Biomedical subjects

W Zhai

Publications and source records attributed to W Zhai.

At least 19 recordsLinked to original sources

PubMed2006

Ischemia/reperfusion injury: The role of CD26/dipeptidyl-peptidase-IV-inhibition in lung transplantation.

UNLABELLED: CD26/Dipeptidyl peptidase (DPP) IV is an integral membrane protein of lymphocytes that modulates the activities of chemokines, interleukins, and neuropeptides. We investigated the effect of enzymatic DPP IV inhibition on ischemia/reperfusion injury after extended ischemia prior to transplantation. MATERIALS AND METHODS: We used a syngeneic rat (Lewis) orthotopic left lung transplantation model. In the control group (group I), donor lungs were flushed and preserved in Perfadex for 18 hours at 4 degrees C, then transplanted and reperfused for 2 hours. Group II donor lungs were perfused with and stored in Perfadex +25mol/L AB192 (bis(4-acetamidophenyl) 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate), a small molecular weight DPP IV inhibitor. After 2-hour reperfusion, we measured blood gas, peak airway pressure, and thiobarbituric acid reactive substances. RESULTS: Grafts from group II versus group I showed a significantly increased oxygenation capacity (II: 298.4 +/- 87.6 mm Hg vs 120.9 +/- 48.0, P < .01), lower peak airway pressure (11.8 +/- 0.9 mm Hg vs 16.0 +/- 1.4, P < .01), and less lipid peroxidation (9.3 +/- 2.0 micromol/L vs 13.8 +/- 1.8, P < .01). CONCLUSION: Inhibition of intragraft DPP IV enzymatic activity significantly reduced ischemia/reperfusion-associated pulmonary injury, allowing for successful transplantation after 18 hours of ischemia.

Animals↗

PubMed2001

Chromosome landing at the bacterial blight resistance gene Xa4 locus using a deep coverage rice BAC library.

Xa4 is a dominantly inherited rice gene that confers resistance to Philippine race 1 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae in rice. In order to isolate the gene by positional cloning, a bacterial artificial chromosome (BAC) library was constructed from genomic DNA isolated from an Xa4-harboring accession, IRBB56. The library contains 55,296 clones with an average insert size of 132 kb, providing 14 rice genome equivalents. Three DNA markers closely linked to Xa4 were used to screen the library. The marker RS13, a resistance gene analogue that co-segregates with Xa4, identified 18 clones, of which four and six, respectively, were simultaneously detected by the other two markers, G181 and L1044. Fingerprinting and Southern analysis indicated that these clones overlapped and define an interval spanning 420 kb. In an F2 population derived from an indica variety, IR24, and its Xa4-containing near isogenic line (NIL), IRBB4, the susceptible plants were screened in order to map the Xa4 gene genetically and physically. Out of 24 insert ends isolated from the BACs in the contig, three revealed polymorphisms between IR24 and IRBB4. Two insert ends, 56M22F and 26D24R, flanked Xa4 on each side. Based on the overlap of the BACs, six overlapping clones were considered to include the Xa4 allele, one of which, 106P13, was chosen for further investigation.

Blotting, Southern↗

PubMed2001

[Expression of telomerase reverse transcriptase and its relationship with tumor suppressor gene p53 in HCC].

