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Within an isospin- and momentum-dependent hadronic transport model, it is shown that the recent FOPI data on the pi;{-}/pi;{+} ratio in central heavy-ion collisions at SIS/GSI energies [Willy Reisdorf, Nucl. Phys. A 781, 459 (2007)10.1016/j.nuclphysa.2006.10.085] provide circumstantial evidence suggesting a rather soft nuclear symmetry energy E_{sym}(rho) at rho> or =2rho_{0} compared to the Akmal-Pandharipande-Ravenhall prediction. Some astrophysical implications and the need for further experimental confirmations are discussed.

Objective: To evaluate the detection method of ELISA and Enhanced Chemiluminescence Immunoassay (ECLIA) in use to determine serum hyaluronate acid (HA), laminin (LN), type IV collagen (IV-C) and type III procollagen (PC III)., Methods: 253 patients with chronic hepatitis B were determined the four liver fibrosis serum markers with both the ECLIA and ELISA, and then compared with pathology results separately., Results: Both the detection results of ELISA and ECLIA can reflect that the patient's liver fibrosis from hepatitis to liver cirrhosis aggravated gradually. Compared with ELISA, the results of ECLIA and pathology have a better correlation., Conclusions: The detection of four liver fibrosis serum markers by ECLIA could indicate the better the response of the state of live fibrosis.

Objective: To explore the significance of Lens culinaris-reactive alpha-Fetoprotein (AFP-L3) detection in primary hepatocellular carcinoma., Methods: AFP-L3 was isolated by using microspin column coupled with lens culinaris agglutinin (LCA), AFP and AFP-L3 were detected with chemiluminescent immunoassay, the proportion of AFP L3 levels were calculated, and the relationship between the elevated AFP-L3 (%) levels and benign and malignant liver disease was analyzed., Results: There were significant differences in positive rate between the patients of HCC, suspected HCC and other liver disease (81.80%, 73.68%, 11.80%, respectively, P < 0.05). Among the undetermined HCC (suspected HCC, liver disease) patients, 12 out of 21 cases of AFP-13 positive were diagnosed to be HCC within 6 months, and 6 of them were diagnosed to be the single small HCC at the early stage through B-Ultrasonic Diagnosis or CT. Among 62 cases of AFP-L3 negative, 3 cases were diagnosed to be HCC within 6 months and the risk of occurrence of HCC for AFP-L3 positive increased 11.9 times., Conclusion: AFP-L3 has no correlation with AFP value, and it can be used as an independent HCC diagnosis factor. The detection of AFP-L3 has a significant implication for the identification of benign or malignant liver disease and the early stage predictive diagnosis of HCC while AFP increases.

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P<0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH.

Background: Severe acute respiratory syndrome (SARS) is a disease with a mortality of 9.56%. Although SARS is etiologically linked to a new coronavirus (SARS-CoV) and functional cell receptor has been identified, the pathogenesis of the virus infection is largely unclear., Methods: The clinical specimens were processed and analyzed using an indirect enzyme-linked immunosorbent assay (ELISA) in-house. Further investigations of target antigen included reviews of phage display technique, rapid amplification of cDNA ends (RACE) technique, protein expression and purification, Western blotting validation, serological and immunohistochemical staining in postmortem tissue., Results: A type of medium or low titer anti-lung tissue antibodies were found in the sera of SARS patients at the early stage of the disease. Human long interspersed nuclear element 1 (LINE1) gene endonuclease (EN) domain protein was one of the target autoantigens and it was aberrantly expressed in the lung tissue of SARS patients. Anti-EN antibody was positive in the sera of 40.9% of SARS patients., Conclusions: Human LINE1 endonuclease domain was identified as a putative target of SARS-associated autoantibodies, which were presented in the serum of SARS patients and may be involved in the pathogenesis of SARS.

Aim: To construct a prokaryotic expression vector containing human interleukin-10 (IL-10) gene, express and identify the protein., Methods: Human IL-10 gene from HepG2 was isolated and identified by DNA sequencing, and then cloned into the expression vector PET-28 a (+) to construct prokaryotic expression vector. The recombinant protein was expressed in BL21(DE3) and identified by Western blot and ELISA., Results: The expressed protein was mainly located in the inclusion body. The relative molecular mass of the expressed product was identical to that of prediction. The expressed protein had binding activity with specific antibody., Conclusion: The human IL-10 gene was successfully cloned and expressed in E.coli. The expressed product had antigenicity which provides foundation for preparation of monoclonal antibodies against IL-10.

