BACKGROUND ＆ AIM: This study was aimed at the detection of novel alternative splicing variants of NRDR in human neuroblastoma. MATERIAL AND METHODS: We obtained 635 bp and 429 bp PCR fragments using NRDR specific primer from human neuroblastoma cell lines SK-N-SH and SK-SY-5Y. The two fragments were then respectively confirmed as NRDR and a novel isoform by alternative splicing. We performed 3'RACE and 5'RACE to obtain the cDNA full sequence of the spliced isoform, and tried to determine its subcellular location by GFP-humNRDRA2 fusion protein. RESULTS: We named the alternative splicing variant as humNRDRA2 with Genbank accesstion No.AY616182. Comparing with NRDR which is composed of eight exons, the novel variant NRDR A2 lost the exon4 and exon6 by alternative splicing, leading to frameshift in coding region starting from exon 5 and emergence of a premature stop codon. Thus, it produced an 188 aa protein with an altered 3'terminal sequence starting from 137 aa. The frameshift generated a bipartite nuclear targeting sequence at 160～176 aa, with the loss of the original peroxisomal targeting signal -SRL tripeptide as in NRDR. Absence of this splice variant in EST pool suggested that NRDR A2 probably was unique to human neuroblastoma. Comparing with the successful subcellular location of another splice variant of NRDR in the control experiment, we found HeLa cells that were transformed by GFP-NRDR A2 fusion protein all floaing rather than sticking to the bottom of flask, suggesting that NRDR A2-encoded protein was probably toxic to cells. CONCLUSION: Human neuroblastoma produces a novel alternative splicing isoform NRDRA2 with frameshift and a bipartite nuclear targeting sequence. Its translated protein is probably cytotoxic.

BACKGROUND ＆ AIM: The cell model of suppressed DNA-PKcs expression by siRNA was constructed and used to study the function of DNA-PKcs. MATERIAL AND METHODS: DNA-PKcs siRNA expression vectors were constructed and HeLa cells were transfected by lipofectamine. Western blot was used to measure the expression of DNA-PKcs and the rate of growth of cells was used to analyze the changes of radiosensitivity. RESULTS: Three stable transfectants were selected from the DNA-PKcs siRNA transfected HeLa cells. Western blot analysis indicated that the expression of DNA-PKcs was suppressed by the siRNA. The sensitivity of cells to UV radiation and ionizing radiation was markedly increased. The growth of tumor cells was inhibited. CONCLUSION: We successfully established the cell model of suppressed DNA-PKcs expression by siRNA. The inhibition of DNA-PKcs could influence the radiosensitivity and growth of tumor cells. DNA-PKcs; siRNA; radiosensitivity; cell proliferation

BACKGROUND ＆ AIM: To explore the effects of Cystathionine beta synthase (CBS) gene polymorphism in parents on occurrence of congenital heart disease (CHD) in offsprings. MATERIAL AND METHODS: 127 CHD case-parent triads were selected from Liaoning Province as case group, and 129 healthy subjects were simultaneously selected together with their biological parents as control group. For all subjects the polymorphism of CBS gene G919A locus was examined by ARMS-PCR method. RESULTS: Significant difference was found in genotype distribution and allele frequency between parents of CHD cases and of controls. Compared with wild type genotype, offsprings of mutant heterozygote and homozygote were at higher risk of CHD, with OR 0.46(95％CI: 0.31～0.69). Further comparison of each type of CHD showed that genotype frequencies in PDA patient, mothers of ASD patients,parents of VSD parents,and patients of other types of CHD were significantly different from their corresponding controls,The results of TDT analysis showed that no allele transmission disequilibrium existed in CHD nuclear families. CONCLUSION: The CBS gene G919A mutation of parents might decrease the risk of CHD in offspring.