OBJECTIVE: To explore the expression of human telomerase reverse transcriptase (HTERT) and its relationship with tumor suppressor gene p53. METHODS: RNA-RNA in situ hybridization (ISH) technique was applied to localize the expression of HTERT mRNA and immunohistochemistry (IHC) was used to observe the p53 protein expression in 34 specimens of HCC and their corresponding non-cancerous tissues. Both the significance of HTERT mRNA expression in the different histopathologic characters and its relationship with p53 protein were investigated. RESULTS: HTERT was detected in tumor tissue in 30 of 34 cases (88.2%), and all the 6 cases of small cancer (diameter<3cm) were positive. The positive rate of HTERT mRNA was higher in the tumor tissues with low-differentiation, extra- or intra-hepatic metastasis, uncompleted fibrotic capsule and/or the non-cancerous tissue with chronic active lesions or positive HBsAg. Intensive signal was seen both in the HCC cells along the front of invasive area and in a few non-cancerous liver cells with dysplasia. Of all 34 HCC, p53 protein was presented in 25 cases. There was a correlation between the expression of HTERT mRNA and of p53 protein. The coincidence rate of the expression (HTERT+/p53+ and HTERT-/p53-) was 79.4%. More intensity of HTERT signal could be seen in the cases of positive p53. HTERT mRNA was not found in normal liver tissues. CONCLUSIONS: High expression of HTERT mRNA is proved in most cases of HCCs. HTERT expression may relate to the pathogenesis and the invasion of HCC. p53 may play a role in regulating the expression of HTERT.

Adult↗

PubMed2000

Structure-activity studies including a Psi(CH(2)-NH) scan of peptide YY (PYY) active site, PYY(22-36), for interaction with rat intestinal PYY receptors: development of analogues with potent in vivo activity in the intestine.

Peptide YY (PYY) is a gut hormone that inhibits secretion and promotes absorption and growth in the intestinal epithelium. We have performed structure-activity studies with the active site, N-alpha-Ac-PYY(22-36)-NH(2), for interaction with intestinal PYY receptors. Investigation of aromatic substitutions at position 27 resulted in analogues that exhibited potent in vitro antisecretory potencies with N-alpha-Ac-[Trp(27)]PYY(22-36)-NH(2) exhibiting even greater potency than intact PYY. In vivo studies in dogs revealed that this analogue also promoted intestinal absorption of water and electrolytes during continuous intravenous and intraluminal infusion. Investigations carried out to identify features that would enhance stability revealed that incorporation of Trp(30) increased affinity for PYY receptors. A "CH(2)-NH" scan revealed that incorporation of reduced bonds at position 28-29 or 35-36 imparted greater receptor affinity. In general, disubstituted analogues designed based on the results of single substitutions exhibited good receptor affinity with N-alpha-Ac-[Trp(27),CH(2)-NH(35-36)]PYY(22-36)-NH(2) having the greatest affinity (IC(50) = 0.28 nM). Conservative multiple substitutions with Nle-->Leu and Nva-->Val also imparted good affinity. An analogue designed to encompass most of the favored substitutions, N-alpha-Ac-[Nle(24,28),Trp(30),Nva(31), CH(2)-NH(35-36)]PYY(22-36)-NH(2), exhibited a proabsorptive effect in dogs comparable to, but longer lasting than, that of intact hormone. Selected analogues also exhibited good antisecretory potencies in rats with N-alpha-Ac-[Trp(30)]PYY(22-36)-NH(2) being even more potent than PYY. However, the potencies did not correlate well with the PYY receptor affinity or the proabsorptive potencies in dogs. These differences could be due to species effects and/or the involvement of multiple receptors and neuronal elements in controlling the in vivo activity of PYY compounds. PYY(22-36) analogues exhibited good affinity for neuronal Y2 receptors but poor affinity for Y1 receptors. Also, crucial analogues in this series hardly bound to Y4 and Y5 receptors. In summary, we have developed PYY(22-36) analogues which, via interacting with intestinal PYY receptors, promoted potent and long-lasting proabsorptive and antisecretory effects in in vivo models. These compounds or analogues based on them may have useful clinical application in treating malabsorptive disorders observed under a variety of conditions.

Animals↗

PubMed2000

Repression of RNA polymerase I transcription by the tumor suppressor p53.

