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Articles by Zhang Yun
Total Records ( 9 ) for Zhang Yun
  HE Ying-ying , LIU Shu-bai , QIAN Jin-qiao , LEE Wen-hui and ZHANG Yun
  βγ-CAT is a naturally existing protein complex of non-lens βγ-crystallin and trefoil factor, purified from Bombina maxima skin secretions. In HUVECs, βγ-CAT can be rapidly endocytosed via intracellular vacuole formation and translocated to the nucleus to regulate cell fuction. In this paper, we found that it contains conserved Walker B motifs (IILYDEPS, residues 6-13) and Walker A motifs (GQSLSGKS, residues 96-103) in the βγ-CAT α-subunit sequence. βγ-CAT showed potential NTP-binding and weak GTPase/ATPase activities in vitro. Through Western blotting analysis, we found that the α- and β-subunits of βγ-CAT participated in a 150 kDa SDS-stable protein complex formation, which also contained positive ubiquitination signals in the βγ-CAT treated HUVEC. Furthermore,under confocal microscopy, the immunofluorescence signals of ubiquitin and βγ-CAT subunits were co-localized in the vacuoles that were distributed in the cytoplasm and nucleus. In addition, βγ-CAT could induce several tumor cell`s detachment and apoptosis, and selectively kill tumor cells. These findings provide a clue to understand the mechanism of βγ-CAT endocysis and nuclear transport, and give an insight to investigate the possible occurrence of similar molecule’s cellular functions and action mechanisms of non-lens βγ-crystallins and trefoil factors in mammals.
  LIU Shu-bai , HE Ying-ying , QIAN Jinqiao and ZHANG Yun
  In vertebrates, non-lens βγ-crystallins are widely expressed in various tissues and their functions are not well known. The molecular mechanisms of trefoil factors (TFFs), which involved in mucosal healing and tumorigenesis, have remained elusive. βγ-CAT is a novel multifunctional protein complex of non-lens βγ-crystallin and trefoil factor from frog skin secretions. Here we report that βγ-CAT could induce sustained contraction of isolated rabbit aortic rings in dosage (2-35 nmol/L) and endothelium dependent manners (P<0.01). In addition, in situ immunofluorescence indicated that positive TNF-α signals were mainly detected at the endothelial cell layer of βγ-CAT (25 nmol/L) treated rings. Furthermore, βγ-CAT induced primary cultured rabbit thoracic aortic endothelial cells (RAECs) rapidly to release TNF-α. After βγ-CAT (25 nmol/L) treated for 10 and 30 min, the levels of the endothelial cells released TNF-α were 34.17±5.10 pg/mL and 98.01±4.67 pg/mL (P<0.01), respectively. In conclusion, βγ-CAT could induce sustained contraction of isolated aortic rings, and the contractile effect might be partially explained by the release of TNF-α. These findings will give new insight into understanding the functions and physiological roles of non-lens βγ-crystallins and trefoil factors.
  LIU Shu-bai , HE Ying-ying , QIAN Jin-qiao and ZHANG Yun
  βγ-CAT is a naturally existing multifunctional protein complex of non-lens βγ-crystallin and trefoil factor from Bombina maxima skin secretions. In this paper, we first analysed the time-response curve of hemolysis and intracellular potassium efflux induced by βγ-CAT on human erythrocytes. After βγ-CAT (3 nmol/L) in human erythrocytes was treated at 37¡æ for 5 min, we found that about 93.31±5.89% (P<0.01) of the intracellular potassium ions leaked, whereas the percentage of hemolysis was only13.12±1.92 % (P<0.05). We employed electron microscopy to observe the erythrocytes’ morphological changes and found erythrocytes were swollen and some had globular structures occurring on the cell surfaces, and fast releasing hemoglobins. These results indicated that the hemolytic effect of βγ-CAT on human erythrocyte membranes was due to intracellular potassium ion¡¯s rapid efflux. These results demonstrate morphological proof in understanding the mechanism of βγ-CAT pore-forming effect on human erythrocytes.
  Chen Xin-xin , Yu guo-yu , ZHAN Yan , Zhang Yun , Shen Ji-hong and Lee Wen-hui
 

OH-CATH is a novel cathelicidin identified from king cobra. It showed strong antibacterial activity against various bacteria in the presence of 1% NaCl and no haemolytic activity toward human red blood cells even at a high concentration. OH-CATH might serve as model molecules for the development of antimicrobial drugs. Understanding the action mechanism of OH-CATH and the reason for its selectivity against microbes is very important for this purpose. The bactericidal effect of the king cobra antimicrobial peptide OH-CATH on Gram-negative Escherichia coli (ATCC 25922) is observed by scanning electron microscopy (SEM) and transmitted electron microscopy (TEM). The SEM and TEM results suggested that the bactericidal mechanism of OH-CATH against Escherichia coli happened in three steps. Firstly, OH-CATH attached to the negatively charged bacterial wall by positively charged amino acid residues. In the second step, the accumulated OH-CATH aggregated and damaged the bacteria membrane in a pore-forming manner. In the last step, with the damage of cell permeability, the contents of the cells were released and eventually cells died.