Background: To explore the significance of hepatitis B virus PreS1-Ag, PreS2-Ag, large protein (LP) detection and the prediction of viral replication., Methods: PreS1-Ag, PreS2-Ag, LP and HBV markers were measured by enzyme linked immunosorbent assay (ELISA) in 201 cases of infected serum. Serum HBV DNA level was quantitatively detected by real-time polymerase chain reaction (PCR)., Results: There were significant differences in positive rate between the PreS1-Ag, PreS2-Ag, LP, and HBsAg; the positive rate of PreS2-Ag and LP were higher than that of the HBeAg. No significant differences were found in the positive rates between LP and the levels of HBV DNA and there was a positive correlation between quantitations of HBV DNA and HBV-LP., Conclusion: Serum PreS1-Ag, PreS2-Ag and LP were laboratory markers that can accurately reflect HBV DNA reproduction, and were helpful complementarity to traditional HBV M. There is a close correlation between the number of copies of HBV DNA and the levels of HBV-LP.

Background: To investigate the biological functions of a novel hepatitis B virus e antigen (HbeAg) interacting protein AK026018, and to use cDNA microarray technique to screen genes regulated by the protein., Methods: The AK026018 coding DNA fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) technique from HepG2 cell. The expressive vector of pcDNA3.1-AK was constructed by routine molecular biological methods. The HepG2 cells were transfected with pcDNA3.1 and pcDNA3.1-AK, respectively by using lipofectamine. The total RNA was isolated and reverse transcribed. The cDNA of each sample was subjected to microarray screening with 8,464 cDNA probes and analyzed by bioinformatics., Results: The expressive vector was constructed and confirmed by DNA sequencing analysis and restriction enzyme digestion. High quality mRNA and cDNA of transfected HepG2 cells had been prepared and successful microarray screening conducted. From the scanning results, there were 122 differential expression genes, of which 36 genes were down-regulated, and 16 genes were up-regulated., Conclusion: Microarray technique was successfully used to screen the genes trans-regulated by AK026018. The expression of AK026018 protein affects the expression spectrum of HepG2 cells.

Objective: To screen and clone the genes in hepatocytes which encode protein that can interact with hepatitis B e antigen(HBeAg) by yeast-two hybridization., Methods: Recombined HBeAg bait plasmid (pGBKT7-eAg) was transformed into yeast AH l09, followed by mating with yeast Yl87 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) which contains X-a-gal for selecting positive blue clones. Then positive clones were selected and plasmids were prepared and sequenced. Finally, bioinformatics analysis was performed., Results: Totally 245 positive colonies were selected and 101 colonies were sequenced. Through sequences alignment, 6 novel genes and 35 recorded genes were screened., Conclusion: Genes of HBeAg interacting proteins have been cloned successfully, which brings some new clues for further studies on the biological functions of HBeAg and the related proteins.

Objective: Test the cytokines secretion by peripheral blood mononuclear cells (PBMC) from hepatitis C patients after stimulation with highly variable region (HVR1) synthetic peptides., Methods: ELISA was used to test the cytokines secretion, flow cytometry to group the proliferated cells, and the antigenicity of synthetic peptide was predicted by the computer modeling., Results: (1)There were PBMC proliferation when stimulated by HVR1 antigen synthetic peptides. (2) There was a rise of IFN-gamma, IL-4 and IL-10. But no rise of IL-2 and TNF-gamma was found. (3) The proliferated cells were mainly CD4+ lymphocytes., Conclusion: PMBC from hepatitis C patients tend to secret Th2 cytokines after stimulation with HVR1 designed by the authors.

