The only difference of primary structure between single-chain prourokinase (pro-UK or scu-PA) and two-chain urokinase (UK or tcu-PA) is the cleavage of a single peptide bond (Lys158-Ile159) and transform scu-PA into its active two-chain form. A 13-peptide (Thr-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Cys), which spans the cleavage peptide bond, was synthesized and linked to KLH (Keyhole limpet hemocyanin). The Balb/c mice were immunized by the conjugated protein with proper adjuvant. According to the Kohler and Milstein's methods, a hybridoma cell line G7 secreting monoclonal antibody specific for scu-PA was obtained. The anti-scu-PA McAb, purified from the supernatant of porous microcarrier hybridoma cell culture, was conjugated to CNBr-activated Sepharose 4B to prepare an immuno-affinity chromatography column. The u-PA was purified only by this affinity column from the supernatant of cultivating the u-PA-producing recombinant CHO cell, the u-PA recovery ratio is 90.4%, the purification factor was about 50, with the specific activity of 1.2 x 10(5) IU/mg, the scu-PA ratio in the u-PA product was 96.3%. Compared to immuno-affinity chromatography, the 3-step process for purifying u-PA (cation-exchange column, gel filtration column and benzamidine affinity column) has a u-PA recovery ratio of about 65%, with a specific activity of 1.0 x 10(5) IU/mg, and an scu-PA ratio of about 90%. These results showed that immuno-affinity chromatography is simple to recover u-PA and effective to separate scu-PA from tcu-PA.
Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; immunology ; isolation & purification ; Urokinase-Type Plasminogen Activator ; immunology ; isolation & purification

Animals ; Carcinoma, Hepatocellular ; blood supply ; virology ; Cell Transformation, Neoplastic ; Endothelial Growth Factors ; biosynthesis ; genetics ; Gene Expression Regulation, Viral ; Hepatitis B virus ; pathogenicity ; physiology ; Humans ; Liver Neoplasms ; blood supply ; virology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Trans-Activators ; physiology

This study was purposed to investigate the molecular basis for RhD negative phenotype in Yiwu population in Zhejiang Province of China. The RhD negative samples were screened by saline agglutination test in blood donors. Some real RhD negative and RhDel phenotypes were identified using anti-human globulin test and absorbtion elution test. Ten exons of RHD gene in these samples were amplified by PCR-SSP, and positive exons were DNA sequenced. The results indicated that 30 real RhD negative and 8 RhDel phenotypes were identified in 38 initial RhD negative samples. Ten exons were complete negative in 28 real RhD negative samples and only exon 1, 2 and 10 were positive in 2 real RhD negative samples amplified by PCR. All 10 exons in 8 RbDel samples were positive and a DNA variant (1227G > A) was found in 8 RhDel samples. It is concluded that all exons are absence in most real RhD negative phenotypes, and the partial exons absence is also found in some real negative phenotypes among Yiwu population in Zhejiang province of China. The G to A mutation at position 1227 is found in all RhDel phenotypes.
Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Exons ; Genotype ; Humans ; Molecular Sequence Data ; Phenotype ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; genetics ; immunology

OBJECTIVETo investigate the effects of HBx gene on proliferation activity of hepatoma cells in vitro and in vivo.

METHODSThe plasmid pHA-HBx carrying HBx gene was transfected into HepG(2) cells, and the positive clones were screened and identified with G418 and RT-PCR, respectively. The growth curve and population doubling time were calculated, and the cell cycle was analyzed by flow cytometry (FCM). The proliferation activity of transformed cells was measured with (3)H-TdR incorporation rate and nude mice model in vitro and in vivo.

RESULTSThe result of RT-PCR indicated that HBx gene was integrated into the genome DNA of HepG(2) cells and transcripted. The growth curve and population doubling time showed a high proliferation activity of transformed cells. The amount of cells at stage S and G(2)/M were significantly higher, and cells at stage G(0)/G(1) were lower than those in control group. The tumors developed from transfected cells grew much quicker than those developed from HepG(2) cells in nude mice model.

CONCLUSIONHBx gene can facilitate the proliferation of hepatoma cells both in vitro and in vivo.