The tumor suppressor protein p53 is frequently inactivated in tumors. It functions as a transcriptional activator as well as a repressor for a number of viral and cellular promoters transcribed by RNA polymerase II (Pol II) and by RNA Pol III. Moreover, it appears that p53 also suppresses RNA Pol I transcription. In this study, we examined the molecular mechanism of Pol I transcriptional inhibition by p53. We show that wild-type, but not mutant, p53 can repress Pol I transcription from a human rRNA gene promoter in cotransfection assays. Furthermore, we show that recombinant p53 inhibits rRNA transcription in a cell-free transcription system. In agreement with these results, p53-null epithelial cells display an increased Pol I transcriptional activity compared to that of epithelial cells that express p53. However, both cell lines display comparable Pol I factor protein levels. Our biochemical analysis shows that p53 prevents the interaction between SL1 and UBF. Protein-protein interaction assays indicate that p53 binds to SL1, and this interaction is mostly mediated by direct contacts with TATA-binding protein and TAF(I)110. Moreover, template commitment assays show that while the formation of a UBF-SL1 complex can partially relieve the inhibition of transcription, only the assembly of a UBF-SL1-Pol I initiation complex on the rDNA promoter confers substantial protection against p53 inhibition. In summary, our results suggest that p53 represses RNA Pol I transcription by directly interfering with the assembly of a productive transcriptional machinery on the rRNA promoter.

Genes, Tumor Suppressor↗

PubMed2000

Identification and chromosomal location of a new tandemly repeated DNA in maize.

Two clones of a new family of tandemly repeated DNA sequences have been isolated from a maize random genomic DNA library. MR68 is 410 bp, representing a monomeric unit and MR77 is 1222 bp, containing three units. The copy number was estimated to be about 3000 per 1C maize genome. Its methylation pattern was also determined. Fluorescent in situ hybridization (FISH) indicates that the sequence is located on the subtelomeric region of the long arm of chromosomes 3 and 6, as well as on the satellite of chromosome 6.

Base Sequence↗

PubMed2000

[Effects of hypoxia on expression and activity of matrix metalloproteinase-2 in hepatic stellate cell].

OBJECTIVE: To study the effect of hypoxia on the expression and activity of matrix metalloproteinase-2 (MMP-2) in the hepatic stellate cell (HSC). METHODS: The expression of MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) in cultured rat HSC was detected by immunocytochemistry (ICC). The relative amount of MMP-2 and TIMP-2 in the culture supernatant was assayed by ELISA and the activity of MMP-2 in supernatant was examined by zymography. RESULTS: The expression of MMP-2 was enhanced in hypoxia group as compared with that in control one (t' 3.4685, P<0.05 ), while the expression of TIMP-2 was decreased (t'=2.5673, P<0.05). The activity of MMP-2 was lower in hypoxia group than in control one (t'=4.270, P<0.01). Comparing the varied duration of hypoxia, the relative amount of MMP-2 protein was the highest in 6 h group (6h vs 12h, t=4. 0945, P<0.001; 6h vs 24h, t=2.4652, P<0.025), with a contrary curve in TIMP-2. CONCLUSION: Hypoxia promotes the expression of MMP-2 and inhibits the expression of TIMP-2 in HSC. It is more notable at early stage of hypoxia.

Animals↗

PubMed1999

Broadly cross-reactive, high-affinity antibody to hypervariable region 1 of the hepatitis C virus in rabbits.

The HCV hypervariable region 1 (HVR1) of the main E2 envelope protein is critically important in HCV neutralization but its extreme variability makes immune therapy and vaccine development particularly difficult. To explore the hypothesis that HVR1 carries a common epitope susceptible of eliciting cross-reactive neutralizing and inhibitory antibodies, rabbits were immunized with a series of synthetic HVR1 peptides. The anti-HVR1 produced were purified and characterized. Several lines of evidence supported the working hypothesis: (1) although injected only once, a boosting effect from poorly homologous peptides was observed; (2) purified rabbit IgG reacted with high affinity with immunizing peptides and cross-reacted with 16 of 17 unrelated HVR1 peptides; (3) antibodies appeared of restricted diversity irrespective of the linear HVR1 peptide sequences; (4) anti-HVR1 peptides effectively captured HCV in 22 of 33 plasmas from random infected patients; (5) anti-HVR1 IgG blocked the binding of antibody-captured HCV to MOLT-4 cells. These findings suggest that with an appropriate HVR1 peptide immunization scheme, high titer, broadly cross-reactive, blocking antibodies to HCV can be produced. Antibodies to the putative ubiquitous HVR1 epitope may have important clinical uses.

