construct and the other with the thaumatinII-nosnptII construct to the third and fifth generation, respectively. Expression of the nptII gene was more stable and predictable in the progeny of the PR-2d lines than that of thaumatin lines. In T2 and T3 generations representing seven PR-2d primary transformants, 78% of the hemizygous progenies exhibited a segregation ratio consistent with Mendelian inheritance; non-Mendelian ratio occurred in the remaining 22%. Mendelian segregation was observed in 46% of the hemizygous progenies derived from 11 thaumatin primary transformants. The segregation ratio for two and three independent loci appeared in single PR-2d and thaumatin lines, respectively.

Crosses, Genetic↗

PubMed2004

Transcriptional expression of a Solanum sogarandium pGT::Dhn10 gene fusion in cucumber, and its correlation with chilling tolerance in transgenic seedlings.

The expression pattern of a Solanum sogarandinum pGT::Dhn10 gene fusion encoding a dehydrin DHN10 protein and the potential role of that protein in cold tolerance in cucumber were analysed in three T1transgenic lines. An accumulation of Dhn10 mRNA was detected in the leaves, cotyledons, hypocotyls and roots of the transgenic seedlings both under the control conditions and after a cold treatment at 6 degrees C for 24 h. This was confirmed by RT-PCR. However, no DHN10 protein was detected by the alkaline phosphatase-conjugated antibody. The transgenic lines exhibited different levels of chilling tolerance. The TCC5/1 line showed a significant increase in its chilling tolerance compared to the non-transgenic line. No chilling injury was observed when the cold hardened (6 degrees C, 24 h) TCC5/1 plants were subsequently exposed to a temperature of 2 degrees C for 6 h. The other two transgenic lines, TCC2/1 and TCC3/2, exhibited a comparable level of chilling tolerance to that of the non-transgenic control. The transgenic lines showed similar or significantly decreased freezing tolerance compared to the non-transgenic control, as evaluated by an electrolyte leakage test. We concluded that the S. sogarandnium GT promoter is functional in the chilling sensitive species Cucumis sativus L., and that the pGT::Dhn10 gene fusion is expressed at the transcriptional level.

Artificial Gene Fusion↗

PubMed2003

[Molecular chaperone GRP75 reprove cells from injury caused by glucose deprivation].

Glucose-regulated proteins 75(grp75) is a member of hsp70 family. The expression of grp75 is upregulated during glucose starvation (such as ischemia). To evaluate grp75 function, CHL cells were cultured with glucose-free media for 20 h (A) and glucose-free media for 12 h + glucose-containing media for 8 h (ischemia reperfusion) (B). A constructed rat grp75 cDNA expression vector (pcDNA/grp75) was transfected into CHL cells and a cell strain that stably overexpressed grp75 was obtained. The transfected cells and untransfected cells(control group) were cultured with A or B. By MTT, LDH leakage measurement and flow cytometry analysis, growth rate of untransfected cells in B is significantly lower than that in glucose-containing media for 20 h (C) (p < 0.05) and A (p < 0.05). Growth rate of transfected cells is apparently higher than that of control group in B (p < 0.01). LDH liberation percentage of untransfected cells in B is obviously higher than that in C(p < 0.01) and it is not different from A(p > 0.05). LDH liberation percentage of transfected cells is apparently lower than that of control group in B(p < 0.01). Apoptosis of transfected cells is obviously lower by flow cytometry analysis. These results provide evidence for the cytoprotective function of grp75 during glucose starving and ischemia reperfusion.

Animals↗

PubMed2002

[Synergetic effect of mitogen-activated protein kinase pathway on expression of tumor necrosis factor alpha gene in RAW 264.7 cells induced by lipopolysaccharide].

OBJECTIVE: To explore the molecular mechanism of the synergetic effect of mitogen-activated protein kinase (MAPK) pathway on expression of tumor necrosis factor alpha (TFG-alpha) gene in murine monocyte (RAW264.7 cells) induced by lipopolysaccharide (LPS). METHODS: RAW264.7 cells were cultured, transfected with vectors with mutants of different constitutively active MAPK kinases, i. e., ERK, JNK, and p38, vectors with inactivated mutants of MAPK kinase, and murine TNF-alpha driven report gene pGL2TNFLuc, and then stimulated by LPS. Protein kinase assay was used to analyze the activity of MAPKs. Western blotting was used to detect the amount of target proteins. RT-PCR was used to detect the influence of the MEK/REK inhibitor PD98059, JNK inhibitor SP600125, and p38 inhibitor SB203580 on the expression of TNF-alpha gene induced by LPS. RESULTS: ERK1, JNK1, and p38 MAPKs were transiently activated by LPS. After the vectors with activated MAPK mutants were co-transfected with murine TNF-alpha driven report gene pGL2TNFLuc into RAW264.7 cells, the transcription activity of TNF-alpha promoter was induced to different degrees. After the vectors with inactivated MAPK mutants were co-transfected with murine TNF-alpha driven report gene pGL2TNFLuc into RAW264.7 cells, the transcription activity of TNF-alpha promoter was inhibited. CONCLUSION: The transactivity of TNF-alpha promoter stimulated by LPS involves ERK, JNK, and p38 pathways. These three major MAPK pathways exert synergetic effects on the gene regulation of TNF-alpha expression.

Animals↗