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Biomedical subjects

Z Zhai

Publications and source records attributed to Z Zhai.

At least 19 recordsLinked to original sources
PubMed2001

Effects of phosphocreatine on apoptosis in a cell-free system.

The characteristic morphological and biochemical changes during caspase-mediated apoptosis can be reproduced to a large extent in a Xenopus laevis egg extract cell-free system by addition of mouse liver nuclei and exogenous cytochrome c. We show that in this system phosphocreatine accelerated the apoptotic morphological changes of the nuclei, but selectively inhibited DNA fragmentation. Western blot showed that the degradation of lamins A and C is accelerated, which is possibly responsible for the nuclear changes during cell apoptosis. However, the degradation of ICAD/DFF45-like protein in the egg extracts is inhibited in a time-dependent manner. Exogenous creatine, ATP, and several organic acids have no effect on DNA fragmentation, excluding the possibility that creatine, ATP, or acidic conditions resulting from phosphocreatine are responsible for inhibiting DNA fragmentation. Lithium chloride, a kinase inhibitor, can overcome the phosphocreatine effects and can restore DNA fragmentation. Our results indicate that phosphocreatine protects ICAD/DFF45-like protein from proteolysis, probably through kinase actions, resulting in its resistance to caspase cleavage and leading to an inhibition of DNA fragmentation.

Adenosine Triphosphate↗
PubMed2001

CSN3 interacts with IKKgamma and inhibits TNF- but not IL-1-induced NF-kappaB activation.

The transcription factor nuclear factor kappaB (NF-kappaB) plays a pivotal role in immune and inflammatory responses. Activation of NF-kappaB requires the activity of IKK, a kinase complex that contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit IKKgamma. To understand how IKK activity is regulated, we searched for IKKgamma-interacting proteins by the yeast two-hybrid system. These screenings identified CSN3, a component of the COP9 signalsome, as a protein specifically interacting with IKKgamma. Overexpression of CSN3 inhibits NF-kappaB activation triggered by tumor necrosis factor (TNF), but not interleukin-1 (IL-1). Moreover, overexpression of CSN3 also inhibits NF-kappaB activation triggered by proteins involved in TNF signaling, including TNF-R1, TRAF2, RIP, and NIK, but not by TRAF6, a protein involved in IL-1 signaling. These data suggest that CSN3 is a specific negative regulator of TNF- but not IL-1-induced NF-kappaB activation pathways.

Blotting, Western↗
PubMed2001

A new method for protein coexpression in Escherichia coli using two incompatible plasmids.

It is commonly believed that incompatible plasmids carrying the same replicon cannot coexist stably in one Escherichia coli cell. However, we found that two incompatible plasmids carrying different antibiotic resistance genes, if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding regions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors-pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli BL21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Induced by isopropyl beta-d-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression product contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhibited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately.

Apoptosis Regulatory Proteins↗
PubMed2001

Analysis of nuclear apoptotic process in a cell-free system.

We report an analysis of the apoptotic process of mouse liver nuclei induced in a cell-free carrot cytosol system by cytochrome c. Typical characteristics of apoptosis were observed, such as chromatin condensation, margination, apoptotic bodies and DNA ladders. Furthermore, transmission and scanning electron microscope analysis of the apoptotic nuclei detected chromatin-free nuclear vesicles before apoptotic bodies appeared at a comparatively late phase. When AC-YVAD-CHO, an inhibitor of caspase 6, was introduced into the system, these vesicles and apoptotic bodies disappeared completely within our study sections. We confirmed the results using whole-mount electron microscopy, and found that although the nuclear lamina was destroyed early, the nuclear matrix largely remained intact during the course of apoptosis. The nuclear matrix played an important role in maintaining the integrity of apoptotic cells and connecting the apoptotic bodies and apoptotic nucleus.

Animals↗
PubMed2001

[Study on variation and interrelations of characteristics at ginseng germplasms].

The variation and interrelations of some characteristics of Panax ginseng germplasms(lines), which were five selfmating generations of some single plants were studied. Methods of multivariate analysis, such as correlation, regression, or clustering, and coefficient of variation were used. The results were of benefit to the breeding and standardized planting of Panax ginseng.