Amino Acid Sequence↗

PubMed1999

Hypervariable region diversity of hepatitis C virus and humoral response: comparison between patients with or without cirrhosis.

To investigate the potential clinical utility of antibody response to HVR1 of HCV, the genomic and amino acid diversity of HVR1 was compared between two groups of four chronic HCV carriers with or without liver cirrhosis. Peptides corresponding to the deduced COOH- and NH2-terminal amino acid sequences of HVR1 were synthesised to assess the reactivity of patient sera to autologous and homologous HVR1 epitopes by enzyme-linked immunosorbent assay. HCV chronic carriers had significantly more frequent cross-reactivity with homologous C- than N-terminal HVR1 peptides. Twelve cirrhotic and eleven noncirrhotic patients had a similar frequency of cross-reactivity with either C- or N-terminal HVR1 peptides. However, noncirrhotic patients had a significantly higher level of C-terminal HVR1 antibody cross-reactivity than cirrhotic patients. In HCV chronic carriers, the magnitude of the immune response to but not the frequency of cross-reactivity with C-terminus HVR1 peptides differ between patients with and without liver cirrhosis.

Adult↗

PubMed1999

Human recombinant single-chain antibody fragments, specific for the hypervariable region 1 of hepatitis C virus, from immune phage-display libraries.

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) may contain a potential neutralization site and the generation of human single-chain antibody fragments (scFv) to HVR1 may therefore provide a useful tool for the study of HCV. In this report, we have isolated and characterized three anti-HVR1 scFv clones from two patient-derived phage-displayed libraries and HCV HVR1 peptides. scFv S52/20 and S53/6 were selected with serologically cross-reactive HVR1 peptides. scFv p3f10 was obtained by screening the library from patient MH with an autologous HVR1 peptide. Nucleotide sequencing showed that the VH chains and Vkappa chains of all three scFv antibodies were derived from VH3 and Vkappa1 family germline V-genes, respectively. The specificity and affinity of the recombinant scFv antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and an affinity biosensor, using HVR1 peptides. S52/20 scFv binding to S52 HVR1 peptide was blocked by preincubation with soluble peptide S52 and was partially competed by one of three HCV-infected patient sera. In addition, scFv S52/20 blocked the binding of HCV-susceptible Molt-4 cells to immobilized S52 peptide. This study demonstrates that recombinant human scFv antibodies to HCV HVR1 can be produced in vitro and directly confirms that HVR1 of HCV elicits highly specific antibodies. The very high specificity of these antibodies to HVR1 may limit their potential use in passive immunization therapy against HCV, and further engineering of the scFvs needs to be performed to generate broad-spectrum blocking scFvs.

Amino Acid Sequence↗

PubMed1999

A kinase activity associated with simian virus 40 large T antigen phosphorylates upstream binding factor (UBF) and promotes formation of a stable initiation complex between UBF and SL1.

Simian virus 40 large T antigen is a multifunctional protein which has been shown to modulate the expression of genes transcribed by RNA polymerase I (Pol I), II, and III. In all three transcription systems, a key step in the activation process is the recruitment of large T antigen to the promoter by direct protein-protein interaction with the TATA binding protein (TBP)-TAF complexes, namely, SL1, TFIID, and TFIIIB. However, our previous studies on large T antigen stimulation of Pol I transcription also revealed that the binding to the TBP-TAFI complex SL1 is not sufficient to activate transcription. To further define the molecular mechanism involved in large T antigen-mediated Pol I activation, we examined whether the high-mobility group box-containing upstream binding factor (UBF) plays any role in this process. Here, using cell labeling experiments, we showed that large T antigen expression induces an increase in UBF phosphorylation. Further biochemical analysis demonstrated that UBF is phosphorylated by a kinase activity that is strongly associated with large T antigen, and that the carboxy-terminal activation domain of UBF is required for the phosphorylation to occur. Using in vitro reconstituted transcription assays, we demonstrated that the inability of alkaline phosphatase treated UBF to efficiently activate transcription can be rescued by large T antigen. Moreover, we showed that large T antigen-induced UBF phosphorylation promotes the formation of a stable UBF-SL1 complex. Together, these results provide strong evidence for an important role for the large T antigen-associated kinase in mediating the stimulation of RNA Pol I transcription.