Animals ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Cycle ; genetics ; Cell Division ; genetics ; Cell Line, Tumor ; Female ; Flow Cytometry ; Humans ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; genetics ; Transfection ; Transplantation, Heterologous

OBJECTIVETo study the anti-endotoxin effect of beta-1, 2, 3, 4, 6-penta-O-galloyl-D-glucopyranose (PGG) in vitro.

METHODSThe affinity of PGG with lipid A was determined with biosensor technology, and the endotoxin-neutralizing effect was assayed with LAL. Human peripheral blood mononuclear cells (hPBMC) were separated from healthy donors and cultured in vitro. The effect of different concentrations of PGG on the release of TNF-alpha and hIL-6 from LPS-stimulated hPBMC were measured by ELISA method.

RESULTSLipid A was combined with different concentrations of PGG. The combination speed was shortened with the increase in PGG concentration. The KD value between PGG and Lipid A was 5.2 x 10(-7) mol/L. The release of TNF-alpha and IL-6 of hPBMC under LPS stimulation (958 +/- 234 ng/L vs 1 351 +/- 99 ng/L) was obviously inhibited by PGG in the concentration of higher than 20 mg/L compared with that without PGG treatment (1 788 +/- 171 ng/L vs 1 965 +/- 232 ng/L, P < 0.05).

CONCLUSIONPGG show an anti-endotoxin effect in vitro, which may be associated with its ability to combine and neutralize endotoxin.

Biosensing Techniques ; Cells, Cultured ; Dose-Response Relationship, Drug ; Drug Antagonism ; Endotoxins ; pharmacology ; Humans ; Hydrolyzable Tannins ; pharmacology ; In Vitro Techniques ; Interleukin-6 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Lipid A ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo evaluate the significance of the expression of matrix metalloproteinases (MMPs) in prostate cancer (PCa) and its clinical association.

METHODSFifty one cases of PCa and 10 cases of BPH were studied by immunohistochemical method using monoclonal antibodies to MMP2 and MMP9.

RESULTSThere was significant correlation between MMP2 or MMP9 and pathological grade, Gleason score and PCa metastasis.

CONCLUSIONThe expression of MMP2 and MMP9 may play an important role in the development and metastasis of PCa.

Aged ; Humans ; Immunohistochemistry ; Male ; Matrix Metalloproteinase 2 ; biosynthesis ; Matrix Metalloproteinase 9 ; biosynthesis ; Middle Aged ; Neoplasm Metastasis ; Prostatic Neoplasms ; metabolism ; pathology

OBJECTIVETo compare the in vitro inhibitory effect of expolysaccharides from Streptomyces, polysaccharides of Ganoderma lucidum and rice bran on six-alpha-helix bundle formation of HIV gp41 protein.

METHODSThe amount of six-alpha-helix bundle formed in the presence of N36 and C34 was tested by ELISA in response to treatments with different doses of polysaccharides.

RESULTSExpolysaccharides from Streptomyces potentially inhibited six-alpha-helix bundle formation with the effective concentration (IC(50)) of 145.48-/+7.25 mg /L. Polysaccharides of Ganoderma lucidum and rice bran showed no effect on the six-alpha-helix bundle formation.

CONCLUSIONExpolysaccharides from Streptomyces can inhibit the six-alpha-helix bundle formation of HIV gp41, whereas polysaccharides of Ganoderma lucidum and rice bran do not exhibit such activity.

HIV Envelope Protein gp41 ; chemistry ; Kinetics ; Oryza ; chemistry ; Polysaccharides ; pharmacology ; Protein Structure, Secondary ; drug effects ; Reishi ; chemistry ; Streptomyces ; chemistry

OBJECTIVETo carry out epidemiological study on an outbreak caused by E. coli O157:H7 infection in Jiangsu province in 1999.

METHODSEpidemiological, microbiological and moleculebiological methods were used to find out the source, route of transmission and risk factors.

