Optimization of SSR-PCR Non-denatured Polyacrylamide Gel Electrophoresis Conditions in Kernelled Apricot

[Objective] The aim was to optimize the SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Method]25 accessions of kernelled apricot and three accessions of edible apricot were selected as experimental materials to screen the repeatable SSR loci with high polymorphism by the use of SSR markers combined with non-denatured polyacrylamide gel electrophoresis.And the effect of different factors on electrophoresis conditions was compared to explore the optimal SSR-PCR non-denatured polyacrylamide gel electrophoresis conditions in kernelled apricot.[Result]The optimal non-denatured polyacrylamide gel electrophoresis conditions for SSR-PCR were established as follows：polyacrylamide gel concentration 6%,the ratio of acrylamide to bisacrylamide 29∶1,electrophoresis at 1 000 V for 2-3 h,and staining for 15 min within 0.1% AgNO3.[Conclusion]The optimum electrophoresis system has provided some technical foundations to further study the phylogenetic relationship of kernelled apricots by SSR markers.

Analysis of Gene Differential Expression of Setcreasea purpurea Boom under Copper Stress by cDNA-AFLP

[Objective] The aim was to explore biological function of related genes in Setcreasea purpurea Boom under copper stress and to study the mechanism of its copper-resistance from standpoint of molecular biology in order to solve the problem of copper pollution. [Method] Setcreasea purpurea Boom was taken as experimental material which enjoys enrichment ability to cupric ion. About 90 fragments of differential expression were obtained by cDNA-AFLP and silver staining technique, among which, 17 fragments were amplified. After purification and identification, sequences of 6 differential fragments were got and used for BLAST X contrast. [Result] Six differential expressed fragments may play roles when Setcreasea purpurea Boom was under copper stress. The homology achieved 49% between differential sequences of E5MG-3 and of Arabidopsis thaliana mRNA （accession numbers： AAM62956.1）, homology was 53% between sequences of E4MB-2 and Solanum tuberosum mRNA （accession numbers： A5A717.1）, and 65% between sequences of E6MG-1 and Colocasia esculenta （L.） Schott mRNA （accession numbers： AAO62313.1）. It can be concluded that differential expressed genes are related to cell signaling, antioxidation, metabolism and protein modification. [Conclusion] The study has laid foundation for further exploration of regulatory network about response of Setcreasea purpurea Boom to copper stress.

Identification of differentially expressed genes in dorsal root ganglion in early diabetic rats

Objective To screen and identify differentially expressed genes in the dorsal root ganglion （DRG） in early experimental diabetic rats. Methods Diabetic model rats were induced by single intraperitoneal injection of streptozotocin （STZ）. At the second week after STZ injection, the sensory nerve conduction velocities （SNCV） of sciatic nerve were measured as an indicator of neuropathy. The technique of silver-staining mRNA differential display polymerase chain reaction （DD-PCR） was used to detect the levels of differentially expressed genes in rat DRG. The cDNA fragments that displayed differentially were identified by reverse-hybridization, cloned and sequenced subsequently, and then confirmed by Northern blot. Results The SNCV in the diabetic model group [n = 9, （45.25±10.38） m/s] reduced obviously compared with the control group [n = 8, （60.10± 11.92） m/s] （P 〈 0.05）. Seven distinct cDNA clones, one was up-regulated gene and the others were downregulated ones, were isolated by silver-staining mRNA differential display method and confirmed by Northern blot. According to the results of sequence alignment with GenBank data, majority of the clones had no significant sequence similarity to previously reported genes except only one that showed high homology to 6-pyruvoyl-tetrahydropterin synthase mRNA （accession No., BC059140）, which had not been reported to relate to diabetic neuropathy. Conclusion These differentially expressed genes in the diabetic DRG may contribute to the pathogenesis of diabetic peripheral neuropathy.

