Oxidative DNA damage induced by high glucose and its suppression in human umbilical vein endothelial cells.

In order to investigate the mechanism of the production of oxidative DNA damage by hyperglycemia, we measured formamidopyrimidine N-glycosylase (FPG)-sensitive sites by the comet assay in human umbilical vein endothelial cells (HUVECs) cultured under various conditions including high glucose. Mean values of FPG-sensitive sites were higher in HUVECs cultured for 5 days in high glucose (45 mM) compared with normal glucose (5mM) medium (P<0.001). FPG-sensitive sites increased in a time-dependent manner under high glucose treatment (3 days: P<0.05, 5 days: P<0.001), whereas L-glucose, which is taken up poorly into the cells, gave a slight increase in FPG-sensitive sites (P<0.05). Flow cytometric analysis using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) showed that incubation with L-glucose produced more reactive oxygen species than incubation with D-glucose. However, these increases were slight (1.22- and 1.12-folds, respectively). Incubation of HUVECs with aminoguanidine (100 microM) or pyridoxamine (1mM), which are inhibitors of glycation, decreased the levels of FPG-sensitive sites (P<0.001). However, these inhibitors did not suppress the intracellular generation of reactive oxygen species induced by high glucose. These results indicate that FPG-sensitive sites induced by high glucose are not due to intracellular reactive oxygen species. In order to clarify what caused the induction of FPG-sensitive sites, we investigated the effect of glyoxal and 3-deoxyglucosone (3-DG) on the induction of FPG-sensitive sites and the intracellular production of reactive oxygen species in HUVECs. Glyoxal and 3-DG at a concentration of 100 microg/ml induced FPG-sensitive sites (P<0.001, P<0.01, respectively). In contrast, glyoxal did not generate reactive oxygen species inside HUVECs. The results shown in this study suggest that glyoxal formed intracellularly or extracellularly during high glucose treatment might induce FPG-sensitive sites by a mechanism not involving reactive oxygen species.

Soluble myelin-associated glycoprotein-immunoglobulin G1 chimera protein promotes neurite outgrowth from mouse cerebellar neurons.

A soluble chimera protein consisting of the extracellular region of mouse myelin-associated glycoprotein (MAG) and the Fc region of human immunoglobulin G1, MAG-IgG, was prepared by transfecting an expression vector, BCMGSneo, containing the corresponding fused gene into mouse myeloma P3U1 cells. It was found that this MAG derivative promoted neurite growth of mouse cerebellar neurons in culture. In the presence of MAG-IgG the rates of neurite-bearing cells were significantly increased, and the neurites were distinctly lengthened. It was also observed that the anti-MAG monoclonal antibody inhibited this stimulation of neurite extension, supporting the specificity of the effect of MAG-IgG. These results suggest that soluble MAG derivative might act as a neurotrophic factor in the developing brain.

Direct molecular analysis of myotonic dystrophy in the German population: important considerations in genetic counselling.

Myotonic dystrophy (DM) is associated with the expansion and instability of a trinucleotide (CTG) repeat at the DM locus on chromosome 19. Direct genomic analysis in the German population was carried out on 18 DM families, six families with equivocal diagnosis, 69 subjects with equivocal clinical diagnosis, and 100 controls using the polymerase chain reaction (PCR) and a refined Southern protocol. In the majority of the cases molecular analysis confirmed the clinical diagnosis. These included seven cases of congenital DM (CDM) with widely differing gene expansions and instabilities. In most DM families the expanded fragment became larger in successive generations, but we also identified four families with contractions and two families that showed stability of the enlarged fragment during transmission. In four clinically defined DM patients we were unable to detect enlarged CTG repeats. Sequencing of each exon of the DM gene in two of these patients failed to show any mutations. Our cases have important implications for genetic counselling of DM families, highlighting both the diagnostic value of direct genomic analysis and its limitations.

The expression of mmp-7 messenger-RNA in primary and metastatic human colorectal adenocarcinoma.

MMP-7 mRNA expression was examined by RT-PCR and Southern blot hybridisation following cDNA synthesis of extracted tissue RNA in a total of 33 colorectal cancer patients. Expression was studied in tumour tissues and compared to adjacent non-neoplastic tissues. MMP-7 mRNA was detected in all tumour samples, with no qualitative difference between primary and metastatic tumours and there was no relation to Dukes' clinical stage. Adjacent nonneoplastic colon and rectum (65%) and liver (100%) also expressed MMP-7, although the signal intensity was weaker. In contrast, only 31% of adjacent non-neoplastic lymph nodes expressed MMP-7. The results suggest that MMP-7 is probably expressed early in the neoplastic transformation in patients with colorectal adenocarcinoma.

Expression of novel DNA-binding protein with zinc finger structure in various tumor cells.

We have isolated from B16 mouse melanoma cells a complementary DNA (Mel-18), whose deduced amino acid sequence possesses a characteristic zinc finger structure. Immunostaining with antibodies raised against partial Mel-18 peptide sequences demonstrated nuclear localization of the gene product. We have also demonstrated that this protein has DNA-binding capacity, and the zinc finger is responsible for the DNA binding. At the transcriptional level the Mel-18 mRNA was detected in all tumor cells examined as well as melanoma cells (ontogenically of neural origin) but was scarcely present in normal tissues except neural organs. The transcript is developmentally regulated. These data suggest that Mel-18 may play a role in transcriptional regulation and also in control of cell proliferation and/or neural cell development.

The poliovirus receptor protein is produced both as membrane-bound and secreted forms.

Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1----13.2 of human chromosome 19.

Isolation of genomic DNA controlling mouse melanoma antigen defined by monoclonal antibody.

We have isolated the genomic DNA controlling the expression of murine specific melanoma antigen by employing cosmid shuttle vector and monoclonal antibody. Transfection of the cosmid library derived from mouse melanoma cells into human melanomas and repeated cell sortings of the fluorescence-bright population enabled us to enrich the antigen-positive transfectants. We rescued a 34.8 kb DNA fragment from the transfectants by in vitro packaging and showed it to be responsible for the antigen expression. However, we noticed instability of the antigen expression when the selection pressure imposed by the cell sorting was removed. This seemed to be due to the fact that the insert DNA was preferentially deleted from this cosmid vector without loss of the vector sequence itself.

Method of genomic DNA cloning by the combination of cosmid shuttle vector and monoclonal antibody.

We report here the strategy to isolate the DNA fragment of any species origin which encodes cell surface antigen by using cosmid library transfection and cell sortings with a monoclonal antibody. We took the mouse melanoma antigen defined by monoclonal antibody as a model system and rescued the genomic DNA by in vitro packaging, showing the feasibility of this procedure.