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A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin dependant kinase. Inhibitory activities against the enzyme ranged from 34nM to >20microM and were dependant upon both the nature of the aryl group and the hydrogen bond donating potential of the maleimide ring. Key interactions with the kinase ATP site and hinge region were found to be consistent with homology modeling predictions.

Objective: To investigate the possible antitumor mechanism of polysaccharide from medicinal fungus Penicillium jiangxiense., Methods: The cytotoxic effect was measured by MTT assay, and the cell cycle was analyzed by flow cytometry with Propidium iodide (PI) staining. To apoptotic detections, Hoechst 33258 staining for chromatin, annexin-V FITC/PI double staining for early phase cell apoptosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for late phase cell apoptosis., Results: Both MPPJ4 and MPPJ5, the fine polysaccharide fractions from P. jiangxiense, showed slight cytotoxic effects to inhibit human gastric adenocarcinoma SGC-7901 cells proliferation, but significantly caused cell cycle arrest at the S phase, and the rate of cell population at the subG1 phase was evidently increased. In apoptotic assays, MPPJ5 was more potent than MMPJ4 in inducing tumor cells in a time-dependent manner at the range from 0 to 72 hours, comparable to the negative control., Conclusion: The antitumor acting mechanism of P. jiangxiense polysaccharide is associated with the induction of apoptotic cell death and cell cycle arrest.

This paper described a rapid and sensitive HPLC method to analyze (E)-3,5,4'-trimethoxystilbene (BTM-0512) in rat plasma and tissues. The analysis used a BDS Hypersil C18 analytical column (250 mm x 4.6 mm ID, 5 microm) and acetonitrile/water as the mobile phase. The UV detection wavelength was 319 nm. Proteins were precipitated with acetonitrile and diethylstilbestrol as internal standard. The method was validated according to State Food and Drug Administration of China and ICH of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines. The limit of detection (S/N: 3/1) for BTM-0512 was 0.005 microg x mL(-1) for plasma. The method performances were shown to be selective for BTM-0512 and the linearity of the assay method was up to 10.0 microg x mL(-1) and 40.0 microg x g(-1) for plasma and tissues, respectively. At 0.1, 1 and 5 microg x mL(-1) (n=5), intraday and interday precision values (% RSD) were in the range of 2.6% - 5.1% and 2.4% - 4.8%, respectively. Mean accuracy and absolute recoveries of BTM-0512 ranged from 95.3% - 100.1% and 95.9% - 100.9% for plasma and tissues, respectively. This method can be quite useful for BTM-0512 pharmacokinetic and tissue distribution studies, for purpose which multiple plasma and tissue samples can be analyzed quickly with high reproducibility.

With spatial autocorrelation analysis, this paper studied the temporal and spatial variations of soil organic carbon (SOC) storage in Henan Province during the period from the 1st national soil survey (1958) to the 2nd national soil survey (1985). The results showed that spatial auto-correlation indices could better describe the spatiotemporal variation of the SOC storage between the two soil surveys. The total SOC storage was 54.93 x 10(8) t in 1958 and 69.65 x 10(8) t in 1985, with an annual increase of 1%, and the SOC density had a trend of higher in southeast and west Henan, followed by north and central Henan, and east Henan. The SOC storage per unit area decreased more quickly in southeast Henan, with the maximum of 1.57% per year, while increased greater in east Henan, with an average rate of 5.27% per year. The spatiotemporal variation of SOC storage had a close correlation with the original SOC content, i. e., the higher the original SOC content, the more greatly the SOC storage decreased.

Background: The pharmacokinetics of duloxetine hydrochloride have been well studied after its approval for clinical use. However, few such data have been reported in the English language literature. We developed a method to determine the pharmacokinetics of duloxetine enteric-coated capsules in healthy Chinese volunteers., Methods: A rapid and sensitive liquid chromatography-mass spectrometric (LC/MS) method for the determination of duloxetine in human plasma using flupentixol as the internal standard (I.S.) was developed and validated. Sample preparation of the plasma involved deproteination with acetonitrile twice, repeatedly. Samples were then analyzed by HPLC on a Thermo Hypersil-Hypurity C18 column (150 x 2.1 mm, 5 microm). A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at 298 m/z for duloxetine and at 435 m/z for the internal standard., Results: Pharmacokinetics were measured in 12 healthy Chinese male volunteers (6 males and 6 females) who received a single regimen with 3 different dosages at 22.4, 44.8 and 67.2 mg of duloxetine enteric-coated capsules., Conclusion: A sensitive and specific method for quantifying duloxetine levels in human plasma has been devised and successfully applied to a clinic pharmacokinetic study of an enteric-coated capsule of duloxetine hydrochloride administered as a single oral dose.