RESULTS95 severe O157:H7 infected patients with acute renal failure in 9 counties and districts of 2 municipalities were reported in Jiangsu province, 1999 while 83 of the patients died with a death rate of 87.37%. Most patients were seen in mid or late June. The ratio of male to female was 1 to 1.44 and 88.42% of the patients were over 50 years old. 38 patients occurred in 2000 with 34 deaths. Major factors contributing to the outbreak would include without drinking tap water, eating leftover food, poor sanitary status in kitchen, not washing hands before meal and after bowl movement. 2 strain of O157:H7 was isolated from severe patients and 3 from diarrhea cases. Carrier rate among animals was up to 9.62% and 99.41% of the strains carried toxic gene. Strains isolated from feces of patients and animals belonged to the same colonies.

CONCLUSIONThis outbreak was severe which caused by O157:H7 and was first seen in China, which was closely related to the high carrier rate of O157:H7 in animals and to the positive rate of high toxic gene of the strains. There were various routes of transmission and the main factors of infection would include poor personal health habits and poor sanitation of the household.

Acute Kidney Injury ; epidemiology ; etiology ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Bacterial ; immunology ; Case-Control Studies ; Child ; Child, Preschool ; China ; epidemiology ; Diarrhea ; epidemiology ; microbiology ; Disease Outbreaks ; Escherichia coli Infections ; complications ; epidemiology ; Escherichia coli O157 ; isolation & purification ; Escherichia coli Proteins ; immunology ; Female ; Hemolysin Proteins ; immunology ; Humans ; Infant ; Male ; Middle Aged ; Seroepidemiologic Studies

OBJECTIVETo find out the differentially expressed genes in the hippocampus of the rats with genetic epilepsy so as to lay a foundation for exploring the pathogenesis of epilepsy by means of cDNA array technology.

METHODSGene expression patterns in the hippocampus of the genetic epilepsy-prone P77PMC rats and normal Wistar rats were established using the alpha-32P-labeled cDNA probes hybridized with the Atlas Rat cDNA Expression Array, and then were analyzed by an image analysis instrument to get the differentially expressed genes.

RESULTSFifteen genes were found having differential expression patterns in hippocampus between the P77PMC rats and the Wistar rats, while there may be many other differentially expressed genes left undiscovered due to having no appropriate image analysis software. And among the fifteen genes, the expression levels of twelve genes in the P77PMC rats were higher than those in the Wistar rats, while the expression levels of the other three genes were lower. The results of reverse transcription-polymerase chain reaction(RT-PCR) have demonstrated the reliability of cDNA arrays method.

CONCLUSIONcDNA array is a powerful tool for identifying differential expression genes of epilepsy on large scales. There are several differentially expressed genes in hippocampus of the P77PMC rats and the Wistar rats. All these identified genes could play potentially important roles in the pathogenesis of epilepsy.

Animals ; Calmodulin ; biosynthesis ; genetics ; DNA, Complementary ; genetics ; metabolism ; Epilepsy ; genetics ; Gene Expression Profiling ; Genetic Predisposition to Disease ; Hippocampus ; chemistry ; metabolism ; Oligonucleotide Array Sequence Analysis ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the application of biosensor technology in the determination of endotoxin-neutralizing materials.

METHODSAfter mixing polymyxin B (PMB) with endotoxin in certain concentration, the neutralizing ratio of PMB to endotoxin was assessed by biosensor technique and limulus amebocyte lysate test respectively. The results from the two methods were compared.

RESULTSThe neutralizing ratio of PMB to endotoxin as assessed by biosensor technology was 0.35 microg to 1 ng, while that by dynamic turbidimetric and chromogenic limulus amebocyte lysate (LAL) technique was 0.5 mg to 1 ng and 1 mg to 1 ng, respectively. The results obtained by biotechnology were similar to that by biosensor technique.

CONCLUSIONBiosensor technology was an accurate, convenient and rapid method for the determination of potency of endotoxin-neutralizing materials.

Bacterial Proteins ; analysis ; Biosensing Techniques ; methods ; Endotoxins ; analysis ; Lipid A ; analysis ; Polymyxin B ; analysis ; Reproducibility of Results ; Sensitivity and Specificity