Antigens, Viral, Tumor↗

PubMed1999

Recruitment of TATA-binding protein-TAFI complex SL1 to the human ribosomal DNA promoter is mediated by the carboxy-terminal activation domain of upstream binding factor (UBF) and is regulated by UBF phosphorylation.

Human rRNA synthesis by RNA polymerase I requires at least two auxiliary factors, upstream binding factor (UBF) and SL1. UBF is a DNA binding protein with multiple HMG domains that binds directly to the CORE and UCE elements of the ribosomal DNA promoter. The carboxy-terminal region of UBF is necessary for transcription activation and has been shown to be extensively phosphorylated. SL1, which consists of TATA-binding protein (TBP) and three associated factors (TAFIs), does not have any sequence-specific DNA binding activity, and its recruitment to the promoter is mediated by specific protein interactions with UBF. Once on the promoter, the SL1 complex makes direct contact with the DNA promoter and directs promoter-specific initiation of transcription. To investigate the mechanism of UBF-dependent transcriptional activation, we first performed protein-protein interaction assays between SL1 and a series of UBF deletion mutants. This analysis indicated that the carboxy-terminal domain of UBF, which is necessary for transcriptional activation, makes direct contact with the TBP-TAFI complex SL1. Since this region of UBF can be phosphorylated, we then tested whether this modification plays a functional role in the interaction with SL1. Alkaline phosphatase treatment of UBF completely abolished the ability of UBF to interact with SL1; moreover, incubation of the dephosphorylated UBF with nuclear extracts from exponentially growing cells was able to restore the UBF-SL1 interaction. In addition, DNase I footprinting analysis and in vitro-reconstituted transcription assays with phosphatase-treated UBF provided further evidence that UBF phosphorylation plays a critical role in the regulation of the recruitment of SL1 to the ribosomal DNA promoter and stimulation of UBF-dependent transcription.

Binding Sites↗

PubMed1999

Resistant mechanisms of Candida albicans to fluconazole.

OBJECTIVE: To detect the resistant mechanisms of Candida albicans to fluconazole (FCZ) at molecular biology level, since the resistance mechanisms of azole antifungal agents have been the focus of attention these years. METHODS: Thirty-two FCZ-resistant C. albicans were selected as our test strains (MICs > or = 64 micrograms/ml). With 14-alpha-demethylase gene (ERG16 gene, target enzyme encoding gene of azoles) as our object, we chose six sets of primers from the ERG16 gene to amplify the interested fragments, and conducted Southern blot hybridization, restriction fragment length polymorphism (RFLP), and single strand conformation polymorphism (SSCP) analysis for the fragments which were amplified by the six sets of primers, and pre-resistant sensitive strains were used as controls. Three representative fragments, A66, D66 and E78, were selected to be cloned and sequenced. RESULTS: The polymerase chain reaction (PCR) amplification showed that several tested strains were negative for some primers. However, our Southern blot analysis reminded that their resistance did not result from the lack of target enzyme coding gene. SSCP analysis showed that differences were noted between the resistant and sensitive strains and inter-resistant strains. Statistical analysis showed that the most variable sequence lied in the amplifier of the sixth pair of primer, and all the tested 32 strains showed positive results. In the 11 mutation points we found, five resulted in amino acid alterations. It is likely that one or more mutational alterations (alone or in combination) might lead to the expression of an enzyme highly resistant to the inhibitory action of FCZ which in turn is responsible for the FCZ resistant trait in these strains. CONCLUSION: One or more mutational alterations might lead to the azole resistant trait in this strains.

