Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

Extracting Human Reaction Time from Observations in the Method of Constant Stimuli

We consider the psychophysical experiments in which the test subject’s binary reaction is determined by the prescribed exposure duration to a stimulus and a random variable subjective threshold. For example, when a subject is exposed to a millimeter wave beam for a prescribed duration, the occurrence of flight action is binary (yes or no). In experiments, in addition to the binary outcome, the actuation time of flight action is also recorded if it occurs;the delay from the initiation time to the actuation time of flight action is the human reaction time, which is not measurable. In this study, we model the random subjective threshold as a Weibull distribution and formulate an inference method for estimating the human reaction time, from data of prescribed exposure durations, binary outcomes and actuation times of flight action collected in a sequence of tests. Numerical simulations demonstrate that the inference of human reaction time based on the Weibull distribution converges to the correct value even when the underlying true model deviates from the inference model. This robustness of the inference method makes it applicable to real experimental data where the underlying true model is unknown.

Mycoplasma infections and different human carcinomas

AIM: To explore relationships between human carcinomas and mycoplasma infection. METHODS: Monoclonal antibody PD4, which specifically recognizes a distinct protein from mycoplasma hyorhinis, was used to detect mycoplasma infection in different paraffin embedded carcinoma tissues with immunohistochemistry. PCR was applied to amplify the mycoplasma DNA from the positive samples for confirming immunohistochemistry. RESULTS: Fifty of 90 cases (56%) of gastric carcinoma were positive for mycoplasma hyorhinis. In other gastric diseases, the mycoplasma infection ratio was 28% (18/49) in chronic superficial gastritis, 30% (14/46) in gastric ulcer and 37% (18/49) in intestinal metaplasia. The difference is significant with gastric cancer (chi(2) = 12.06, P 【 0.05). In colon carcinoma, the mycoplasma infection ratio was 55.1% (32/58),but it was 20.9% (10/49)in adenomarous polyp (chi(2)=13.46, P 【 0.005). Gastric and colon cancers with high differentiation had a higher mycoplasma infection ratio than those with low differentiation (P 【 0.05). Mycoplasma infection in esophageal cancer, lung cancer, breast cancer and glioma was 50.9% (27/53), 52.6% (31/59), 39.7% (25/63) and 41% (38/91), respectively. The mycoplasma DNA was successfully amplified with the DNA extracted from the cancer tissues that were positive for mycoplasma infection (detected with antibody PD4). CONCLUSION: There was high correlation between mycoplasma infection and different cancers, which suggests the possibility of an association between the two. The mechanism involved in oncogenesis by mycoplasma remains unknown.

Rapid detection of sepsis complicating acute necrotizing pancreatitis using polymerase chain reaction

INTRODUCTIONAcute narcotizing pancreatitis usually takes a severe clinical course and is associated with multiple organ dysfunction .With the further understanding of pathophysiological events of acute pancreatisis and the therapeutic measuses taken by the clinicians ,the patients can pass through the critical carry stages ,and then the septic complication caused by rtanslocated bacteria, mostly gram-negative microbes from the intestines ensues[1].

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

p16 gene methylation in colorectal cancers associated with Duke's staging

AIM: To explore the association of methylation of the CpG island in the promotor of the p16 tumor suppressor gene with the clinicopathological characteristics of the colorectal cancers. METHODS: Methylation-specific PCR (MSP) was used to detect p16 methylation of 62 sporadic colorectal cancer specimens. RESULTS: p16 methylation was detected in 42% of the tumors.Dukes'staging was associated with p16 methylation status.p16 methylation occurred more frequently in Dukes'C and D patients (75.9%) than in Dukes'A and B patients (12.1%). CONCLUSION: p16 methylation plays a role in the carcinogenesis of a subset of colorectal cancer, and it might be linked to poor prognosis.

Identification of differentially expressed genes in normal mucosa,adenoma and adenocarcinoma of colon by SSH

AIM: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC). METHODS: Suppression subtractive hybridization(SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. RESULTS: By this way, there were about 3-4 X 10(2) clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database. CONCLUSION: All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.

Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block

The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section.The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a defined cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.

PCR analysis of Yq microdeletions in infertile males, a study from South India

AIM: To estimate the frequency of microdeletions in the long arm of Y-chromosome of 20 infertile males from South India. METHODS: Polymerase chain reaction (PCR) amplification using Y-specific STS of azoospermia factor (AZF) regions i.e., SY 84 for AZFa, SY 127 for AZFb and SY 254 for AZFc. RESULTS: Of the 20 infertile subjects 3 (15 %), one azoospermic and two oligozoospermic, showed microdeletions in the AZF region of Y-chromosome. CONCLUSION: The frequency of deletions involving AZF region of the Y-chromosome is 15 % in azoospermic and severely oligozoospermic infertile men. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.

HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV-infection: does it really mean viral replication?

AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.

Expressions of ICAM-1 and its mRNA in sera and tissues of patients with hepatocellular carcinoma

INTRODUCTIONThe increased expression of ICAM-1 on a widerange of cells and in the sera of patients withmalignancies, chronic liver diseases andinflammation diseases has been described since thelate 1980s[1-22]. Recently rapid progress in studieson expression of ICAM-1 in patients withhepatocellular carcinoma ( HCC ) have beenachieved, including clinical and experimentalresearches[23-31].

Virulence and potential pathogenicity of coccoid Helicobacter pylori induced by antibiotics

AIM: To explore the virulence and the potential pathogenicity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to antibiotic. METHODS: Three strains of H. pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid from by exposure to metronidazole. Both spiral and coccoid form of H. pylori were tested for the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells, and the differences of the protein were analysed by SDS-PAGE and Western blot. The mutation of the genes including ureA, ureB,hpaA, vacA and cagA, related with virulence, was detected by means of PCR and PCR-SSCP. RESULTS: In the coccoid H. pylori,the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells all decreased. In strain F44, the rate and index of adherence reduced from 70.0% +/- 5.3% to 33% +/- 5.1% and from 2.6 +/- 0.4 to 0.96 +/- 0.3 (P 【 0.01), respectively. The invasion of coccoid H. pylori into Hep-2 cell could be seen under electronmicroscope. SDS-PAGE showed that the content of the protein with the molecular weight over Mr 74000 decreased, and the hybriditional signal in band M(r) 125000 weakened, while the band M(r)110000 and M(r)63000 strengthened in coccoid H.pylori as shown in Western blot. The results of PCR were all positive, and PCR-SSCP indicated that there may exist the point mutation in gene hpaA or vacA. CONCLUSION: The virulence and the proteins with molecular weight over M(r)74000 in coccoid H.pylori decrease, but no deletion exists in amplification fragments from ureA, ureB, hpaA, vacA and cagA genes, suggesting that coccoid H.pylori may have potential pathogenicity.

Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens

AIM:To comparatively evaluate PCR and other diagnostic methods (the rapid urease test and/or culture) in order to determine which of the three PCR methods (ureA,glmM and 26-kDa,SSA gene) was most appropriate in the diagnosis of Helicobacterpylori(Hpylori) infection and also to evaluate the detection of a putative virulence marker of H pylori,the cage,gene,by PCR in biopsy specimens. METHODS:One hundred and eighty-nine biopsy specimens were collected from 63 patients (three biopsies each) undergoing upper gastroduodenal endoscopy for various dyspeptic symptoms.The PCR methods used to detect H pylori DNA directly from biopsies were the glmM,26-kDa, ureA and then cagA was used to compare the culture technique and CLO for urease with the culture technique being used as the gold standard. RESULTS:Thirty-five percent of the biopsies were positive for H pylori DNA using the 3 PCR methods,while 68% of these were positive for the cagA gene.Twenty-four percent of the biopsies were negative for H pylori DNA in all PCR methods screened.The remaining 41% were either positive for ureA gene only,glmM only,26-kDa only,or ureA+glmM, ureA+26-kDa,glmM+26-kDa.Out of the 35% positive biopsies,41% and 82% were positive by culture and CLO respectively,while all negative biopsies were also negative by culture and cagA.Cag A+ infection was also predominantly found in H pylori DNA of the biopsies irrespective of the clinical diagnosis. CONCLUSION:This method is useful for correctly identifying infections caused by H pylori and can be easily applied in our laboratory for diagnostic purposes.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

The detection of micrometastases in the peripheral blood of patients with breast cancer for hSBEM mRNA and CD44V6 mRNA

