The effects of sulfated polysaccharides from brown seaweeds GS201 on neuronal survival of cultured brain neurons were investigated in this paper.Results indicated that GS201 at concentrations of 0.01 0.1 1 10 mg?L -1 significantly enhanced the neuronal survival of both hippocampus and neurocortex.The mechanisms underlying the neurotrophic effect exerted by GS201 need to be further elucidated.

OBJECTIVETo investigate the expression of cyclooxygenase-2 (COX-2) in bladder transitional cell carcinoma tissues, and understand its clinical significance.

METHODSReversal transcription polymerase chain reaction (RT-PCR) was used to assess the expression of COX-2 mRNA in 52 cases of bladder transitional cell carcinoma tissues and 17 cases of normal bladder tissues far from neoplasm; Western blot was used to assess the expression of COX-2 protein in 49 cases of bladder cancerous tissues and 17 cases of normal tissues.

RESULTSPositive expression of COX-2 mRNA was detected in 83% (43/52) of bladder cancer tissues and in 29% (5/17) of normal tissues by RT-PCR and there was significant difference in expression of COX-2 mRNA between cancer tissues and normal tissues. Western blot analysis showed that expression of COX-2 protein was correlation with the stage and grade of cancer.

CONCLUSIONCOX-2 is overexpressed in bladder transitional cell carcinoma. COX-2 maybe play a certain role in carcinogenesis and progression of bladder cancer and turn into a useful target of chemoprevention of bladder cancer.

Adult ; Aged ; Aged, 80 and over ; Blotting, Western ; Carcinoma, Transitional Cell ; enzymology ; pathology ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Urinary Bladder ; enzymology ; Urinary Bladder Neoplasms ; enzymology ; pathology

OBJECTIVEThe current available assays for HIV subtyping, such as sequence-based phylogenetic analysis or heteroduplex mobility assay (HMA), are labor-intensive and time-consuming. The authors have just developed a simple and rapid subtype-screening assay for subtypes B, C, and CRF01-AE using a single nested multiplex PCR.

METHODSProviral DNA from HIV-positive samples was extracted and subjected to first round PCR with universal primers for gag region that can detect HIV-1 M group isolates. In the second round PCR, three pairs of subtype-specific primers, respectively detecting subtype B, C and CRF01-AE, were added into one tube. The PCR products of different subtypes could be distinguished in agarose-gel electrophoresis. Another pair of primers exclusively detecting the prevalent recombinant B/C strains CRF07-BC and CRF08-BC were designed and used. Additionally, all of these samples were sequenced and analyzed phylogenetically.

RESULTSPhylogenetic analysis showed that out of 119 samples, there were 43 subtype B samples (Euro-American B 11, Thailand B 32), 54 subtype C, 17 CRF01-AE, 3 subtype A and 2 subtype D samples. The subtype B, C, and CRF01-AE specific primer sets detected 35 (81.4%), 46 (85.2%), and 13(76.5%) samples with accuracy and specificity. Non-specific bands occasionally appeared but did not interfere with interpretation of the results. The primer pairs for CRF07-BC and CRF08-BC amplified target sequences were confirmed by sequencing and phylogenetic analysis. The specificity of all these subtype-specific primers was found to be 100%.

CONCLUSIONA simple and rapid assay was developed for screening subtypes B, C, CRF01-AE, CRF07-BC and CRF08-BC in China. This assay may have potential application in HIV laboratories in China and worldwide.

Acquired Immunodeficiency Syndrome ; virology ; Base Sequence ; China ; DNA Primers ; HIV-1 ; classification ; genetics ; isolation & purification ; Humans ; Phylogeny ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Animals ; Ginsenosides ; pharmacology ; Hypertension, Pulmonary ; etiology ; metabolism ; Hypoxia ; complications ; metabolism ; Lung ; drug effects ; metabolism ; MAP Kinase Signaling System ; Male ; Panax ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo establish a new function method for the analysis of a-fetoprotein (AFP) and beta-hCG in testicular tumors.

METHODSWe reexamined the serum levels of AFP and beta-hCG after radical orchiectomy, and calculated the measured coordinate, with the abscissa representing the number of the half-lives of tumor markers, and the ordinate representing the measured value of tumor markers. Referring to the measured value of tumor markers before surgery as a, the number of half-lives as x, and their theoretical value over a period of x elimination half-lives as y (logarithm to the base 2 of y), we calculated the predicted coordinate according to the formula y = log2(a/2x) ==> x + y = log2a (function 1). Then we assessed tumor residue and metastasis by analyzing the relationship between the measured and predicted coordinates.