BACKGROUND ＆ AIM：Methionine synthase(MS) catalyzes the tetrahydrofolate transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, producing methionine (Met) and tetrahydrofolate. Met is the precursor of S-adenosylmethionine, which is a crucial methyl group donor in DNA methylation. Vitamin B12 (B12) is a co-factor of MS and plays an important role in folate metabolism. This experiment aimed to evaluate the genotoxic effects of Met and B12 deficiency in human lymphoblast cell line carrying BRCA1 mutation. MATERIAL AND METHODS: Lymphoblast cells were cultured under 10 different combined of vitamin B12 (100,300,600,800,1 200 pmol/L) and methionine (15 μmol/L and 50 μmol/L) concentration medium for 9 days. Routine RPMI 1640 medium was set as the control. The frequencies of micronucleated binucleated cell (MNBN) were evaluated by cytokinesis-block micronucleus assay(CBMN). RESULTS: The frequencies of MNBN were significantly higher in 15 μmol/L of Met combined with all concentrations of B12 than that of 50 μmol/L of Met with all B12 concentrations(P<0.05). The frequencies of MNBN were makedly reduced when 50 μmol/L of Met and 600 pmol/L of B12 were used. CONCLUSION: A significant inhibitory effect of MNBN was found at 600 pmol /L of B12. There were no significant differences found among 600,800,1 200 pmol/L of B12 and RPMI1640 medium groups. We concluded that 50 μmol/L of Met and 600 pmol/L of B12 formed an optimal combination for stabilizing the genome of the test cell line. This preliminary investigation suggested that Met and B12 deficiency caused genomic instability. One might consider supplying adequate amounts of Met and B12 to maintain genome stability.

BACKGROUND ＆ AIM: To study the effect of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) on proliferation and apoptosis of human-derived fetal hepatocytes(L-02) cells. MATERIAL AND METHODS: L-02 cells were treated with MX (10，30，100，300 μmol/L)for 3 hours and further incubated for 0, 3, 6 and 9 hours. Proliferation and apoptosis of L-02 cells were analyzed by using flow cytometric analysis and microscopic analysis after staining with ethidium bromide and acridine orange. RESULTS: The distribution of cells at different phases of the cell cycle was associated with the dose of MX and the incubation time. When L-02 cells were treated with MX for 3 hours and not further incubated, S phase and G2 phase cells increased significantly at MX concentration of 30 μmol/L and 10,100, 300 μmol/L,respectively. At incubation time of 9 hours , G2 phase and S phase cells increased significantly at MX concentration of 10 μmol/L and 30,100, 300 μmol/L,respectively. The percentage of apoptotic L-02 cells increased at first and decreased subsequently with further incubation time. Peak value of apoptotic percentage appeared at the incubation time of 6 hours and showed statistical significance in comparison with solvent control (P<0.01). The highest apoptotic percentage was observed at the MX concentration of 30 μmol/L. CONCLUSION: MX induced apoptosis and arrested cycling of L-02 cells at G2 phase and S phase. The type of cell cycle being arrested was associated with the dose of MX and the incubation time.

BACKGROUND ＆ AIM: To study the toxicity of hexavalent chromium on rat hepatocytes . MATERIAL AND METHODS: Rat hepatocytes BRL－3A were treated with hexavalent chromium at concentrations of 0, 0.1, 1, 10 and 100 μmol/L Viability was assessed by 3_[4,5_dimethylthiazol_2_yl]_2,5 diphenyl tetrazolium bromide(MTT) method and flow cytometry was used to analyze cell apoptosis. RESULTS: Viability decreased with the increasing concentration of hexavalent chromium. Cell apoptosis induced by hexavalent chromium also showed in a dose_dependent pattern. CONCLUSION: These data indicated that hexavalent chromium had a damaging effect on hepatocytes and induced apoptosis at the concentrations used in the present study.