Perospirone is a novel atypical antipsychotic with a unique combination of 5-HT(1A) receptor agonism as well as 5-HT(2A) and D(2) receptor antagonism. A simple rapid and selective LC-MS method utilizing a single quadrupole mass spectrometer was developed and validated for the determination of perospirone hydrochloride in human plasma. N-hexane was used to extract perospirone hydrochloride and amlodipine benzenesulfonate (internal standard (IS)) from an alkaline plasma sample. LC separation was performed on a XTerra MS C(18) column (100mmx2.1mm, i.d. 3.5microm) using methanol -10mM ammonium acetate (84:16, v/v) as a mobile phase. The quantification of target compounds was obtained by using a selected ion monitoring (SIM) at m/z 427.5 [M+H](+) for perospirone hydrochloride, and at m/z 431.4 [M+Na](+) for IS (amlodipine benzenesulfonate). Perospirone and IS eluted as sharp, symmetrical peaks with retention times of 3.11+/-0.01min and 4.15+/-0.2min, respectively. Calibration curves of perospirone hydrochloride in human plasma at concentrations ranging from 0.10 to 21.1ng/mL exhibited excellent linearity (r(2)=0.9997). The mean absolute recovery of the drug from plasma was more than 85%. Intra- and inter-day relative standard deviations were less than 6.43% and 11.9% for perospirone hydrochloride at the range from 0.32 to 10.6ng/mL. Stability characteristics of the drug-containing plasma were thoroughly evaluated to establish appropriate conditions to process, store and prepare for chromatographic analysis without inducing significant chemical degradation. The following pharmacokinetic parameters were elucidated after administering a single dose of 8mg perospirone hydrochloride. The area under the plasma concentration versus time curve from time 0 to 24h (AUC(0-24)) was 15.48+/-4.23microg/Lh; peak plasma concentration (C(max)) was 2.79+/-0.78microg/L; time to C(max) (T(max)) was 1.79+/-0.45h; and elimination half-life (t(1/2)) 6.78+/-1.38h. The described assay method showed acceptable precision, accuracy, linearity, stability, and specificity and can be used for pharmacokinetic studies, therapeutic drug monitoring, and drug abuse screening.

Aim: This study was designed to investigate whether the activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is required for thermal preconditioning to protect rat cerebellar granule neurons (CGN) against apoptosis induced by low potassium, and to explore the possibility of a link between the upregulated heat shock protein (HSP)70 expression and Akt activation in the acquisition of neuroprotection induced by thermal preconditioning., Methods: CGN cultured for 8 d in vitro were switched to 5K medium for 24 h after thermal preconditioning (TP; 43.5 degree for 90 min, then 37 degree for 1 h). To study the role of the PI3-K/Akt pathway, a PI3-K inhibitor, LY294002 (20 micromol/L) was added into the cultures 1 h before TP. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay and fluorescein diacetate staining were used to determine cell viability. Hoechst 33258 staining and agar gel electrophoresis were used to test the morphological and biological characters of CGN. Western blot analysis was employed to detect the levels of phospho-Akt, phospho-glycogen synthase kinase 3beta (GSK3beta) Akt, GSK3beta, and HSP70., Results: TP protected CGN against apoptosis induced by low potassium. LY294002 inhibited the neuroprotective effect on CGN induced by TP. TP induced a robust activation of Akt and the inactivation of GSK3beta via PI3-K. Furthermore, the activation of the PI3-K/Akt pathway by TP persisted for 24 h in the 5K cultures. LY294002 (20 micromol/L) failed to inhibit the upregulated HSP70 expression induced by TP., Conclusion: The activation of the PI3-K/Akt pathway is required for TP to protect CGN against apoptosis induced by low potassium, but the neuroprotective effect by Akt activation is not mediated through the downstream induction of HSP70 expression.