RESULTSThe pathological examination of case 1 revealed a germ cell tumor of a mixed histological pattern of syncytiotrophoblast and yolk sac tumor. The measured coordinates of AFP and beta-hCG were (2.22, 6.21) and (10, 8.38), and the predicted coordinates (2.22, 6.34) and (10, 4.41) , indicating the elimination of the yolk sac tumor and metastasis of the syncytiotrophoblast tumor. Case 2 demonstrated the mixed pathological nature of teratocarcinoma and yolk sac tumor. The measured coordinates of AFP and beta-hCG were (2.67, -1.03) and (12, -3.32), and the predicted coordinates (2.67, 1.41) and (12, -5.80). But the review times of AFP and beta-hCG were out of the effective range of half-lives, with the measured values below the normal, which suggested no tumor residue or metastasis. Case 3 was found to be embryonal carcinoma. The measured coordinate of AFP was (0.22, 9.25) , and the predicted coordinate (0.22, 9.55) , indicating the elimination of tumor.

CONCLUSIONThe change of the tumor markers predicted by the function method coincided with the natural course of disease in the three cases. The coincidence of the measured with the predicted coordinate after radical orchiectomy indicates no metastasis, while their disagreement suggests possible residue and metastasis of the tumor.

Adult ; Biomarkers, Tumor ; blood ; Chorionic Gonadotropin, beta Subunit, Human ; blood ; Humans ; Male ; Models, Statistical ; Orchiectomy ; Testicular Neoplasms ; metabolism ; pathology ; alpha-Fetoproteins ; analysis

OBJECTIVETo characterize CRF01_AE strains of recombinant human immunodeficiency virus type-1 (HIV-1) found in the Second National Molecular Epidemiology Study on HIV in China and to analyze its sequence variation in the env V3-C3 region during the First National Molecular Epidemiology Study (NMES1, 1996 - 1998) to the Second National Molecular Epidemiology Study (NMES2, 2001 - 2002).

METHODSDNA was extracted from peripheal blood mononuclear cells of the subjects with HIV infection. The env C2-V4 region of HIV-1 was amplified with nested polymerase chain reaction (n-PCR). PCR products were directly sequenced using ABI 377 DNA sequencer, then the gene-based phylogenetic tree was constructed and its variation of amino acids was analyzed with GCG software.

RESULTSTotally, 169 strains of recombinant HIV-1 CRF01_AE were identified from blood samples collected from different high risk groups in 17 of 31 provinces, municipalities and autonomous regions all over China by the end of 2002. Although sexual transmission still dominated during NMES1 (62.2%, 23/37) and NMES2 (55.3%, 73/132), prevalence of HIV-1 CRF01_AE in intravenous drug users (IDUs) increased to 41.6% (57/137) during NMES2 from 27% (10/37) during NMES1. Phylogenetic tree analysis showed that HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 did not cluster with those prevalent in the subjects infected by sexual transmission during NMES2 and those in IDUs during NMES1. The amino acid residues of V3 region of HIV-1 CRF01_AE in IDUs were relatively conservative, but the sixth, eighth, ninth, tenth, twelfth, fifteenth, sixteenth amino acid residues of C3 region displayed regular changes.

CONCLUSIONSHIV-1 CRF01_AE strain has been introduced into inland provinces from southeastern coast areas and southwestern border areas, with an increasing prevalence in IDUs. The sequence of env V3-C3 region of recombinant HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 was obviously different from that during NMES1, suggesting that HIV-1 CRF01_AE strains prevalent in IDUs during NMES2 might come from a new source and have a potential to spread.

Amino Acid Sequence ; China ; epidemiology ; Genes, env ; genetics ; Genetic Variation ; Genome, Viral ; HIV Infections ; epidemiology ; genetics ; virology ; HIV-1 ; classification ; genetics ; Humans ; Molecular Epidemiology ; Molecular Sequence Data ; Phylogeny ; RNA, Viral ; genetics ; Recombination, Genetic ; Sequence Homology, Amino Acid ; Substance Abuse, Intravenous ; virology

Adult ; Aged ; Apoptosis ; Carcinoma, Transitional Cell ; chemistry ; pathology ; Female ; HSP70 Heat-Shock Proteins ; analysis ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Urinary Bladder Neoplasms ; chemistry ; pathology

BACKGROUNDBladder cancer is a relatively common tumor in the urinary system, in which mitomycin C (MMC)-based chemotherapy or combination chemotherapy has been mainly used to treat patients with advanced bladder cancer. The prognosis of patients with advanced bladder cancer is still extremely poor in spite of recent therapeutic advances. To improve the prognosis, the sensitivity of tumor cells to mitomycin C by the induction of apoptosis with the abating heat shock protein 70 (HSP70) expression in human bladder cancer cell lines of BIU-87 was investigated.

METHODSHSP70 expression was abated in BIU-87 cells by HSP mRNA antisense oligomers. MTT assay and the clone-forming test were used for evaluating the sensitivity of cells to MMC. Apoptosis was assessed using both fluorescent microscopy after staining the cells with Hoechst 33258 and DNA fragment ladder agarose electrophoresis. Thirty-two male six-week-old BALB/c nude mice, at the beginning of the experiment, were used to evaluate the effect of antisense oligomers (ASO) on the tumor formation in vivo.

