An autosomal genomic scan for loci linked to type 2 diabetes in northern Han Chinese.

We report the results of a genome-wide scan conducted in 219 individuals from 34 large multiplex nuclear pedigrees from the northern Han Chinese population at an average resolution of about 10 cM. Nonparametric two-point and multipoint linkage analyses were performed to detect evidence of linkage with type 2 diabetes in this study. On chromosome 1 four regions showed evidence of linkage with type 2 diabetes in northern Han Chinese. Of these regions a marker D1S193 (73 cM) showed evidence of linkage (two-point nonparametric linkage 2.409), and another region (around 190 cM) was a replication of several other studies performed in different ethnic populations. Evidences of linkage have been confirmed by typing additional markers (average distance 1-5 cM) flanking these two positive regions on chromosome 1. We also found indication of linkage with type 2 diabetes on chromosomes 2, 10, 12, 18, 20, and 22 by two-point linkage analyses.

Carcinogenesis or tumourigenicity testing of animal cell lines for vaccine preparation by colony formation on soft agar and by agglutination under plant lectins.

The carcinogenic or tumourigenic testing of seven animal kidney cell lines (F-81, CRFK, MDCK, Vero, Vero-2 cell line, MA-104 and BHK-21) established in China, were carried out in more than 700 nude mice for colony formation in soft agar and for agglutination under different density of plant lectins. Tests showed that there were correlation between cell line chromosome number variations and anchorage independence in soft agar, agglutinability under lectins and tumour-forming ability in nude mice. Since testing in vitro was more economical, simpler and faster and thus thought to be more reliable, we recommend measuring agglutinability, followed by anchorage independence or analysis of karyotype as the initial means for monitoring tumourigenicity of animal cell lines in nude mice.

[Analyses of chromosomal karyotypes and cytogenetic variations of animal cell lines].

After the master cell stock(mcs) and working cell bank of more than 30 different strains of 7 animal kidney cell lines (F-81 or CRFK cell line, MDCK cell line, Vero or Vero-2 cell line, MA-104 cell line and BHK-21 cell line) were established in China, the chromosomal number variations and structural aberrations of the above lines, primary feline or canine kidney cell (FKC or CKC) and HeLa cell line were investigated and their karyotypes of routine or Giemsa chromosomal bands were analyzed. The carcinogenesis or tumorigenicity testing of these cells in about 700 nude mice and for colony formation in soft agar (SA) and for agglutination under different concentration of plant lectins was carried out. Both tumorigenicity-negative strains of F-81, CRFK, Vero or Vero-2 lines and very-low-tumorigenicity strains of MDCK line were successfully selected and evaluated for the production of canine or feline combination viral vaccines, which are free of infectious agents, and described with respect to cytogenetic characteristics and tumorigenicity. Rate of modal chromosome number represents the ratio of cell number having modal chromosome number to all the split cell number analyzed at random. Rate of difference represents the ratio of difference of the rate of modal chromosome number between mcs (master cell stock) + n and mcs passages. The chromosomal analysis results showed that the ratio of difference of the rate of modal chromosome number between mcs + n and mcs passages was not more than 5%-15% and the structure aberrations was generally 0%-3%, not more than 5%-10%, thus the hereditary character of cell lines is comparatively stable without significant difference between different passages. The genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species specific. Experimental models were established for the researches on the prevention and prophylaxis of malignant tumors or cancers and their genetically biological characteristics. Tests showed that there was correlation among cell line chromosome number variations, anchorage independence in soft agar, agglutination under plant lectins and tumor-forming ability in nude mice. Since testing in vitro is more economic, simpler, faster, and is thought to be reliable, we recommend plant lectins followed by SA or analysis of karyotypes as the initial means for monitoring tumorigenicity of animal cell line in nude mice.

[Fudenine, relative to blood glucose-control, is a novel membrane protein].