Surface molecules are important biomarkers for cell proliferation and differentiation and play important roles in cell function and cell interaction. Notch is a transmembrane receptor that regulates developmental processes and cell-fate decision. Histamine is used as an adjunct to immunotherapy in myelogenous leukemia, and regulates hematopoietic cell development. Thus, we investigated the effects of histamine on immunophenotype and Notch signaling in human HL-60 leukemia cells. Histamine (0.1-10 microM) inhibited the colony-forming efficiency of HL-60 cells in a dose-dependent fashion and shifted the growth curve to the right. HL-60 cells were treated with histamine 0.1-1.0 microM for 6 days, and surface molecules were analyzed by flow cytometry. Histamine decreased CD49d positive cells by 74% while increasing CD31 positive cells by 53% as compared to controls. Histamine did not affect the expression of CD11b, CD14, CD34, CD44, CD54, CD49e, and CD62L. To examine Notch signaling in histamine-induced immunophenotype alterations in HL-60 cells, total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. The expressions of Notch1, Notch4, the ligands Jagged1, Delta4, and the downstream hairy enhancer of split 1 gene (HES1) were not significantly altered by histamine. In summary, this study demonstrated that histamine inhibited HL-60 cell growth and regulated immunophenotypes of CD49d and CD31. These effects are not mediated through the Notch signaling.

The aim of this study was to establish the standard protocols for isolating and enriching hematopoietic stem/progenitor cells (HSPC) from human placenta tissue (PT). Single-cell suspension from of human PT was prepared by mechanical method combined with collagenase digestion. Mononucleated cells (MNC) derived from PT were separated by hydroxyethyl starch (6% HES), then the three cell subsets of different immunophenotypes (CD34(-), CD34(+)CD38(-), CD34(+)CD38(+)) contained in MNC were isolated by Magnetic Activated Cell Sorting (MACS). The cell immunophenotype of each sorting steps was analyzed by flow cytometer (FCM). The cell enrichment and recovery rate of each sorting step were calculated. The results showed that MNC could be harvested up to (12.30 +/- 3.51) x 10(8) from a single-cell suspension of human PT by mechanical method and collagenase digestion, no significant difference existed as compared with umbilical cord blood (UCB) initial sample [(8.86 +/- 5.38) x 10(8)], but the percentage of CD34(+) cells in MNC of human PT was (3.93 +/- 2.31)%, much higher than that in UCB [(0.44 +/- 0.29)%] (P < 0.001). recovery rate of MNC and CD34(+) cells from PT after separation with 6% HES were (45.3 +/- 11.7)% and (51.1 +/- 9.8)%, respectively. After MNC being sorted by MACS, the enrichment and recovery rate of CD34(+) cells in CD34(+) group were (73.4 +/- 14.1)% and (52.7 +/- 11.7)% respectively. It is concluded that the protocols established here for isolating and enriching hematopoietic stem/progenitor cells from human placenta can acquire HSPC with high abundance, enrichment and viability and may be a useful reference of isolating methods for future related study.

The aim of this study was to evaluate the homing capabilities of hematopoietic stem/progenitor cells (HSPCs) derived from human placenta tissues (PT). Single cell suspension of human PT was prepared by mechanical method. The expression levels of homing-related adhesion molecules (HRAM) including CD11a, CD49d, CD44, CD49e, CD62L and CD54 on CD34(+) cells and the percentages of CD34(+) cells and their subpopulations in nucleated cells (NC) from fresh human PT, umbilical cord arterial blood (UCAB) and umbilical cord venous blood (UCVB) were detected by using flow cytometry. The results showed that the percentage of CD34(+) cells and CD34(+)CD38(-) cells in placenta were higher than those in UCAB and UCVB. There were no significant difference in percentage of HSPC between UCAB and UCVB. Placenta-derived CD34(+) cells strongly expressed CD11a, CD49d, CD44, CD49e and CD54, among which expression levels of CD49e and CD54 on placenta-derived CD34(+) cells were significantly higher than those on UCAB and UCVB-derived CD34(+) cells. While the percentage of CD34(+)CD62L(+) cells in placenta was only lower than that in UCVB. It is concluded that human placenta is rich in HSPC. Moreover, the expression levels of most HRAM in CD34(+) cells from PT are higher than those from UCAB and UCVB or are close to them. It suggested that HSPCs derived from PT might have stronger homing capabilities than those from UCB.