Objective： Successful treatment of breast cancer greatly depends on the early detection of its metastasis, therefore a sensitive and specific biomarker for detecting dissemination of the cancer cells will help to achieve this goal. This study was to evaluate the prognostic significance of human small breast epithelial mucin （hSBEM） and CD44V6 in breast cancer. Methods： The expressions of hSBEM mRNA and CD44V6 mRNA were detected with nested reverse transcription polymerase chain reaction （nested RT-PCR） in 67 samples of breast cancer and adjacent normal breast tissue, 16 samples of breast benign lesions tissue, and 67 specimens of peripheral blood from patients with breast cancer, 16 specimens of benign breast lesions, 20 specimens of healthy volunteers, and 25 （each five cases） other carcinomas tissue samples, including those of gastric carcinoma, colorectal carcinoma, esophageal carcinoma, lung carcinoma, and ovary carcinoma, were analyzed for hSBEM mRNA expression by nested RT-PCR. Results： hSBEM mRNA expression was observed in 62/67 （92.54%） of breast cancer, 14/16 （87.50%） of breast benign lesions and 59/67 （88.05%） of normal breast tissue, with no significant differences between them （P 〉 0.05）. None of the samples from other cancer tissues were positive. In peripheral blood the expression of hSBEM mRNA was detected in 34/67 （50.75%） from patients with breast cancer, with significant increasing （P 〈 0.05） in the cases of metastatic disease （stage Ⅳ） and those with lymph node metastasis compared with localized disease （stage Ⅰ, Ⅱ and Ⅲ） and without lymph node metastasis, but its expression was not found in peripheral blood of patients with benign breast lesions or healthy volunteers. Although CD44V6 mRNA was significantly higher in breast cancer than in benign breast lesions tissue and normal breast tissue, its expression in peripheral blood show no significant difference （P 〉 0.05） in the patients with breast cancer （82.09%）, benign breast lesion （75.00%）, or healthy volunteers （70.00%）. The expressions of hSBEM mRNA and CD44V6 mRNA had no correlation with the age of the patients, size of primary tumor, histological type and estrogen or progestin receptor status （P 〉 0.05）. Conclusion： hSBEM mRNA, as assessed by nested RT-PCR, shows a mammary-specific and mammary-sensitive expression, and is a sensitive indicator of hematogeneous spread of breast cancer cell, while CD44V6 shows low sensitivity and specificity in detecting dissemination of breast cancer cell in peripheral blood, hSBEM mRNA is a promising molecular biomarker for detecting breast cancer micrometastases.

福建省两株HTLV-Ⅰ前病毒外膜区部分env核苷酸序列测定及分析

目的 :了解我国福建省HTLV Ⅰ感染的来源和亚型。方法 :对福建省发现的两株HTLV Ⅰ ,用PCR扩增HTLV Ⅰ部分env 5 2 2bp的片段 (gp46 5’末端和 gp2 1的大部分 ) ,全自动测序仪测序 ,并分析此两株HTLV Ⅰ与HTLV Ⅰ各亚型代表株的核苷酸、氨基酸的同源性。结果 :获得福建省两株HTLV Ⅰ的部分env核苷酸序列 (6 0 6 5～ 6 5 44 )和编码氨基酸序列 (2 96～ 45 5 )。结论 :福建省的这两株HTLV Ⅰ与日本的HTLV Ⅰ (MT2和H5 )核苷酸、氨基酸序列接近 ,属A亚型。

轿车悬架橡胶衬套对人体主观感受的影响研究

为了研究悬架橡胶衬套对人体主观感受的影响,在SIMPACK多体软件系统下,建立悬架有、无衬套的多体整车模型。螺旋弹簧及橡胶衬套部分参数采用试验获取特性曲线;基于相关车辆平顺性道路试验方法,对整车多体模型进行离线时间积分,分别获取座椅处X、Y、Z三轴向加速度时间历程,利用MATLAB软件编制程序,计算三轴向总加权加速度均方根值,比较发现该值在有橡胶衬套情况下较小;进一步以控制臂的第二个橡胶衬套为对象,分别屏蔽其六向刚度中的一向,积分并计算总加权加速度均方根值,结果发现在屏蔽X轴向刚度、Z轴径向刚度时,该值变化较明显,即依据国际标准ISO2631中总加权加速度均方根值与人体感受之间的关系,当橡胶衬套某向刚度发生改变时,人体舒适性主观感受将发生变化。

Multichannel piezoelectric genesensor for the detection of human papillom a virus

OBJECTIVE: To establish a method for rapid detection and sub-typing of human papilloma virus (HPV). METHODS: We utilized the piezoelectric genosensor (PG) technique, which is a combination of the piezoelectric biosensor and gene chips for HPV identification in 22 recurrent biopsy specimens and 22 corresponding original biopsy specimens. The control samples came from normal tissue of healthy persons. A combined reaction took place on the sensor surface between the target genes and probes. The frequency of the piezoelectric sensor will decrease when such reactions occur, and the frequency decrease depends on the concentration of the target gene. Specimens were also analyzed with conventional PCR and dot blot. RESULTS: Of the 22 recurrent specimens, 15 contained HPV6 DNA, 2 HPV11 DNA, and 4 HPV16 DNA. Only one specimen was negative. All the 22 original specimens were positive: 17 harbored HPV6 DNA, 3 sequence homologous HPV11 DNA, and 2 HPV16 DNA. No HPV18 DNA was detected in any specimen. When compared with PCR and dot blot analysis, the results were essentially the same except for one specimen, which was shown to contain other sub-types of HPV. CONCLUSION: Our results show that the piezoelectric genosensor technique is a rapid and specific method to analyze HPV.

对人与自然的关系再思考

科学发展的历史,既是科学方法的发展历史,又是观念发展的历史。科学既有自身的发展规律,又越来越外化。科学与自然、社会的相互包容,相互同态关系正是20世纪科学发展史向我们昭示的。科学理性与人文精神的统一,既是时代精神的要求和体现,又是历史发展的必然。