OBJECTIVE: To determine whether Fudenine is a novel membrane protein. METHODS: Green fluorescence protein(GFP) was used to localize Fudenine in vivo. GFP, as a control, was targeted to cytoplasm. Epithelial cell, CBRH7919, and non-epithelial cell, L-6TG, were cultured and transiently transfected by using the lipofectamine reagent. After 48 h, intact cells were examined with fluorescence microscope for Fudenine. RESULTS: Reporter plasmid pEGFP-N1, as a control, was expressed and localized to cytoplasm. But Fudenine, driven by the cytomegalovirus promoterenhancer contained in the pEGFP-N1 vector, was overexpressed and targeted to cellular membrane. CONCLUSIONS: Fudenine is a novel membrane protein. It may play the similar role with its homologues AC133 antigen and prominin in human and mouse, respectively. It might be involved in signaling transduction and regulate blood glucose metabolism in vivo.

[Advances in genetic epidemiology].

We have discussed some advances in genetic epidemiology, which include the effect of gene-environment interaction on diseases. The developing genetic tests and genetic information will benefit the health care and disease prevention.

[Statistical approaches to gene mapping of human diseases].

Gene Mapping of human diseases, especially the complex genetic diseases, has been the difficulty and hotspot in medical genetics and gene study. In this article, the principle and application of several gene mapping methods are discussed, including linkage analysis (parametric and nonparametric linkage analysis), association study and linkage disequilibrium analysis.

Formation of glycidyl methacrylate-DNA adducts in vivo.

In in vivo test, rats were orally administrated with glycidyl methacrylate (GMA) at respective doses of 250 mg/kg, 125 mg/kg and 62.5 mg/kg, 31.25 mg/kg and solvent as control for 14 days. DNA adducts produced in the liver, kidney, blood and testis were analyzed by RP-HPLC and nuclease P1 mediated 32P-postlabelling method. Results showed that several potential GMA-DNA adducts were formed in various organs (4 adducts in blood, 3 adducts in liver and kidney, 1 adduct in testis). A linear dose-response relationship was observed within certain dose levels. The relative adduct labeling values failed to further increase any more when the concentration went up to 125 mg/kg. The order of adduct level with GMA was kidney, liver, blood and testis. The GMA adduct N3-methacrylate-2-hydroxypropyl-dCMP was found in kidney, liver and blood. These results indicated that GMA could react with negatively charged centers on DNA and form GMA-DNA adducts. If carcinogen induced DNA damage exceeds the ability of repair systems, gene mutation is induced. Therefore, study on molecular mechanism of gene mutation induced by DNA adducts is not only an important part of chemical-carcinogenesis, but also provides information on critical biomarkers for monitoring human exposure to genetic toxins.

Role of dNTPs in mutagenesis.

The induced mutation frequency by alkylating mutagen glycidyl methacrylate (GMA) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated with or without perturbation of deoxyribonucleoside triphosphate (dNTP) pools; the influence of short treatment at different concentrations of GMA or MNNG on dNTP pools was also explored. The results indicated that the induced mutation frequency increased greatly at high dosages of mutagen (GMA approximately 64 micrograms/ml, MNNG approximately 8 micrograms/ml) and the perturbation on dNTP pools was carried out before the treatment of mutagen; the short treatment with mutagen could induce distinct fluctuations of dNTP pools, but different mutagen might have different effects on dNTP pools. According to the results of the present study and other reports in literature, we conclude that dNTP pools may be the targets of alkylating mutagens and the fluctuations of dNTP pools are closely associated with mutagenesis.

Role of deoxyribonucleoside triphosphates (dNTPs) in cell transformation.