We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties and an aminocyclohexanecarboxylate residue at P2. This combination of binding elements resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors. Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone resorption IC50 after 24 h indicated a correlation between functional cellular and in vivo assays. Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action may be useful in osteoporosis and other indications involving bone resorption.

Aim: To determine whether insulin could promote sinusoidal endothelial cell (SEC) proliferation mediated by upregulation of vascular endothelial growth factor (VEGF) in regenerating rat liver after partial hepatectomy (PHx)., Methods: Adult male Sprague-Dawley rats undergoing 70% PHx were injected with insulin (300 MU/kg) or saline via the tail veins every 8 h after surgery for 7 d and killed at 0, 24, 48, 72, 96, 120, 144, and 168 h after surgery. Proliferation of both hepatocytes and SECs was monitored by evaluating the proliferating cell nuclear antigen (PCNA) labeling index (LI). The expression of VEGF protein was evaluated by immunohistochemistry. The mRNA expressions of VEGF and its receptors Flt-1 and Flk-1 were evaluated by semi-quantitative reverse transcription-PCR., Results: Insulin markedly increased the expression of VEGF mRNA between 24 and 120 h after hepatectomy compared to controls. Similarly, insulin significantly increased the expression of Flt-1 between 24 and 96 h. However, insulin had no significant effect on Flk-1. Furthermore, the immunohistochemical staining revealed that expression of VEGF protein increased in the insulin groups. Insulin significantly increased the PCNA LI of hepatocytes and SECs compared to controls., Conclusion: Exogenous insulin may promote SEC proliferation with an enhanced expression of VEGF and its receptor Flt-1 in regenerating rat liver after PHx.

Objective: To develop a new kind of portable ambulatory ECG monitor., Method: The hardware and software were designed based on RCA-CDP1802. New methods of ECG data compression and feature extraction of QRS complexes were applied to software design. A model for automatic arrhythmia analysis was established for real-time ambulatory ECG Data analysis. Compact, low power consumption and low cost were emphasized in the hardware design., Result: This compact and light-weight monitor with low power consumption and high intelligence was capable of real-time monitoring arrhythmia for more than 48 h. More than ten types of arrhythmia could be detected, only the compressed abnormal ECG data was recorded and could be transmitted to the host if required., Conclusion: The monitor meets the design requirements and can be used for ambulatory ECG monitoring.

Objective: To evaluate the specificity and sensitivity of immunodiagnosis with the antigen with molecular mass of 32 000 of Angiostrongyliasis cantonensis(AC32)., Methods: The major antigenic protein AC32 was purified from the antigens of A.cantonensis adult worm by electroelution from SDS-polyacrylamide gels. AC32-enzyme linked immunosorbent assay (ELISA) and adult worm antigen (AWA)-ELISA were used to detect the specific IgG in the sera of normal rats (n=5) and rats with angiostrongyliasis (n=61), sera of healthy individuals (n=50) and patients (n=50) with angiostrongyliasis, schistosomiasis and other parasitosis., Results: The positivity rate in AC32-ELISA of the sera from rats with angiostrongyliasis was 100%; without false positive results or cross reaction between AC32 and the sera of the normal control and patients infected with parasites other than A.cantonensis., Conclusion: AC32 of A.cantonensis is a valuable candidate antigen for immunodiagnosis of angiostrongyliasis with high sensitivity and specificity.

Background: A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj)., Methods: A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients., Results: Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07 x 10(6) individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%., Conclusions: The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

Aim: To analyze the structure and expressions of the protein encoded by an HCC-associated novel gene, lysosome-associated protein transmembrane 4 beta (LAPTM4B)., Methods: Primary structure and fundamental characteristics of LAPTM4B protein were analysed with bioinformatics. Expressions of LAPTM4B in HCC tissues and various cell lines were detected using polyclonal antibodies and Western blot., Results: LAPTM4B encoded two isoforms of proteins with molecular masses 35-ku and 24-ku, respectively. The expression level of LAPTM4B-35 protein in HCC tissues was dramatically upregulated and related to the differentiation status of HCC tissues, and it was also high in some cancer cell lines. Computer analysis showed LAPTM4B was an integral membrane protein with four transmembrane domains. LAPTM4B showed relatively high homology to LAPTM4A and LAPTM5 in various species., Conclusion: LAPTM4B gene encoded two isoforms of tetratransmembrane proteins, LAPTM4B-35 and LAPTM4B-24. The expression of LAPTM4B-35 protein is upregulated and associated with poor differentiation in human HCC tissues, and also at high levels in some cancer cell lines. LAPTM4B is an original and conserved protein.