Acquired Immunodeficiency Syndrome↗

PubMed1999

[Development of healthy beverage from Eucommla ulmoides foliage].

Effective compositions of Eucommla ulmoides foliage, sampled from the south side west section of the Qinling Mountain area, are analyzed qualiatively and quantitatively. It is shown that the foliage contains plenty of mineral elements, seventeen amino acids, proteins, sugars, alkaloids, flavonoids, aucubin, chlorogenic acid, pinoresinol diglucoside, coffeic acid juice and Jingniping glucoside. Based on the analysis and the main material of Eucommla ulmoides foliage, a healthy beverage functioning especially on resisting tire actions is formulated and produced. This work has provided a dependable new idea for further exploitation and utilization of the natural resources.

Amino Acids↗

PubMed1998

[Expression of integrin alpha 5, beta 1 subunit and fibronectin mRNAs in human hepatocellular carcinoma].

OBJECTIVE: To study the biologic significance of the expression of integrin alpha 5, beta 1 subunit and fibronectin (FN) mRNAs in hepatocellular carcinoma (HCC). METHODS: Integrin alpha 5, beta 1 subunit and FN mRNAs were detected by using Northern blot (alpha-32P-labeled cDNA probes) and in situ hybridization (Dig-labeled cDNA probes) in 15 cases of HCC, 5 cases of paracancerous cirrhotic liver and 3 cases of normal liver tissue. RESULTS: In well-differentiated HCC, the level of integrin alpha 5, beta 1 or FN mRNA expression was similar to those in normal liver and paracancerous cirrhotic liver tissues, but was markedly decreased, even absent in the poorly- and moderately-differentiated HCC tissues (P < 0.01, respectively). The expression of integrin alpha 5 and FN mRNAs was mainly detected in the cytoplasm of HCC cells. In all the HCC cases tested, the expression of integrin alpha 5, beta 1 and FN mRNAs was lower in 8 cases of HCC with focal invasion and/or intrahepatic metastasis than that in the others without invasion or metastasis(P < 0.05). In addition, the heterogeneous expression of FN mRNA was found in 5 cases of HCC with intrahepatic metastasis. CONCLUSIONS: Combined with the immunohistochemical observation before, it might be deduced that the changes of integrin alpha 5 beta 1 and FN protein expression in HCC tissues are because of changing of the mRNA levels related, which might deeply influence the cell differentiation, tumor invasion and metastasis of HCC. The appearance of the heterogeneous FN mRNA and its encoded FN protein might be associated with the development of HCC metastasis.

Carcinoma, Hepatocellular↗

PubMed1998

Experimental study on pathogenicity of precore mutants in Hepadnaviridae.

OBJECTIVE: To study the replicative competency and pathogenicity of precore gene mutants of duck hepatitis B virus (DHBV) in the duck model. METHODS: Three site-directed point mutations in the precore region of cloned DHBV were constructed. Head-to-tail dimers were formed. The three plasmids were named: pEDM1-2 (initiation codon ATG mutated to TTG), pEDM2-2 (an "A" was inserted down stream of codon 12, leading to frame shift in the distal end of precore region), pEDM3-2 (codon 38 was changed from TAT to TAA, leading to a stop codon at the 3'-end). Mutants and wild-type cloned DNA dimers were first separately used to transfect LMH cells (a chicken hepatoma cell line) and viruses were collected from supernatant and used to infect 6 one-day-old ducklings per group. Serum duck hepatitis B surface antigen (DHBsAg) and DHBV DNA were assayed. Six weeks after infection, ducks were killed and liver tissues were studied for histopathological changes. RESULTS: After transfection, pEDM1-2, pEDM2-2 and pEDM3-2 expressed similar level of DHBsAg. Replication of pEDM1-2 and pEDM3-2 was similar to that of the wild type clone, while pEDM2-2 replicated at a significantly decreased level. Infection study employing the supernatant of transfected cells was as follows: pEDM1-2 infected 5/6 ducklings, pEDM2-2 non infected, pEDM3-2 infected 2/6 ducklings, wild type virus infected 6/6 ducklings. Positive serum samples from both pEDM1-2 and pEDM3-2 were at a lower serum DHBV level compared to that of the wild type virus. Pathological changes were more significant in pEDM3-2 infected duck livers, with numerous inflammatory cells in portal tract and infiltration into parenchyma. CONCLUSIONS: Mutations in the initiating codon or generation of a stop codon at the 3'-end of the precore region resulted in decreased replication competency of DHBV, while frame-shift mutation of the precore region, covering the epsilon encapsidation signal abolished the replication of DHBV. When the mutants replicated in hosts, more severe pathological changes were observed in ducks infected with mutant harboring a stop codon at the 3'-end. Data suggest that replicative-competent DHBV precore mutant can be more pathogenic than wild-type DHBV.