Effects of Zn^（2＋） on root growth，cell division，and nucleoli of Allium cepa L．

The effects of different concentrations of Zn￣(2+)ion on root growth,cell division,and nucleoli of Allium cepa were studied. The test Zn￣(2+) ion concentration was made up from zinc sulphate (ZnSO4. 7H2O) ranging from 10￣(-7) to 10￣(-2) mol/L. The solutions were prepared in tap water (pH =6. 5).The results indicated that Zn￣(2+) could obviously inhibit root growth at concentrations from 10￣(-4)to 10￣(-2) mol/L.Roots treated with zinc sulphate showed the presence of c-mitosis, anaphase bridges,including sticky and fluidized bridges (at 10￣(-3) to 10￣(-2) mol/L) , chromosome stickiness, irregularly shaped nuclei, broken nuclei and micronuclei. A toxicity effect was also observed on the nucleoli using silver staining technique after 48h of treatment with 10￣(-4)to 10￣(-2) mol/L Zn￣(2+), e. g,the nucleolar particulate material scattered around the nucleoli in the nucleus of root tip cells.

Interferon plus ribavirin and interferon alone in preventing hepatocellular carcinoma: A prospective study on patients with HCV related cirrhosis

AIM:To determine the role of interferon(IFN)with or withoutribavirin in preventing or delaying hepatocellular carcinoma(HCC)development in patients with hepatitis C virus(HCV)related cirrhosis.Data on the preventive effect of IFN plusribavirin treatment are lacking.METHODS:A total of 101 patients(62 males and 39 females,mean age 55.1±1.4 years)with histologically proven HCVrelated liver cirrhosis plus compatible biochemistry andultrasonography were enrolled in the study.Biochemistryand ultrasonography were performed every 6 mo.Ultrasoundguided liver biopsy was performed on all detected focallesions.Follow-up lasted for 5 years.Cellular proliferation,evaluated by measuring Ag-NOR proteins in hepatocytesnuclei,was expressed as AgNOR-Proliferative index(AgNOR-PI)(cut-off=2.5).Forty-one patients(27 males,14 females)were only followed up after the end of anyearly treatment with IFN-alpha2b(old treatment controlgroup=OTCG).Sixty naive patients were stratified accordingto sex and AgNOR-PI and then randomized in two groups:30 were treated with IFN-alpha2b+ribavirin(treatmentgroup=TG),the remaining were not treated(control group=CG).Nonresponders(NR)or relapsers in the TG receivedfurther IFN/ribavirin treatments after a 6 mo of withdrawal.RESULTS:AgNOR-PI was significantly lowered by IFN(P<0.001).HCC incidence was higher in patients withAgNOR-PI>2.5(26% vs3%,P<0.01).Two NR in the OTCG,none in the TG and 9 patients in the CG developed HCCduring follow-up.The Kaplan-Mayer survival curves showedstatistically significant differences both between OTCG andCG(P<0.004)and between TG and CG(P<0.003).CONCLUSION:IFN/ribavirin treatment associated with re-treatment courses of NR seems to produce the best resultsin terms of HCC prevention.AgNOR-PI is a useful markerof possible HCC development.

Chromosomal localization of ribosomal DNA sequences in an apple rootstock using a digoxygenin detection system

A 6kb rDNA probe comprising the 18S coding plusspacer sequences has been hybridized to the metaphase chromosomes of apple rootstock cultivar MM106 demon-strating the localization of ribosomal gene arrays in the vicinity of the telomeric regions of the short arms of chro-mosomes 6 and 14. The in situ results using digoxygenin labelling coupled to an alkaline phosphatase immunoassay were confirmed by silver staining for NORs and nucleoli. This study demonstrates the feasibility of molecular cyto-genetic analysis of very small chromosomes (1.0-2.7μm)of apple.

Structural components of the nuclear body in nuclei of Allium cepa cells

Nuclear bodies have long been noted in interphase nuclei of plant cells, but their structural component, origin and function are still unclear by now. The present work showed in onion cells the nuclear bodies appeared as a spherical structure about 0.3 to 0.8 microm in diameter. They possibly were formed in nucleolus and subsequently released, and entered into nucleoplasm. Observation through cytochemical staining method at the ultrastructural level confirmed that nuclear bodies consisted of ribonucleoproteins (RNPs) and silver-stainable proteins. Immunocytochemical results revealed that nuclear bodies contained no DNA and ribosomal gene transcription factor (UBF). Based on these data, we suggested that nuclear bodies are not related to the ribosome or other gene transcription activities, instead they may act as subnuclear structures for RNPs transport from nucleolus to cytoplasm, and may also be involved in splicing of pre-mRNAs.