  GAO Zhen-hua , ZHAO Hui , YU Guo-yu , ZHANG Yun , SHEN Ji-hong and LEE Wen-hui
 

SgI-29 is a newly characterized antibacterial peptide derived from human semenogelin I. Using SgI-29 as model, 4 peptides with different length were synthesized. Physico-chemical characteristics and structure prediction of SgI-29 and its derived peptides were analyzed by software packages and helix-wheel plot. The antibacterial activities of SgI-29 and its derived peptides against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were determined. The structure-function relationship of SgI-29 and its derivatives was analyzed. Our results indicated that SgI-22 has the strongest antibacterial activities against the tested bacteria among the synthetic peptides and might be used as a good model for the structure optimization.

  Du Ting-Yi , Zhang Yong and Zhang Yun
  Trefoil factor (TFF) family is a group of peptides with one or several trefoil factor domains in their structure, which are highly conserved in evolution, and are characterized by heat and enzymatic digestion resistance. The mammalian TFFs have three members (TFF1-3) , and the gastrointestinal tract and the airway system are major organs of their expression and secretion. At certain physiological conditions, with a tissue-specific distribution,TFF plays an important role in mucosal protection and wound healing. But in the malignant tissues, TFF is widely expressed, correlated strongly with the genesis, metastasis and invasion of tumor cells. These phenomena indicated that TFF may be a possible common mediator of oncogenic responses to different stimuli. The biological functions of TFF involve complex regulatory processes. Single chain TFF may activate cell membrane receptors and induce specific signaling transduction. On the other hand, TFF can form a complex with other proteins to exert its biological effects. In clinical medicine, TFF is primarily applied as drugs in the mucosal protection, in the prevention and the treatment of mucosal damage-related diseases and as pathological biomarkers of tumors. At present the first hand actions and the molecular mechanisms related to TFFs are still the major challenges in TFF research. Furthermore, the discovery of the naturally occurring complex of TFF and crystallins is highly valuable to the understanding of the biological functions and action mechanisms of TFF.
  Yu guo-yu , ZHANG Yong , JIANG Ping , Lee Wen-Hui and Zhang Yun
  Bm-TFF2, an amphibian trefoil factor, which is isolated from skin secretions of frog Bombina maxima, has much stronger biological activities than human TFFs. In the present study, Bm-TFF2 gene was amplified by polymerase chain reaction (PCR) from its cDNA and cloned into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and α-factor leader sequence. Multi-copies insertion transformants were screened on G418 plates. After the induction by 1% methanol for 72 hours, the expression of Bm-TFF2 came up to the best quantity which was about 50 mg in 1L medium, and 80% saturation ammonium sulfate was suitable to collect the Bm-TFF2 protein, as identified by SDS-PAGE and Western blotting assay. The results showed that the plasmid of Bm-TFF2-pPIC9K was constructed successfully and expressed abundantly in eukaryotic expression system, which lies basis for researching further the biological activities and the relationship of structure and functions of Bm-TFF2.
  Yu Guo-Yu , XIANG Yang , ZHANG Hong-Yun , JIANG Ping , Lee Wen-Hui , ZHANG Yun and ZHANG Yong
  Bm-TFF2, a trefoil factor from the large-webbed bell toad (Bombina maxima), can stimulate cell migration and inhibit cell apoptosis. To study the structure-function relationship of Bm-TFF2, we constructed wild-type and mutated Bm-TFF2 plasmids and expressed recombinant proteins in E. coli. The wild-type Bm-TFF2 gene encoding mature peptide was obtained by RT-PCR, while the N-terminal, C-terminal and two arginine mutated Bm-TFF2 clones were constructed, and ligated into pET-32a(+) expression vectors. The fusion proteins were induced by IPTG at 37°C. The mutant Bm-TFF2 fusion proteins expressed mainly in the inclusion bodies. The mutant (TRX)/Bm-TFF2 could be purified by using Ni2+-chelating chromatography and reverse-phase HPLC from the inclusion body supernatant. The fusion proteins were analyzed by SDS-PAGE and Western blotting. The yield of mutant Bm-TFF2 fusion proteins of above 95% purity was about 20 mg/L. All three recombinant mutant proteins can promote the migration of AGS cells in a dose-dependent manner with no obvious activity difference.
  LI Sheng-An , LEE Wen-Hui and ZHANG Yun
  Animal models are essential for the development of new anti-infectious drugs. Although some bacterial infection models have been established in rodents, small primate models are rare. Here, we report on two bacterial infection models established in tree shrew (Tupaia belangeri chinensis). A burnt skin infection model was induced by dropping 5x106 CFU of Staphylococcus aureus on the surface of a wound after a third degree burn. This dose of S. aureus caused persistent infection for 7 days and obvious inflammatory response was observed 4 days after inoculation. A Dacron graft infection model, 2x106 CFU of Pseudomonas aeruginosa also caused persistent infection for 6 days, with large amounts of pus observed 3 days after inoculation. These models were used to evaluate the efficacy of levofloxacin (LEV) and cefoperazone (CPZ), which reduced the viable bacteria in skin to 4log10 and 5log10 CFU/100 mg tissue, respectively. The number of bacteria in graft was significantly reduced by 4log10 CFU/mL treatment compared to the untreated group (P<0.05). These results suggest that two bacterial infection models were successfully established in tree shrew using P. aeruginosa and S. aureus. In addition, tree shrew was susceptible to P. aeruginosa and S. aureus, thus making it an ideal bacterial infection animal model for the evaluation of new antimicrobials.
 
 
 
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