[KCNQ1, KCNH2, KCNE1 and KCNE2 potassium channels gene variants in sudden manhood death syndrome].

OBJECTIVE: To investigate KCNQ1, KCNH2, KCNE1 and KCNE2 gene variants in the cases of sudden manhood death syndrome (SMDS). METHODS: One hundred and sixteen sporadic cases of SMDS and one hundred and twenty-five healthy controlled samples were enrolled. Genomic DNA was extracted from blood samples. Gene variants of KCNQ1, KCNH2, KCNE1 and KCNE2 were screened by direct sequencing. RESULTS: A total of 14 mutations and 14 SNP were detected. Two non-synonymous mutations of them were newfound. There was no non-synonymous mutation found in the control group. CONCLUSION: There are KCNQ1, KCNH2, KCNE1 and KCNE2 gene variants found in Chinese SMDS cases. KCNQ1, KCNH2, KCNE1 and KCNE2 gene mutation may correlate partly with the occurrence of some cases of the SMDS in China.

[Estimation of postmortem interval by detecting thickness of cornea using ultrasonic method].

OBJECTIVE: To explore the postmortem changes of cornea thickness measured by ultrasonic pachymetry. METHODS: Eleven rabbits were randomly divided into two groups: one group with intact corneal epithelium and another group without intact corneal epithelium. In the later group, the corneal epithelium of the rabbit was scraped using mechanical elimination method. The corneal thickness was monitored continuously by ultrasonic pachymetry at several postmortem interval points in rabbits of the two groups. The changes of corneal thickness and postmortem interval were explored by relative regression analysis. RESULTS: The thickness of the cornea showed a strong non-linear correlation with the postmortem interval in the group with intact corneal epithelium. The group with intact corneal epithelium showed the correlation coefficient 0.922 and the group without intact corneal epithelium showed the correlation coefficient 0.822, respectively. CONCLUSION: The corneal thickness measured by ultrasonic pachymetry shows a potential value for estimating early postmortem interval. The intact corneal epithelium is a crucial factor for the measurement of cornea thickness by ultrasonic pachymetry.

[A study of gene amplification and expression of cyclin D1 in hepatocellular carcinoma].

OBJECTIVE: To investigate gene amplification of CCND1 and expression of cyclin D1 in hepatocellular carcinoma (HCC) and to explore the possible relationship between CCND1 gene status and carcinogenesis of HCC. METHODS: Differential PCR, RT-PCR and immunohistochemistry were used to detect gene amplification, mRNA and protein expression of cyclin D1 in 20 HCC cases respectively. The relationship between the gene amplification rate and the expression level of cyclin D1 and the histological grades of HCC was analyzed. RESULTS: CCND1 gene amplification was detected in 30% of the cases HCC. An overexpression of cyclin D1 mRNA and protein could be demonstrated in 45% and 70% cases respectively. The expression of cyclin D1 mRNA correlated with its gene amplification status (P < 0.05) and was responsible for the protein expression level (P < 0.05). There was a close relationship between the expression level of cyclin D1 protein and HCC histological grades (P < 0.05). CONCLUSIONS: CCND1 gene amplification is a common phenomenon in HCC and may be directly responsible for the cyclin D1 mRNA and protein overexpression. Cyclin D1 protein expression level is directly related to HCC histological grades. Therefore, CCND1 amplification and cyclin D1 overexpression may play an important role in development and differentiation of HCC.

[Relationship between TIMP-3 expression and promoter methylation of TIMP-3 gene in hepatocellular carcinoma].

OBJECTIVE: To investigate further the possible mechanism of carcinogenesis and portal invasion of hepatocellular carcinoma (HCC). METHODS: Samples of the primary tumors, cancer cells emboli in the portal veins and normal liver tissues adjacent to the tumor were collected from 20 cases of primary HCC. Expression of TIMP-3 (tissue inhibitor of metalloproteinases-3) protein was detected using Western blot. Expression of TIMP-3 mRNA was detected by RT-PCR. Methylation of TIMP-3 gene promoter was detected using methylation-specific PCR (MSP). RESULTS: Expression of TIMP-3 protein and mRNA were obtained in all of the normal liver tissues adjacent to tumor. However, loss of TIMP-3 protein expression was found in 5 and 36 cases respectively in the primary tumors and tumor cell emboli in portal veins. Expression of TIMP-3 protein and mRNA in primary tumors and tumor emboli were significantly lower than that in the normal liver tissues. Promoter methylation of TIMP-3 gene could be detected in primary tumors (7 cases) and cancerous emboli (9 cases) in HCC, while no methylation found in normal liver tissues. In all the HCC cases with promoter gene methylation including primary tumors and cancerous emboli in portal veins, 13 cases showed complete loss and 6 cases showed low expression of TIMP-3 protein and mRNA. Promoter methylation of TIMP-3 was noticed not related with the histological grading of HCC. CONCLUSIONS: There is a close relationship between loss or low expressions of TIMP-3 and carcinogenesis and portal invasion of HCC. The loss and low expression of TIMP-3 gene and protein were caused by methylation of the gene promoter.