Deoxyribonucleoside triphosphate (dNTP) pools were measured in normal BALB/c3T3 cells, transformation-treated cells and transformed cells with reverse-phase HPLC. The fluctuation of dNTP pools was similar after transformation treatment with alkylating mutagen glycidyl methacrylate (GMA) or Nmethyl-N'-nitro-N-nitrosoguanidine (MNNG). However, the gap between deoxyguanosine triphosphate + deoxyadenosine triphosphate (dGTP + dATP) pools and deoxythymidine triphosphate + deoxycytidine triphosphate (dTTP + dCTP) pools was greatly intensified. The measurements also indicated that the dNTP pools in transformed cells were quite different from those in normal cells. The results suggested that dNTP pools may play an important role in cell transformation.

Spectrum of glycidyl methacrylate-induced mutation in plasmid-Escherichia coli system.

In order to characterize the spectrum of mutation induced by glycidyl methacrylate (GMA), the plasmid pBR322 was modified with this mutagen in vitro, transfected into appropriate Escherichia coli host HB101. The mutants were then screened and defined by DNA sequencing. Sequence analysis reveals that GMA induces two classes of mutations: deletion of the mono-, di- or tetra-base or the insertion of mono- or di-base. Both types of mutations, with about 10% frequency, occur predominantly at C-G runs and at 5'-CNCCN-3' sequence, which are hotspots for GMA damage and may cause frameshift mutation.

Analysis of the phenotype and the restriction enzyme mapping level of mutations induced by the new mutagen glycidyl methacrylate.

Glycidyl methacrylate (GMA) is a recently recognized chemical mutagen. In order to explore the mutagenicity and mutagenic process of GMA, plasmid pBR322 was used for in vitro binding, mutant screening, and restriction enzyme mapping. The binding between GMA and DNA in vitro has been verified by means of a spectrophotometric method. When pBR322 and GMA-bound pBR322 were used to transform Escherichia coli HB101, the following results were obtained: (1) The transformation efficiency of GMA-bound pBR322 was much lower than that of pBR322 alone. (2) GMA-bound pBR322 induced phenotype changes in competent cells (i.e., tetracycline-resistance inactivation or ampicillin-resistance inactivation). There were two mutants of pBR322, ApRTCS and ApSTcR, in the transformants and a deductive mutant ApsTcs in the nontransformants. (3) All of the selected mutants were stable and heritable. (4) When restriction enzyme maps were used to analyze the mutant ApRTcS, four of seven maps were changed, some sites were shifted to other resistant gene regions, for example, sites of Bg/I, EcoRI, HindIII, HincII, etc., and there was a new recognition site for HincII (252). We did not observe any DNA fragment insertion or deletion on any maps. Our results suggest that when GMA is covalently linked to the plasmid DNA, it gives rise to a premutagenic lesion of DNA that is converted in vivo into a point mutation.

Antigenic variation and lentivirus persistence: variations in envelope gene sequences during EIAV infection resemble changes reported for sequential isolates of HIV.

The extent and nature of genomic variation among nine antigenically distinct EIAV isolates recovered during sequential clinical episodes from two experimentally infected ponies were examined by restriction fragment analysis and nucleotide sequencing. Only minor variations in restriction enzyme patterns were observed among the viral genomes. In contrast, env gene sequences of four isolates from one pony revealed numerous clustered base substitutions. Divergence in env gene nucleotide and deduced amino acid sequences between pairs of virus isolates ranged from 0.62 to 3.4% env gene mutation rates for isolates recovered during sequential febrile episodes were calculated to be greater than 10(-2) base substitutions per site per year. The degree and nature of env gene variation in EIAV is remarkably similar to the human immunodeficiency virus, suggesting common mechanisms for env gene variation among lentiviruses.

New evidence for the inhibition by harringtonine in the formation of the first peptide bond in protein synthesis.

New evidence for the inhibition by harringtonine in the formation of the first peptide bond in protein synthesis was obtained by means of experiments in the cell-free system. The antitumour drug strongly blocked dipeptide synthesis in the system without elongation factor G and GTP. Furthermore, it inhibited N-Acetyl-phenylalanyl-puromycin formation from N-Acetyl-phenylalanyl-tRNA, puromycin, and ribosomes.