BACKGROUND ＆ AIM: To study the role of expression of Syk(Spleen tyrosine kinase) in the course of As2O3 induced brain tumor cell cycle defects. MATERIAL AND METHODS: To integrate syk gene into vector pIND and co_transfect tumor cell line U373 with mutated P53 gene together with regulator vector pVgRXR in order to induce the expression of Syk. The expression of cell cycle negative regulative factor P21 was measured by Western blot. The cell DNA content was assessed by FACS. The role of Syk's expression in As2 O3 inducing brain cell cycle defects was examined using the confocal laser scanning microscope system. RESULTS: U373 cells transfected with syk gene(U373 Syk_ind)could express Syk in the presence of inducing agent Pon A.As2O3 could make maintain U373 cells at G2/M stage,but the proportion was decreased when U373 expressed Syk.Syk could upregulate P21. CONCLUSION: As2O3 influenced brain tumor cell line U373 normal cycles,blocking them in G2/M stage.As2O3 could cause tumor cell apoptosis,while also inducing normal cells into tumor cells. The expression of Syk could decrease the proportion of blocked cells,so reducing the side_effects of As2O3 when used in treating cancer.

BACKGROUND ＆ AIM: To detect the germline mutations in E_Cadherin gene (CDH1) and to investigate their possible role in familial gastric cancer. MATERIAL AND METHODS: With PCR, denaturing high_performance liquid chromatography (DHPLC) and direct sequencing, we screened all exons of the E_cadherin gene for mutations in 80 Chinese gastric cancer patients, 62 of whom had positive family history,we investigated 80 healthy individuals and conducted a case_control study to investigate whether such mutations affect the risk of developing gastric carcinoma. RESULTS: Two of 80 gastric cancer patients carried missense mutation Thr340Ala (heterozygosity). This variation was not found in the control population. We confirmed a T→C polymorphism site at position －13 in upstream of exon 13. The polymorphism was identified in 10 of 80 gastric cancer patients and 12 of 80 healthy individuals and the frequency of this polymorphism did not differ between the familial gastric cancer patients and controls. We found a germline E_cadherin nonsense mutation at codon 751 in exon 14 in 19 of 80 patients analyzed. Only 7 of 80 healthy individuals were Asn751Asn carriers, and significant difference existed between gastric cancer patients and control population, especially between the elder patients and controls. CONCLUSION: Though the germline mutations of CDH1 gene did not occur frequently in familial gastric cancer, CDH1 gene Asn751Asn single nucleotide polymorphism might increase the risk of gastric cancer in Chinese population.

BACKGROUND ＆ AIM: To determine whether serum CA_125 and serial β-hCG levels could be used to distinguish between tubal miscarriage and viable ectopic pregnancy (EP). MATERIAL AND METHODS: 86 women with EP who were stable and had B_ultrasound, CA_125 and β_hCG tests at admission were studied. 25 of the 86 patients received conservative treatment and 61 were managed surgically by laparoscopy or laparotomy. of the latter group, 20 had proved tubal miscarrige and 41 had active, viable EPs. RESULTS: The mean CA_125 level was 83.69±36.37 IU/ml for the patients with aborted EP, 32.23±19.30 IU/ml for the patients with viable EP，and 64.10±28.33 IU/ml for the patients receiving conservative treatments. The mean β_hCG level was 3 323±2 908 mIU/L for the patients with aborted EP, 12 056±9 138 mIU/L for the patients with viable EP, and 1 878±1 043.5 mIU/L for the patients receiving conservative treatments. Both of the serum CA_125 and β_hCG levels between the aborted and the viable EP groups as well as between the conservative treatment and the viable EP groups showed statistical differences. Linear regression analysis revealed a significant inverse relation between serum CA_125 and β_hCG levels. Spearman nonparametric correlation analysis showed a statistically significant relation between management strategy and both of CA_125 and β_hCG levels. CONCLUSION: The use of CA_125 levels as an adjunct to serial β_hCG levels holds promise as a means for differentiating tubal miscarriage from viable EP.

BACKGROUND ＆AIM: To study the spectra of trace elements (TE) in tap water to provide the research basis of pathogenesis of esophageal cancer (EC). MATERIAL AND METHODS: Thirteen trace elements (Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, Sr, V, Zn) in tap water of different districts were studied. The contents of TE were analyzed with t_test and correlation analysis and the regression equation was established. RESULTS: The TE spectra of TE in tap water in areas with high and low incidences of EC were discriminated using Fisher method, the accuracy rates were up the 93％.CONCLUSION: The TE in water of different districts could affect the incidence of EC.