Objective: To assess the intestinal barrier function of patients with advanced digestive tract malignant tumors by measuring the changes in intestinal permeability., Methods: Sixteen patients with digestive tract tumors and 16 healthy volunteers were enrolled. After oral administration of mannitol and lactulose in deionized water, the 6-hour urine samples were collected for analyzing mannitol and lactulose concentrations using gas-liquid chromatography., Results: Urine lactulose concentration and lactulose/mannitol (L/M) ratio of the tumor patients were increased in comparison with those of the healthy subjects (P<0.05)., Conclusion: he intestinal permeability increased in these tumor patients, indicating intestinal barrier dysfunction.

In the present work, sensitized near-infrared fluorescence from Yb3+-chrome blue-black R (CBR) complex was studied. It was found that Yb3+ forms 1:2 complex with CBR in natural ethanol solution. The visible fluorescence from CBR was quenched when the complex forms, while the near-infrared fluorescence from Yb3+ was greatly enhanced. A mechanism in which CBR absorbs excitation light and transfers its energy to Yb3+ ions was suggested. Near-infrared fluorescence intensity of Yb3+ is proportional to its concentration under the experimental condition, and was not disturbed by the addition of a small amount of other lanthanide ions, which indicate that near-infrared fluorometric analysis should be promising in the determination of single langthanide ions.

Objective: To study whether human placenta contains hematopoietic stem/progenitor cells (HSPCs), and analyze phenotypes of lymphocyte subpopulations in the placenta., Methods: Nucleated cells from fresh human placenta were analyzed for phenotypes of HSPCs and lymphocyte subpopulations by flow cytometry (FCM). And CD(34)(+) cells were sorted from human placenta nucleated cells by FCM or MiniMACS., Results: (1) CD(34)(+) cells, CD(34)(+)/CD(38)(+) cells, and CD(34)(+)/CD(38)(-) cells from a human placenta were 8.8, 4.6 and 11.9 times higher than those from umbilical cord blood (UCB), respectively. (2) The yields and purity of CD(34)(+) cells isolated from human placenta by FCM sorting system were (63.05 +/- 10.14)% and (86.39 +/- 11.27)%, respectively. (3) Lymphocytes, T cells (CD(3)(+)/CD(2)(+)), B cells (CD(19)(+)), Th cells (CD(3)(+), CD(4)(+)), and Th/Ts ratio in the placenta tissue were apparently lower than those in the UCB, while the CD(8)(+)/CD(28)(-) T suppressor cells were higher in the placenta than in the UCB., Conclusions: Human placenta is rich in HSPCs, and has important hematopoietic function in ontogeny. It is probable that human placenta would be graft resource for HSPCs transplantation. CD(8)(+)/CD(28)(-) T suppressor cells might play an important role in feto-maternal immunologic tolerance.

Aim: To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect., Methods: By ConA-sepharose affinity chromatography, ADP-agarose affinity chromatography, and DEAE anion exchange chromatography, we were able to purify HSP70-associated peptides from mouse hepatoma (HCaF) cells treated in heat shock at 42 degrees. Specific active immunization and adoptive cellular immunization assay were adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes isolated from HcaF., Results: The finally purified HSP-associated peptides had a very high purity and specificity found by SDS-PAGE and Western blot. Mice immunized with HSP70-associated peptide complexes purified from HCaF cells were protected from HCaF living cell challenge. This effect was dose dependent. Adoptive immunization of immune spleen cells of mice immunized with HSP70-associated peptide complexes could elicit immunity against HCaF challenge, and the tumor-free mice could resist repeated challenges. This effect could be continuously enhanced by repeated challenge with HCaF living cells. The tumor-free mice could tolerate the challenge for as high as 1 x 10(7) HCaF cells. The mice immunized once with spleen cells pulsed with HSP70-associated peptide complexes in vitro could also result in a certain adoptive immunity against HCaF., Conclusion: High purity and specificity of HSP70-associated peptides could be achieved from tumor cells by the low-pressure affinity chromatography method used in this study. HSP70-associated peptide complexes derived from the HCaF can elicit non-MHC-I molecule restrictive immunoprotective effect against HCaF. This effect can be transferred by adoptive immunization to mice and enhanced by repeated challenge with HCaF live cells.