[Pollution characteristics of volatile organic compounds from wastewater treatment system of vitamin C production].

Using a portable gas chromatography and mass spectrometry (GC-MS), the volatile organic compounds (VOCs) pollution in each unit of the wastewater treatment system for vitamin C production was studied, and the species characteristics of volatile organic compounds (VOCs) were analyzed and summarized. The results showed that 32 kinds of volatile organic compounds were identified, and the total mass concentration range of volatilizing VOCs was 0.9629-32.0970 mg x m(-3). The most species and the largest concentration (25 and 32.0970 mg x m(-3)) of volatilizing VOCs were found in grit chamber, which was located in the most front-end of the wastewater treatment system and was in semi-closed state. The proportion of molecular sulfide in the grit chamber was as high as 30.02%; Higher proportions of aromatic hydrocarbons were monitored in the subsequent processing units, with percentages of 21.06%-31.48%. The main types of VOCs monitored were chlorinated hydrocarbons and ketones, accounting for 6.39%-55.80% and 10.40%-58.08% of the total amount, respectively; 14 kinds of VOCs were detected in every unit of the wastewater treatment system: acetone, 2-butanone, n-hexane, chloroform, chlorobenzene etc, among which, vinyl chloride, styrene and 1,3-butadiene belong to the highly toxic substances. The vinyl chloride concentration exceeded the standard of "atmospheric pollutants emission standards" (GB 16297-1996), while 1,3-butadiene and other pollutants have no national standard limits. The results of this study provide a scientific basis for the revision of China's pharmaceutical wastewater VOCs emission standards.

[Construction of an eukaryotic expression vector for PRL-2 and its effect on human hepatocellular carcinoma cell invasiveness and migration in vitro].

OBJECTIVE: To construct an eukaryotic expression vector for PRL-2 and evaluate its effect on the invasiveness and migration of a human hepatocellular carcinoma cell line. METHODS: RT-PCR was performed to amplify the complete PRL-2 open reading frame using the total mRNA of hepatocellular carcinoma HepG2 cells as the template. PRL-2 gene was inserted into the pGEM T easy vector and sequenced, and the correct PRL-2 sequence was subcloned into the mammalian expression vector pcDNA3.1+. The constructed PRL-2 vector was transfected into CL1 cells via lipofectamine, and the stable expression of PRL-2 mRNA was detected by RT-PCR, the expressed protein identified by immunohistochemistry and Western blotting, and the effect of PRL-2 on the adhesion ability of CL-1 cell evaluated with MTT assay 20 and 120 min after transfection. The effect of PRL-2 on the invasive migration of CL-2 cells was evaluated according to the number of cells penetrating the Matrigel layer of polycarbonate membrane of Boyden chamber. RESULTS: RT-PCR yielded a fragment of 504 bp and the inserted PRL-2 sequence was verified by sequence analysis. The subclones were identified by restriction endonuclease digestion, and a G418-resistant clone, PRL-2-CL1, was obtained after 8 weeks of selection. RT-PCR showed stable expression of PRL-2 mRNA, and Western blotting confirmed overexpression of PRL-2 protein in the transfected cells. PRL-2 increased the adhesion rate of CL-1 cells to fibronectin at 20 min and 120 min after transfection (P<0.05), and also the number of CL-1 cells penetrating the polycarbonate membrane from 10.0+/-3.7 to 44.8+/-2.6 (P<0.05). CONCLUSION: An eukaryotic expression vector of PRL-2 has been successfully constructed, which allows stable and efficient expression in CL-1 cell line. PRL-2 can promote cell adhesion and invasion activity of human hepatocellular carcinoma cells.