BACKGROUND ＆ AIM: To investigate the immunoregulatory and antitumor activities of Undaria pinnatifida extract. MATERIAL AND METHODS: Effects of Undaria pinnatifida extract on proliferation of spleen cells of mice were analyzed by 3H-TdR incorporation methods, effects of Undaria pinnatifida extract on stimulating tumor necrosis factor -α (TNF-α) secretion from mice monocytes were examined by a biological method, inhibition of tumor cells by Undaria pinnatifida extract with MTT technique. RESULTS: All concentrations of Undaria pinnatifida extract had effects of promoting the proliferation of spleen cells of mice. In vitro, undaria pinnatifida extract could activate mice monocytes to produce more TNF-α, and inhibited proliferation of four types of tumor cells. CONCLUSION: The present study indicateed that Undaria pinnatifida extract had the effects of promoting the proliferation of mice spleen cells, and induced mice monocytes to produce TNF-α.and significantly inhibited the growth of four kinds of tumer cells.

BACKGROUND ＆ AIM:To study the influence of Mian_Zeng_Ling on mouse erythrocyte immunological function and Natural Killer(NK) cell activity. MATERIAL AND METHODS: The ratio of RBC_CR rosette,the ratio of RBC_IC rosette,the ratio of RBC_Ca rosette and NK cell activity were determined. RESULTS: Experiments showed mice erythrocytes could immunologically adhere to tumor cells. After peritoneal injection of cyclophosphamide,the ability of mice erythrocytes adhering to tumor cells was declined,Mian_Zeng_Ling could opposed this action,improving the adherent ability of erythrocytes. The effect of Mian_Zeng_Ling was superior to Zhen-Qi-Fu Zheng granlies sold in the market.Mian-Zeng-Ling also could oppose cyclophosphamide which reduced mouse NK cell activity,therefore improving and raising the activity of moccse NK cell. CONCLUSION: Mian_Zeng_Ling could improve erythrocyte immunological function and NK cell activity suppressed by cyclophosphamide.

BACKGROUND ＆ AIM: To evaluate the effects of different doses of cyclophosphamide (CP) and sampling time on mouse micronucleus frequency in bone marrow. MATERIAL AND METHODS: Young adult NIH mice were treated with a sigle dose of CP(40 or 80 mg/kg) by intraperitoneal injection. The micronucleus freqyuency in bone marrow polychromatic erythrocytes (PCE) at different sampling time 18 , 24 , 48 h after treatment was evaluated. RESULTS: Our data showed: ① The changing trends of the micronucleus frequencies in PCE in the two groups receiving 40 mg/kg and 80 mg/kg of CP were the same at different sampling times. The peak time of the micronucleus incidence was at 24 h after treatment. ② The ratios of PCE to NCE of the two treated groups were not significantly different from the control group. This indicated that the mice bone marrow cells were not suppressed by one CP of 40 or 80 mg/kg by intraperitoneal injection. ③ The micronucleus frequency in PCE of 40 mg/kg was significantly different from that of 80 mg/kg, showing a dose_response relationship. CONCLUSION: CP can be used as a positive control of micronucleus test. The dose range of 40～80 mg/kg was fine, and the optimal sampling time is at 24 h after treatment.

BACKGROUND＆AIM: To study the relationship between serum P53 antibody, gene mutation,protein expression and their roles in the early diagnosis and clinical significance in colorectal tumors. MATERIAL AND METHODS: P53 antibody of preoperative serum from 34 patients with colorectal cancer and 10 healthy persons was measured by enzyme-linked immunosorbent assay (ELISA). At the same time, by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemistry (IHC) (S-P methods), the mutations in exon 5－8 of the P53 gene and protein expression were examined. RESULTS: P53 antibody was positive in 6 of 34 (17.6％) patients with colorectal cancers, but was not found in serum of normal controls. The mutation of exon 5－8 and protein expression of P53 gene were found in 18 of 34(52.9％) and 19 of 34(55.9％) cases, respectively. No mutation and protein expression were positive in normal mucosa. Of 16 patients who were positive for P53 gene mutation in their tumor tissue, 7(43.8％) had evidence of alterations in the P53 gene within the stool. No relationship was found between P53 antibody, protein expression, P53 gene mutation and cliniopathological factors. CONCLUSION: Our data indicate that mutation of P53 gene was one of the main factors which caused its protein expression,hence inducing serum P53 antibody formation. P53 gene mutation could be detected within the stools. Detection of P53 serum antibody and gene mutation in stool may provide new ways for the early diagnosis and survey of population at high risk of colorectal cancers.