Objective: To explore whether autoimmune phenomena exist in SARS patients, and to seek for unusual autoimmune antibodies in SARS patients., Methods: Autoantibodies against cell nuclei (ANA), autoantibodies against smooth muscles (SMA), autoantibodies against parietal cells (PCA), autoantibodies against heart cells (HRA) were detected by using immunofluorescence, and autoantibodies against live-kidney microsomes (LKM) and anti-M2 antibodies were detected by ELISA in sera taken from 27 SARS patients and 18 healthy controls. Immunofluorescence was used to localize the targets antigens in slides with biochips of lung (monkey) of SARS associated antibodies., Results: ANA, AMA, LKM and SMA were found positive in 3, 1, 1, and 1 of the 27 SARS sera. In 18 healthy control sera, one ANA and one AMA were positive. Statistical analysis showed that there were no difference between two groups in every item detected. Twenty-six of 27 SARS patients and 5 of 18 healthy controls had strongly stained columnar epithelia of the bronchiole, especially the lumen border of the epithelia?the difference between two groups was significant., Conclusion: No antibodies against organs but lung were found in SARS patients. There are auto antibodies against lung tissues in sera of SARS patients.

With an isospin- and momentum-dependent transport model, we find that the degree of isospin diffusion in heavy-ion collisions at intermediate energies is affected by both the stiffness of the nuclear symmetry energy and the momentum dependence of the nucleon potential. Using a momentum dependence derived from the Gogny effective interaction, recent experimental data from NSCL-MSU on isospin diffusion are shown to be consistent with a nuclear symmetry energy given by E(sym)(rho) approximately 31.6(rho/rho(0))(1.05) at subnormal densities. This leads to a significantly constrained value of about -550 MeV for the isospin-dependent part of the isobaric incompressibility of isospin asymmetric nuclear matter.

Aim: To investigate the interaction between HBeAg and CD81., Methods: The CD81 gene was amplified by RT-PCR from HepG2 cells. The recombinant expression vector pGADT7-CD81 was constructed by routine molecular biological method. The auxotroph yeast cells were cotransfected with pGADT7-CD81 and pGBKT7-eAg and plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal. The binding of HBeAg to CD81 was also detected in vitro translation and coimmunoprecipitation test., Results: The recombinant expression vector was constructed and confirmed by restriction enzyme (EcoR I and BamH I) digestion and DNA sequencing analysis. The positive yeast clones could grow and from blue colonies on SD/-Trp-Leu-His-Ade/x-alpha-gal medium. The result of immunocoprecipitation showed that HBeAg could bind to CD81., Conclusion: HBeAg can bind to CD81, suggesting that CD81 plays an important role in the pathogenesis of HBV.

Signal regulatory protein (SIRP) alpha1 is a member of the SIRP family that undergoes tyrosine phosphorylation and binds SHP-2 tyrosine phosphatase in response to various mitogens. The expression levels of SIRPalpha1 were decreased in HCC tissues, compared with the matched normal tissues. Exogenous expression of wild type SIRPalpha1, but not of a mutant SIRPalpha1 lacking the tyrosine phosphorylation sites, in SIRPalpha1-negative Huh7 human HCC cells resulted in suppression of tumor cell growth both in vitro and in vivo. Treatment of Huh7 transfectants with EGF or HGF induced tyrosine phosphorylation of SIRPalpha1 and its association with SHP-2, which were accompanied by reduced ERK1 activation. Expression of SIRPalpha1 significantly suppressed activation of NF-kappaB and also sensitized Huh7 cells to TNFalpha or cisplatin-induced cell death. In addition, SIRPalpha1-transfected Huh7 cells displayed reduced cell migration and cell spreading in a fashion that was dependent on SIRPalpha1/SHP-2 complex formation. In conclusion, a negative regulatory effect of SIRPalpha1 on hepatocarcinogenesis is exerted, at least in part, through inhibition of ERK and NF-kappaB pathways., (Copyright 2004 American Association for the Study of Liver Diseases)