RESULTSHSP70 expression in BIU-87 was effectively abated by HSP70 mRNA antisense oligomers. The percentage of apoptotic cells in ASO group was greater than in sense oligomers (SO) [P < 0.05, (18.31 +/- 2.89)% vs (1.89 +/- 0.74)%], nonsense oligomers (NO) [P < 0.05, (18.31 +/- 2.89)% vs (1.78 +/- 0.92)%] and blank groups [P < 0.05, (18.31 +/- 2.89)% vs (1.87 +/- 0.84)%], while the sensitivity of tumor cells to mitomycin C was enhanced. The in vivo tumor inhibition rate of ASO plus MMC (> 50%) was more than that of ASO or MMC group alone (all P < 0.05).

CONCLUSIONSThe abating level of HSP70 expression can strengthen the sensitivity of BIU-87 to MMC. One of this effect might be related to the induction of apoptosis by abating HSP70 expression.

Apoptosis ; drug effects ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; drug effects ; HSP70 Heat-Shock Proteins ; antagonists & inhibitors ; genetics ; physiology ; Humans ; Mitomycin ; pharmacology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Urinary Bladder Neoplasms ; drug therapy ; metabolism ; pathology

OBJECTIVETo identify variations in the env gene of human immunodeficiency virus type 1 (HIV-1) subtype CRF01-AE strains circulating in China and to elucidate the potential relationship between genetic variation and evolutionary pressure.

METHODSFragments of the HIV-1 env gene were amplified by nested-polymerase chain reaction (n-PCR) from the whole blood of HIV-1 infected individuals from four provinces in Southeast China (Guangdong, Hunan, Jiangsu and Jiangxi). The PCR products were then directly sequenced by ABI 377 DNA sequencers. The sequences covering the env V3-V4 region of 34 HIV-1 subtype CRF01-AE strains were selected to analyse phylogenetic trees and amino acid mutations. The accumulation of synonymous (Ks) and antonymous (Ka) substitutions as well as Ks/Ka ratios were calculated using DIVERGE.

RESULTSPhylogenetic trees showed that the 34 HIV-1 subtype CRF01-AE strains from China clustered with the Chinese AE reference strain (AE.97CNGX2F), as well as with the reference strains from Thailand (AE.CM240 and AE.93TH253). The amino acid sequences of the env V4 and C3 regions in the samples were highly variable, compared with those of V3 and V3-downstream regions. The V3 loop central motif in the majority (87.5%) of the strains was GPGQ. The majority of strains did not contain positively charged amino acids at positions 306 and 320 in V3 loop. The N-linked glycosylation sites in the V3-V4 region and flanking regions in these strains were relatively conserved. Analysis of the entire region showed that the mean Ks values were significantly higher than that of the Ka values (P < 0.001), with the Ks/Ka significantly higher than 1.0 (P < 0.001). In contrast, the Ks/Ka ratio in the V4 region was significantly lower than 1.0 (P < 0.01).

CONCLUSIONSOur study indicated that the majority of HIV-1 subtype CRF01-AE strains circulating in China were highly homogeneous. The amino acid sequences of the V4 and C3 regions were significantly more variable than those of the V3 loop. Our analysis also suggested that the phenotype of nearly all strains was likely to be non-syncytium inducing (NSI). Finally, the variation found in the V3-V4 sequence was significantly influenced by functional constraints as opposed to positive selective pressure, while the variability of the lone V4 region was strongly related to positive selective pressure.

Adult ; Amino Acid Sequence ; China ; Evolution, Molecular ; Female ; Genes, env ; genetics ; Genetic Variation ; HIV-1 ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Sequence Homology, Amino Acid

ObjectiveTo explore the feasibility of inducing human umbilical cord mesenchymal stem cells (HUMSCs) to differentiate into Leydig cells in the interstitial tissue of the rat testis.

METHODSHUMSCs were obtained by tissue blocks culture attachment and their purity and multi-lineage differentiation ability were verified by flow cytometry and chondrogenic/adipogenic/osteogenic differentiation. Then the HUMSCs were marked by CM-Dil and transplanted into the interstitial tissue of the rat testis. At 4 and 8 weeks after transplantation, the survival and differentiation status of the HUMSCs were observed by immunofluorescence staining and flow cytometry. The suspension of the rat Leydig cells was obtained at 8 weeks for determining the expression of the Leydig cell marker 3β-HSD in the HUMSCs, the cells labeled with CM-Dil were sorted and cultured, and the medium collected after 3 days of culture for measurement of the testosterone level.

RESULTSThe expression of the Leydig cell marker CYPllal was not observed in the HUMSCs at 4 weeks but found at 8 weeks after transplantation and the differentiation rate of 3β-HSD was about 14.5% at 8 weeks. CM-Dil labeled cells survived after sorting and testosterone was detected in the medium.

CONCLUSIONSHUMSCs are likely to differentiate into Leydig cells in the interstitium of the rat testis.

Animals ; Biomarkers ; metabolism ; Carbocyanines ; Cell Differentiation ; Cholesterol Side-Chain Cleavage Enzyme ; metabolism ; Feasibility Studies ; Humans ; Leydig Cells ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Testis ; cytology ; Time Factors ; Umbilical Cord ; cytology