Aim: To investigate the prevention and therapy of fungal infection in patients with severe acute pancreatitis (SAP)., Methods: Seventy patients with SAP admitted from Jan. 1998 to Dec. 2002 were randomly divided into garlicin prevention group, fluconazole (low dosage) prevention group and control group. The incidence of fungal infection, the fungal clearance and mortality after treatment were compared., Results: The incidence of fungal infection in garlicin group and fluconazole group was lower than that in control group (16% vs 30%, P<0.05 and 9% vs 30%, P<0.01, respectively). Amphotericin B or therapy-dose fluconazole had effects on patients with fungal infection in garlicin group and control group, but had no effects on patients with fungal infection in fluconzole group., Conclusion: Prophylactic dosage of antifungal agents (garlicin or low dosage fluconazole) can reduce the incidence of fungal infection in patients with SAP. But once fungal infection occurs, amphotericin B should be used as early as possible if fluconazole is not effective.

Lysosomal-associated protein transmembrane-4 beta (LAPTM4B), a novel gene upregulated in hepatocellular carcinoma (HCC), was cloned using fluorescence differential display, RACE, and RT-PCR. It contains seven exons and encodes a 35-kDa protein with four putative transmembrane regions. Both the N- and C-termini of the protein are proline-rich, and may serve as potential ligands for the SH3 domain. Immunohistochemical analysis localized the protein predominantly to intracellular membranes. Northern blot showed that the LAPTM4B mRNAs were remarkably upregulated in HCC (87.3%) and correlated inversely with differentiation status. LAPTM4B was also overexpressed in many HCC-derived cell lines. It was also highly expressed in fetal livers and certain adult normal tissues including the heart, skeletal muscle, testis, and ovary. Promoter function assays showed a distinct difference in the gene's activities between BEL7402 and HLE cell lines, suggesting that the transcription factors responsible for regulation of the gene in the two cell lines are different, and that possible negative regulatory cis-elements may exist upstream of the promoter region. It was demonstrated that the N-terminus of LAPTM4B was essential for survival of the cells. Cells harboring the full-length LAPTM4B cDNA expression clone displayed a slightly increased efficiency in colony formation. These results suggest that LAPTM4B is a potential protooncogene, whose overexpression is involved in carcinogenesis and progression of HCC. In normal cells, it may also play important roles such as regulation of cell proliferation and survival.

Objective: To obtain single chain variable fragment(ScFv) against the circulating antigen(CAg) from Schistosoma japonicum(Sj)., Methods: Metabolic antigen of adult worm of Sj (Sj-MAg) was used in the panning of phage library against Schistosoma japonicum. The activity of Sj-MAg-binding phage clones was assayed by ELISA. The specificity of expression products of the positive clones was analyzed by ELISA, SDS-PAGE and Western blotting., Results: Seventy-two randomly selected clones were tested for the presence of anti-Sj-MAg ScFvs, 6 clones showed positive. The specificity of these 6 clones was confirmed by binding them to antigens of other four trematodes. Two clones (B04, C24) were found to bind to Sj-MAg but not to any of the antigens of other four trematodes and their expression products were about 31 kDa in size., Conclusion: ScFv antibodies against the circulating antigens from Schistosoma japonicum Sj-MAg can be selected and manufactured from the antibody library.

The "Taigu" genic sterility gene Ms2 located on the short arm of the 4D chromosome of common wheat (AABBDD) originally incorporated into hexoploid triticale (AABBRR) and durum wheat (AABB) through intergenomic translocation in distant hybridization was introduced back into the genomes of common wheat. The dominant male sterility was expressed normally in the new "Taigu" genic sterile wheat carrying the intergenomically translocated Ms2, and the female fertility mechanism in its male sterile plants was normal as well. Observation of the chromosome configuration at meiosis of pollen mother cells (PMC) of the young ears of the sterile plants showed that they were euploid plants (2n = 42). No configurations different from those of the "Taigu" genic sterility gene located at the original locus were noticed of the Ms2 intergenomically translocated back into the common wheat. In systematic test crosses with marker genes the intergenomically translocated gene Ms2 was found to be linked with the dominant dwarf marker in common wheat Rht3 and, consequently, remapped and located on the short arm of the 4B chromosome of common wheat with a distance of 9.7 cM from Rht3. The new locus was designated as Ms2 (4BS). Discussions are given of the fate of Ms2 during translocation in the hexoploid triticale, the exchange of the names for 4A and 4B chromosomes in common wheat and the possible exploitation of the new locus Ms2 (4BS), and the following speculations are made: (a) In genic genes of allopolyploid organisms the donor chromosomes tend to be intergenomically translocated to their physiologically and evolutionarily close chromosomes with the same order number and the same arm; (b) it is confirmed that the 7th International Conference of Wheat Genetics was right to exchange the names between chromosomes 4A and 4B of common wheat in 1988; and (c) as a new genetic marker and a breeding tool for all the chromosome B-carrying species in the tribe of Triticeae, Ms2(4BS) may have wide application in building and expanding the gene pool of germplasm resources of various species of wheat.