BACKGROUND ＆ AIM: To evaluate the value of three-dimensional ultrasonography(3D-US) in breast cancer diagnosis. MATERIAL AND METHODS: The LOGIQ 7 sonography imaging system was used to perform conventional two-dimensional and three-dimensional ultrasonographic examinations. The study population consisted of 48 randomized breast cancer patients. RESULTS： The positive rate of the breast cancer in 3D-Us was higher than that of 2D-US,even in the tumor's relation with adjacent tissues and the blood distribution. 3D-US could stereoscopically show the tumor's shape,the relations between the tumor and the skin,chest muscle,chest wall,etc. And it could clearlg show the network of blood vessels of the cancer. CONCLUSION： 3D-US could display breast cancer's structure, adjacent relations and blood vessels because of its high sensitivity. It is helpful for the diagnosis and evaluation of breast cancers.

BACKGROUND ＆ AIM: To investigate the genotoxicity of bottom ashes of municipal solid waste incineration from Macao and assess their potential risks on eco-environment and human beings. MATERIAL AND METHODS: The micronucleus (MN) assay was employed to detect their genotoxicity on Vicia faba. RESULTS: The leaching solutions of the bottom ashes could induce significantly increasing frequencies of micronuclei in the root tip cells of Vicia faba under the present conditions, compared with the control group. The MN frequency caused by the leaching solutions 0.5％ Na2EDTA was the lowest in the tested groups. CONCLUSION: It is necessary to remove the toxic elements or compounds of the bottom ashes before recycling. The Na2EDTA solution can decrease the bioavailability and the genotoxicity of the heavy metals on Vicia faba, offering a promising chance of chemical remedy.

BACKGROUND ＆ AIM: To test the safety of pollen pini as a health food. MATERIAL AND METHODS: Acute toxicity test,bone marrow cell micronucleus test,mice sperm abnormality test and 30 d feeding study were performed to study the toxicity of pollen pini. RESULTS: In acute toxicity test, male and female KM mice were fed with 15 g/kg dose of pollen pini, no toxic symptoms were observed. The results of bone marrow cell micronucleus test and mice sperm abnormality test were negative. In the 30 d feeding test, no obvious toxic effects were detected in clinical examination, blood tests, biochemical tests, organ weights and pathological examination in treated Wistar rats. CONCLUSION: Pollen pini passed the toxicological tests and deemed safe as a kind of health food.

BACKGROUND ＆ AIM: To study the acute toxicity and the mutagenicity of pummelo peel and orange peel. MATERIAL AND METHODS: The oral acute toxicity test in rats , Ames test, micronucleus test in bone marrow and sperm abnormality test were carried out. RESULTS: LD50 in rats of the pummelo peel and orange peel were more than 21.5 g/kg. The micronucleus rates and sperm abnormality rates in all doses were not significantly different from the control group. No increase in the number of revertant colonies was found in the Ames test with and without S9 mixture. CONCLUSION: Acute toxicity and the mutagenicity of pummelo peel and orange peel were not observed in this study.

BACKGROUND & ATM: To follow late effects of a victim in a radiation injury accident and observe cytogenetic characteristics of a continual irradiation. MATERIAL AND METHODS: Conventional chromosomal aberrations and G-banded kayrotype analysis. RESULTS: There are little decline in dicentrics. Stable aberrations such as translocations play dominant role in total aberration frequency of 26 %. Clonal aberrations are not yet observed. CONCLUTION: There are more severe cytogenetic damage and slower repair in continual exposure to radiation than in single irradiation.