Genetic Variation↗
PubMed2000

In vitro nuclear reconstitution could be induced in a plant cell-free system.

A cell-free system derived from carrot cell cytosol extract has been developed for reassembling nuclear structure around the added demembranated sperm chromatin of Xenopus. Morphological evidence suggests that reassembled nuclei display the typical characteristics of normal eukaryotic nuclei, such as double-layered nuclear membrane and nuclear pores. Micrococcal nuclease treatment indicates that remodeling of the demembranated sperm chromatin has occurred and the structure of nucleosome is formed during nuclear reconstitution. These data indicate that the nuclear reconstitution can be induced in cell-free systems from plants, and the self-assembly of the nucleus is ubiquitous in both animal and plant cells.

Animals↗
PubMed2000

Ceramides induce apoptosis in HeLa cells and enhance cytochrome c-induced apoptosis in Xenopus egg extracts.

Ceramide has been reported to induce typical apoptotic changes in nuclei incubated in a cell-free system, and that the addition of ceramide bypasses the requirement for mitochondria. Here, we explore the possible pathways by which ceramide induces apoptosis either in intact cells or in a cell-free system which we have developed. We found that in the cell-free system, C2-ceramide is not able to induce apoptosis in nuclei whereas cytochrome c does, but it is able to induce HeLa cells to undergo apoptosis. Ceramide is also not able to induce apoptosis when added into the cell-free system together with purified mitochondria. Further investigation showed that C2-ceramide at certain concentrations greatly increases nuclear apoptosis caused by cytochrome c in the cell-free system. From these results we conclude that the induction of apoptosis by ceramide may require intact cells in which some unknown signal transduction pathways are involved.

Animals↗
PubMed2000

[Molecular cloning and expression of E2 gene of the Chinese classical swine fever virus(shimen strain) and preliminary studies of its DNA vaccine].

A 1.1 bp fragment of E2 gene of Chinese classical swine fever virus(CSFV) Shimen strain, a standard virulent strain, was amplified by RT-PCR from total RNA of cell cultures infected by CSFV, and cloned into pGEM T vector. The nucleotide sequence of this fragment was sequenced by Sanger's method and the amino acid sequence was deduced. Compared with the corresponding region of Alfort, Brescia and C strain of CSFV, the nucleotide sequence homology is 84.7%, 92.6% and 95.2% respectively, and the amino acid sequence 89.4%, 92.6% and 94.6%, respectively, we subcloned 1.1 bp of E2 gene cDNA into baculovirus transfer vector and successfully constructed two recombinant baculoviruses expressing GST-E2 and GST-GFP-E2 fusion protein respectively by homologous recombination in sf-9 cell. Furthermore, we also constructed recombinant eukaryotic expression vector pcE2 containing E2 gene in frame and transfected COS-7 cell by lipofectamine, the indirect immunofluorescence assay (IFA) showed that the expressed E2 protein can be recognized by E2 specific monoclonal antibody the pcE2 DNA was directly injected into BALB/c mice intramuscular(i.m.) and the CSFV E2-specific antibodies was measured by enzyme-linked immunosorbent assay(ELISA) the ELISA results indicated the E2-specific antibodies was induced in inoculated mice and virus neutralization assays also indicate single inoculations of plasmids expressing CSFV E2 glycoprotein raised neutralizing antibody in BALB/c mice. these results will be beneficial to investigate the possibility of DNA vaccine against CSFV.

Amino Acid Sequence↗
PubMed2000

[Effect of amniotic membrane on expressions of TGF-beta 1, collagens I, III and fibronectin in rabbit corneal healing after photorefractive keratectomy].