Background: Using hepatitis B virus e antigen (HBeAg) gene to construct the DNA-binding domain vector, which can express HBeAg in yeast cell, and can be used in yeast double hybrid as "bait plasmid" to look for the gene from the cDNA library, which expresses the protein that can interact with HBeAg., Methods: PCR was performed to amplify the HBeAg gene from a sera of hepatitis B patient. The product of the amplification was inserted into T-vector and was verified by sequencing. Then it was inserted into the "bait" plasmid pGBKT7 after the digestion with the restricted endonuclease of EcoR I and Sal I. The plasmid was transformed into the yeast cell. PCR was used to verify whether the plasmid was transformed into yeast. The HBeAg protein expressed in the cell was confirmed by Western blot. Using nutrition selection assay to verify the constructed plasmid alone could not activate the reporter gene in the yeast cell., Results: Sequenced and digested by two endonucleases, the recombined vectors pGBKT7-eAg produced anticipated fragment. PCR verified that there was HBeAg fragment in the yeast. Having assayed by Western blotting, it was shown that the yeast cell transformed with pGBKT7-eAg vector had positive signal which could not be seen in the control. Tested by the nutrition selection assay, the recombined vectors pGBKT7-eAg could not activate LacZ reporter gene in the yeast., Conclusion: DNA-binding domain plasmid was successfully constructed and could express HBeAg proteins in the yeast cell but could not activate transcription of LacZ reporter gene alone. The recombined plasmid can be used in yeast double hybrid.

Using an isospin-dependent transport model, we study the effects of nuclear symmetry energy on two-nucleon correlation functions in heavy-ion collisions induced by neutron-rich nuclei. We find that the density dependence of the nuclear symmetry energy affects significantly the nucleon emission times in these collisions, leading to larger values of two-nucleon correlation functions for a symmetry energy that has a stronger density dependence. Two-nucleon correlation functions are thus useful tools for extracting information about the nuclear symmetry energy from heavy-ion collisions.

High energy heavy-ion collisions are proposed as a novel means to constrain stringently the high density (HD) behavior of nuclear symmetry energy. Within an isospin-dependent hadronic transport model, it is shown for the first time that the isospin asymmetry of the HD nuclear matter formed in high energy heavy-ion collisions is uniquely determined by the HD behavior of the nuclear symmetry energy. Experimental signatures in two sensitive probes, i.e., pi(-) to pi(+) ratio and neutron-proton differential collective flow, are also investigated.

The neutron-proton differential flow is shown to be a very useful probe of the isospin-dependence of the nuclear equation of state (EOS). This novel approach utilizes constructively both the isospin fractionation and the nuclear collective flow as well as their sensitivities to the isospin-dependence of the nuclear EOS. It also avoids effectively uncertainties associated with other dynamical ingredients of heavy-ion reactions at intermediate energies.

AIM:To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS:The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using (32)P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS:Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively. The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC <5cm was 90% (9/10) and 83.3% (5/6), respectively.CONCLUSION:MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.

Seven healthy macaques were inoculated with the GBV-C/HGV-RNA serum from a non-A-E hepatitis patient. The serology and pathology of the liver in the animals were observed. The results indicated that all inoculated animals were infected with a GBV-C/HGV-RNA viremia and had mildly abnormal alanine transaminase levels during the infectious period. The histology, immuno-histochemistry, and in situ hybridization in the liver tissues of the inoculated animals also showed that there was a very mild hepatitis with the positive antigenic expression and the genome of GBV-C/HGV-NS5 in hepatocytes. The pathological changes in the infected animals appeared to become normal whether or not GBV-C/HGV-RNA viremia persisted. There is a possibility that the mild virulence of the GBV-C/HGV to the host became harmless with time after inoculation. Infection and the transmission of the GBV-C/HGV virus in the macaques provides an appropriate animal model and new information about GBV-C/HGV infection in both humans and animals. It is possible that this virus is a mild and self-limited pathogenic agent to the hepatic cells of primates., (Copyright 2000 Wiley-Liss, Inc.)

Five patients with systemic lupus erythematosus (SLE) were perfused through an extracorporeal shunt filled with DNA-immunoadsorbent (DNA immobilized on carbonized resin beads). High concentrations of anti-DNA antibodies (36.4-67.0%) (binding percentage with 125I-DNA) in the serum of SLE patients were reduced to 13.8-53.0%, respectively. The highest removal percentage was 62.1%. Although the decline levels varied, the symptoms of patients, i.e., long-term severe joint pain, severe edema, hydropericardium, and ascites were all relieved considerably. The immunoadsorbent showed satisfactory blood compatibility.