Animals↗

PubMed1997

SV40 large T antigen binds to the TBP-TAF(I) complex SL1 and coactivates ribosomal RNA transcription.

SV40 large T antigen is a multifunctional regulatory protein that plays a key role in the viral life cycle and can stimulate cell proliferation. To accomplish this, large T antigen has to control the expression of cellular genes involved in cell cycle progression and cell growth. rRNA synthesis by RNA polymerase I (Pol I) is tightly associated with cell growth and proliferation, and previous studies indicated that large T antigen up-regulates RNA Pol I transcription in SV40-infected cells. How this process occurs is currently unclear. To investigate the mechanisms of large T antigen stimulation of RNA Pol I transcription, we have established an in vitro transcription system that is responsive to large T antigen. Here, we show that recombinant large T antigen stimulates Pol I transcription reconstituted with purified RNA Pol I, UBF, and the TBP/TAF complex SL1. Immunoprecipitation experiments revealed that large T antigen directly binds to SL1, in vitro, as well as in SV40-infected cells. In addition, our data indicate that this interaction occurs by direct association with three SL1 subunits, namely TBP, TAF(I)48, and TAF(I)110. Transcription studies with large T antigen deletion mutants show that the 538-amino-acid amino-terminal domain is necessary for full stimulation of Pol I transcription. Importantly, mutants that no longer bind to SL1 are also unable to stimulate Pol I transcription. This indicates that recruitment of large T antigen to the rRNA promoter by SL1 constitutes a crucial step in the activation process. Taken together with recent studies on large T antigen activation of RNA Pol II transcription, these results suggest that viral modulation of genes involved in cell proliferation involves direct targeting of promoter-specific TBP/TAF complexes (i.e., SL1 or TFIID) by large T antigen.

Antigens, Polyomavirus Transforming↗

PubMed1997

[Expression and detection of integrin alpha 5 beta 1, fibronectin and its fragment in patients with hepatocellular carcinoma].

OBJECTIVE: To study the relationship between the expression of integrin alpha 5 beta 1, fibronectin (FN) and its fragment (malignant disease-associated DNA-binding protein 2 MAD2) and the biologic behavior of human hepatocellular carcinoma HCC) as well as the significance of MAD2 detection in plasma. METHODS: Integrin alpha 5 beta 1, FN and MAD2 were localized by immunohistochemical methods and quantified by image analysis in tumor and peripheral liver tissues from 40 HCC cases. The concentration of FN and MAD2 in the plasma samples from 30/40 cases were examined using the ELISA method. RESULTS: The expression of integrin alpha 5 beta 1, FN and MAD2 in well differentiated HCC were not significantly different from that of normal tissues, while they were markedly decreased or undetectable in mode rately and poorly differentiated HCC. Decreased expression of integrin alpha 5 beta 1 and FN at sites where HCC invaded the connective tissue and enhanced expression on the HCC cells around cancerous emboli were also noted. The mean concentration of plasma MAD2 in patients with HCC were significantly increased when compared to those with chronic liver disease and normal subjects. CONCLUSIONS: The changes in integrin alpha 5 beta 1 and FN expression may be associated with HCC differentiation, invasion and metastasis. The detection of plasma MAD2 in patients with HCC may have clinical significance.

Antigens, CD↗