OBJECTIVE: To observed the effect of amniotic membrane transplantation (AMT) on expressions of transforming growth factor-beta 1(TGF-beta 1), collagens I, III and fibronectin (FN) in rabbit corneal healing after photorefractive keratectomy (PRK), and investigate the anti-scarring formation mechanism of AMT. METHODS: Ten rabbits underwent bilateral PRK to correct 8 diopters of myopia. One eye was randomly transplanted with preserved human amniotic membrane, and the other eye served as the control. The expressions of TGF-beta 1, collagens I, III and FN were studied by immunohistochemistry at 4 weeks after operation. RESULTS: The expression of TGF-beta 1 in corneal epithelium and keratocytes and collegans in anterior stroma was significantly less in AMT group than in the control group. The expressions of collagens III and FN in anterior stroma of ablation area were also significantly less in AMT group than in the control group. The staining of collagen I had no significant difference between the AMT group and the control group. CONCLUSION: The expressions of TGF-beta 1, collagens III and FN can be suppressed by amniotic membrane after PRK. These data suggest that part of the anti-scarring effect of AMT may be mediated through the suppression of TGF-beta 1, collagens III and FN expression.

Amnion↗
PubMed1999

Urothelial hinge as a highly specialized membrane: detergent-insolubility, urohingin association, and in vitro formation.

Urothelial surface is covered by numerous plaques (consisting of asymmetric unit membranes or AUM) that are interconnected by ordinary looking hinge membranes. We describe an improved method for purifying bovine urothelial plaques using 2% sarkosyl and 25 mM NaOH to remove contaminating membrane and peripheral proteins selectively. Highly purified plaques interconnected by intact hinge areas were obtained, indicating that the hinges are as detergent-insoluble as the plaques. These plaque/hinge preparations contained uroplakins, an as yet uncharacterized 18-kDa plaque-associated protein, plus an 85-kDa glycoprotein that is known to be hinge-associated in situ. Examination of the isolated, in vitro-resealed bovine AUM vesicles by quick-freeze deep-etch showed that each AUM particle consists of a 16-nm, luminally exposed "head" anchored to the lipid bilayer via a 9-mm transmembranous "tail", and that an AUM plaque can break forming several smaller plaques separated by newly formed particle-free, hinge-like areas. These data lend support to our recently proposed three-dimensional model of mouse urothelial plaques. In addition, our findings suggest that urothelial plaques are dynamic structures that can rearrange giving rise to new plaques with intervening hinges; that the entire urothelial apical surface (both plaque and hinge areas) is highly specialized; and that these two membrane domains may be equally important in fulfilling some of the urothelial functions.

Animals↗
PubMed1999

[The morphological structure of classical swine fever virus and some characteristics of its multiplication].

Some characteristics of the multiplication of classical swine fever virus(CSFV) Thiverval strain were studied by means of the immunofluorescence technique. Under optimum culture conditions, the concentration of CSFV in the culture liquid multiplicated by MPK cells is ten times higher than by PK-15 cells. The half-life period of CSFV at 37 degrees C is 3 hour. The location of CSFV's replication in host cells is detected by the monoclonal antibodies of the structural protein E2 and the non-structural protein p120 of CSFV. Based on the above results, the ultrastructure of CSFV particles inside host cells was further studied using electron microscope and the changes of the ultrastructure of CSFV-infected cells are shown. The possible reasons for lower viral titre are also discussed in this paper.

Animals↗
PubMed1999

[Effect of artemether on nucleoside uptake and nucleic acid content in Schistosoma japonicum].

AIM: To observe the effect of artemether (Art) on nucleoside uptake and nucleic acid content in Schistosoma japonicum. METHODS: RNA and DNA contents of both male and female worms harbored in mice treated intragastrically (i.g.) with Art 300 mg/kg for 24 h or 48 h were determined, respectively. After in vivo drug treatment, the schistosomes recovered were in vitro maintained in drug-free medium containing [3H]adenosine, [5-(3)H] uridine or [methyl-3H]thymidine at a final concentration of 37 MBq/L or 74 MBq/L for 2 h or 4 h, the tritiated nucleoside uptake and incorporation into nucleic acid of schistosomes were measured. RESULTS: The RNA and DNA contents of female worms recovered from the host 48 h after dosing were markedly decreased by 51.6% and 23.5%, respectively, while the RNA content of male worms showed 42.4% reduction. When the above-mentioned schistosomes were in vitro exposed to the tritiated nucleoside for 2 h or 4 h, apparent decrease in tritiated nucleoside uptake with reduction rates of 35.2%-50.1% was seen in female worms. The incorporation of [methyl-3H]thymidine into the female worm DNA 2 h after incubation was reduced by 71.4% while the incorporation of [3H]adenosine into the female worm RNA and DNA 4 h after incubation was reduced by 65.2% and 50.0%, respectively. CONCLUSION: Art exhibited an apparent effect on the nucleic acid metabolism in schistosomes, especially in female worms.