Relationship between the Clinical Features and the Viral Antigen in the Extremity Blood of the Patients with Hemorrhagic Fever with Renal Syndrome

Summary: The direct immunogold silver staining (D IGSS) method was used to detect the viral antigen in the extremity blood of 67 cases of hemorrhagic fever with renal syndrome. The positive rate of viral antigen was the highest during the fever, hypotension and oligouria phrase; and the rate dropped gradually during the polyuria and convalescent phase. It is suggested that clinical staging was positively related with the percentage of the viral antigen positive cells (P<0.001). It is concluded that the positive rate was related to the extent of the injuries by direct viral attack and immune reaction. The D IGSS was proved to be fast, simple, economical, with high sensitivity and specificity.

ESTABLISHMENT OF DIFFERENTIAL DISPLAY mRNA AND ITS APPLICATION IN THE STUDY ON THE MECHANISM OF LUNG CANCER INDUCED BY RADIATION

Objective: A method for separating mRNAs by means of the polymerase chain reaction (differential display mRNA), and identifying the genes related to radiation-induced lung cancer was introduced. Methods: The RNAs were isolated from two pairs of samples, SV40-immortalized human fetal tracheal fibroblast cell (SHTF) versus αSHTF cell (transformed SHTF cell induced by α particles) and lung cancer tissue versus normal lung tissue obtained from one miner, and amplified by RTPCR. The differential expressed gene fragments were displayed by autoradiograph or silver nitrate stain. Results: The differential display mRNA method was established using both cell and tissue samples. The bands stained by silver nitrate were clearer than those on X-ray film. The rate of reamplification of differentially expressed gene fragments stained by silver nitrate is 80%, higher than that by autoradiograph, 50%. Conclusion: Differential display mRNA method was established successfully on both cell and tissue samples. The modified method for staining band increased the rate of reamplification and established the basis for confirming relative genes.

Detection of p53 gene mutations in sputum samples and their implications in the early diagnosis of lung cancer in suspicious patients

Objectives To evaluate the value of detecting p53 gene point mutations in sputum samples and its validity and reliability as a surveillance index in the early diagnosis of lung cancer in suspicious patients.Methods Sputum samples were collected from 54 cases identified as lung cancer and 114 cases identified as pulmonary benign disease. The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was performed for the detection of point mutations at exons 5-8 of the p53 gene, and sputum smears were also used for each sample. Conclusion Detection of p53 gene alterations in sputum samples by PCR-SSCP-silver stain can be used as a follow-up surveillance index for the early diagnosis of lung cancer in suspicious patients.

A clinical comparative study of polymerase chain reaction assay for diagnosis of pneumocystis pneumonia in non-AIDS patients

Background Pneumocystis jirovecii pneumonia （PCP） is one of the most common and fatal infections in non-AIDS immunocompromised patients, which is difficult to diagnose by traditional morphologic methods. This study evaluated polymerase chain reaction （PCR） assays of Pneumocystis jirovecii mitochondrial large subunits ribosomal RNA in sputum and bronchioalveolar lavage fluid （BALF） for diagnosing PCP. Methods Sputum and BALF specimens from two groups were collected： one group （PCP group） included 20 patients definitely diagnosed of PCP by Gomori methenamine silver （GMS） stains of BALF; the other group （non-PCP group） included 40 patients. Each specimen was examined by GMS stains and PCR assays. Results GMS stains of BALF in PCP group were 100% positive （20/20）, GMS stains of sputum in PCP group were 35% positive （7/20）; GMS stains of BALF in non-PCP group were 100% negative （40/40）, GMS stains of sputum in non-PCP group were 100% negative （40/40）. PCR assays of BALF in PCP group were 100% positive （20/20）, PCR assays of sputum in PCP group were 100% positive （20/20）; PCR assays of BALF in non-PCP group were 100% negative （40/40）, PCR assays of sputum in non-PCP group were 100% negative （40/40）. Sensitivity and specificity of PCR assays of sputum and BALF were both 100%; positive and negative predictive values were also both 100%. Conclusion The diagnostic value of PCR assays of Pneumocystisjirovecii mitochondrial large subunits ribosomal RNA on sputum and BALF for pneumocystis pneumonia are both high and equivalent.