AIM:To observe the changes in oxygen free radical (OFR) and the curative effect of traditional Chinese medicine Qing Yi Tang in acute necrotizing pancreatitis (ANP).METHODS:After induction of ANP by injection of sodium taurocholate into pancreatic duct, 16 dogs were randomly divided into control group and Chinese medicine group.Serum amylase, SOD and MDA were determined on postoperative day 1, 2, 4 and 7. The animals were sacrificed on day 7. SOD and MDA in organs were determined, and pathological changes in pancreas were observed.RESULTS: As compared with control group, the serum level of amylase (734U/L vs 2783U/L) and MDA (7.8nmol/ml vs 14.8nmol/ml) in Chinese medicine group were decreased on day 7 (P < 0.05), while SOD increased significantly (281nU/ml vs 55nU/ml, P < 0.01), and similar changes occurred in MDA and SOD in organs, especially in the pancreas; the pathological changes in the pancreas were alleviated as well.CONCLUSION: Qing Yi Tang is effective in clearing OFRs and alleviating pathological changes in ANP.

AIM:To investigate the potential role of intestinal microflora barrier in the pathogenesis of pancreatic infection.METHODS:Fifteen dogs were colonized with a strain of E.coli JM109 bearing ampicillin resistance plasmid PUC18.The animals were divided into two groups. In experimental group (n = 8), acute necrotizing pancreatitis (ANP) was induced by injection of 0.5ml/kg of sodium tarocholate with 3000U/kg trypsin into the pancreatic duct. The control group (n = 7) underwent laparotomy only. All animals were sacrificed 7 days later. Mucosal and luminal microflora of intestine were analyzed quantitatively, and various organs were harvested for culturing, blood samples were obtained for determination of serum amylase activities and plasma lipopolysaccharide (LPS) concentrations.RESULTS:In the experimental group, the number of E. coli in the intestine was much higher than those of the controls, while bifidobacterium and lactobacillus were decreased significantly (Jejunum, 1.75 ± 0.95 vs 2.35 ± 0.79,P <0.05; 1.13 ± 0.8 vs 1.83 ± 0.64,P <0.05; ileum, 2.89 ± 0.86 vs 3.87 ± 1.05,P <0.05; 1.78 ± 0.79 vs 3.79 ± 1.11,P <0.01;cecum, 2.70 ± 0.88 vs 4.89 ± 0.87,P <001; 2.81 ± 0.73 vs 3.24 ± 0.84,P <0.05. Content of Cecum, 3.06 ± 0.87 vs 5.15 ± 1.44,P <0.01; 2.67 ± 0.61 vs 4.25 ± 0.81,P <0.01), resulting in reversal of bifido-bacterium/E. coli ratio as compared with the control group (jejunum,0.51 ± 0.76 vs 1.23 ± 0.53, P <0.05; ileum, 0.62 ± 0.68 vs 1.16 ± 0.32,P <0.05; cecum,0.46 ± 0.44 vs 1.03 ± 0.64, P < 0.05). In addition, intestinal bacteria were isolated from organs of all animals in the experimental group, and JM109 was also detected in most cases. Positive blood culture was 75.0% and 62.5% on day 1 and 2 after induction of ANP, respectively, but no bacterium was found in the controls. As compared with the control group, blood LPS levels and serum amylase activities increased 1-3 times and 3-8 times respectively.CONCLUSION: Microecological disturbance could occur in ANP, and overgrowth of intestinal gram-negative bacteria may lead to translocation to the pancreas and other organs, becoming the source of pancreatic and peripancreatic infection.