Animals↗
PubMed1998

The role of keratin filaments during nuclear envelope reassembly in Xenopus egg extracts.

We report here a new structure, named 'strings-of-pearls', which are seen to form in Xenopus egg extracts after incubation, as 200 nm membrane vesicles attach to 10 nm filaments. These membrane vesicles fuse together along the filaments to form annulate lamellae (AL) or attach to demembranated sperm chromatin to initiate assembly of a nuclear envelope. Immunoassay with anti-keratin antibodies AE3 showed that the filaments were mainly composed of a 56 kDa keratin-like protein. Addition of AE3 to the extracts resulted in inhibition of AL formation and defective assembly of NE. These results suggest a function of keratins in the assembly of nuclear envelopes during Xenopus development.

Animals↗
PubMed1998

[The envelope formation of AcMNPV and the location of envelope protein gp64 in host cell].

Electron Microscopic study on the recombinant AcMNPV infected Sf9 cell showed that the virus has at least two forms of envelope formation: one is budding from plasma membrane and the other is enveloping in the nucleus by membrane structure. An additional possible pathway is budding from nuclear membrane. The envelope formation of AcMNPV in Sf9 cell line and its relationship with the envelope protein gp64 were investigated by using immunofluorescence techniques. The results showed gp64 was mainly located on the plasma membrane and nuclear membrane of infected cell. Whereas, there is no gp64 on the envelope of OV virus particles in the nuclei. This pattern cause the differences in envelope composition between the virus particles that budding from plasma membrane and nuclear membrane and those that enveloping in the nucleus.

Animals↗
PubMed1997

Association of DNA with nuclear matrix in in vitro assembled nuclei induced by rDNA from Tetrahymena shanghaiensis in Xenopus egg extracts.

Nuclei assembled in vitro from Xenopus egg extracts and DNA display many properties known from in vivo nuclei. However, the distribution pattern of DNA in such nuclei remains unknown. We introduced rDNA from Tetrahymena macronuclei into Xenopus egg extracts and examined the association of specific DNA sequences with the nuclear matrix (NM) of the nuclei formed. We found that the 5' non-transcribed spacers (5'-NTS) specifically bound to the NM as previously shown in normal Tetrahymena macronuclei.

Animals↗
PubMed1997

[Immunohistochemical study of p53, c-erbB-2 and nm23 proteins in human gastric cancer].

OBJECTIVE: To evaluate p53, c-erbB-2 and nm23 oncoproteins in diagnosis of gastric cancer. METHODS: The expression of p53, c-erbB-2 and nm23 oncoproteins was detected immunohistochemically in 141 surgically resected specimens and 93 endoscopic biopsy specimens of gastric cancer. RESULTS: The positive rate of p53, c-erbB-2 and nm23 immunoreactivity was 45.8-56.3%, 18.3-31.2% and 72.9%-81.3% respectively. No immunoreactivity was observed in the tumor-adjacent normal mucosa and non-tumor biopsy specimens. The presence of c-erbB-2 was correlated with the size of tumor, grade of tumor differentiation and Lanren classification. Patients with c-erbB-2 positive tumor had lower 5-year survival rate. The expression of nm23 was negatively correlated with the depth of invasion, and lymphnode metastasis. The positive rates of p53, c-erbB-2 and nm23 had no statistical difference between biopsy and resected specimens. CONCLUSION: Determination of p53, c-erbB-2 and nm23 oncoproteins immunohistochemically might be useful in the diagnosis and differential diagnosis, clinical staging, and predicting prognosis of gastric cancer.

Adenocarcinoma↗