A detailed linkage map of medaka, Oryzias latipes: comparative genomics and genome evolution.

We mapped 633 markers (488 AFLPs, 28 RAPDs, 34 IRSs, 75 ESTs, 4 STSs, and 4 phenotypic markers) for the Medaka Oryzias latipes, a teleost fish of the order Beloniformes. Linkage was determined using a reference typing DNA panel from 39 cell lines derived from backcross progeny. This panel provided unlimited DNA for the accumulation of mapping data. The total map length of Medaka was 1354.5 cM and 24 linkage groups were detected, corresponding to the haploid chromosome number of the organism. Thirteen to 49 markers for each linkage group were obtained. Conserved synteny between Medaka and zebrafish was observed for 2 independent linkage groups. Unlike zebrafish, however, the Medaka linkage map showed obvious restriction of recombination on the linkage group containing the male-determining region (Y) locus compared to the autosomal chromosomes.

The enhancement of cell lethality and changes in production and repair of DNA damage by hypertonic treatment after exposure to X rays, bleomycin, and neocarzinostatin.

Chinese hamster ovary cells were treated with hypertonic 0.5 M NaCl solution after exposure to X rays or the radiomimetic drugs bleomycin or neocarzinostatin. The cytotoxicity of these agents was greatly enhanced by the hypertonic treatment. On the other hand, no effect was observed after exposure to ultraviolet light, and a significant effect was obtained with mitomycin C (MMC), adriamycin (ADR), and ethyl methanesulfonate (EMS). Assays by filter elution revealed that hypertonicity had various effects on the damage produced by the different agents. Strand breaks resulting from exposure to X rays and radiomimetic agents were sensitive to hypertonic treatment. Hypertonicity caused the production of new lesions and inhibited the rejoining of DNA strand breaks, both of which may be responsible for the enhanced cytotoxicity. On the other hand, the formation of crosslinks by MMC or protein-associated double-strand breaks by ADR, the major forms of damage by which these agents cause cytotoxicity, was not affected by hypertonic treatment. As strand breaks are known to be produced by MMC or ADR, they could account at least partly for the sensitization. However, various kinds of damage are produced by MMC, and any of these could be involved in the sensitization. To our knowledge EMS produces only base damage. Thus hypertonic treatment may have an effect on various types of damage.

Cultured cells from a severe combined immunodeficient mouse have a slower than normal rate of repair of potentially lethal damage sensitive to hypertonic treatment.

The effects of hypertonic 0.5 M NaCl treatment after irradiation on the repair of DNA damage were examined in fibroblasts of the severe combined immunodeficient (scid) mouse. These cells are hypersensitive to ionizing radiation because of a deficiency in the repair of double-strand breaks. Hypertonic treatment caused radiosensitization due to a fixation of potentially lethal damage (PLD) in scid cells, demonstrating that scid cells normally repair PLD. To assess the kinetics of the repair of PLD, hypertonic treatment was delayed for various times after irradiation. Potentially lethal damage was repaired during these times in isotonic medium at 37 degrees C. It was found that the rate of repair of PLD was much slower in scid cells than in BALB/c 3T3 cells, which have a "wild-type" level of radiosensitivity. This fact indicates that the scid mutation affects the type of repair of PLD that is sensitive to 0.5 M NaCl treatment. In scid hybrid cells containing fragments of human chromosome 8, which complements the radiosensitivity of the scid cells, the rate of repair was restored to a normal level. An enzyme encoded by a gene on chromosome 8 may also be connected with PLD which is sensitive to hypertonic treatment.

Apoptosis induced by X-rays and chemical agents in murine fibroblastic cell lines with a defect in repair of DNA double-strand breaks.

PURPOSE: To investigate apoptosis in murine fibroblasts that are deficient in DNA double-stand breaks (dsb) repair. MATERIALS AND METHODS: BALB/c 3T3 cells and cells from a severe combined immunodeficient (scid) mouse were exposed to X-rays, UV light, bleomycin, etoposide or cisplatin. After exposure, the rate of apoptosis was assayed by propidium iodide staining based on characteristic morphological change. RESULTS: The scid cells, defective in dsb repair, were extremely sensitive to apoptosis induced by X-rays and bleomycin compared with BALB/c 3T3 cells. The scid cells were slightly sensitive to apoptosis induced by etoposide, an agent producing protein-associated dsb, and to apoptosis induced by agents not causing dsb. CONCLUSIONS: These studies show that sensitivity of cells to apoptosis induced by X-rays or bleomycin is enhanced by the scid mutation and that apoptosis induced by etoposide, which causes protein-associated dsb, is not greatly affected. There may be different processes involved in the induction of apoptosis by X-rays or bleomycin and etoposide.

[Inhibitory factors of calcium oxalate crystal growth in urinary macromolecules].

We studied the inhibitory effect of urinary glycosaminoglycans (GAGs), in the natural form, on calcium oxalate crystals. Control urine was collected from five healthy subjects and filtered by 5 microns and 0.22 micron membranes. The urine was cut off by a membrane of 30,000 M.W. and the remaining urine was fractionated into three groups by anion-exchange chromatography on DEAE-cellulose: peak A contained only protein, peak B both protein and GAGs and peak C only GAGs. Peak A and peak B were fractionated again through a Sephacryl S-200 gel column. Each fractionated sample was examined for the inhibitory effect on calcium oxalate crystal growth using the 14C-oxalate seeded crystal growth assay in a metastable solution developed by Koide et al. GAGs were determined by two-dimensional electrophoresis on cellulose acetate membrane. SDS-polyacrylamide electrophoresis was used to determine the molecular weights of proteins. Peak A was found to have many kinds of proteins which had low inhibitory effects. Peak B had three kinds of proteins such as those with molecular weights of 130,000, 45,000 and 35,000 as well as uronic acid. These proteins and uronic acid had high inhibitory effect and the highest inhibitory effect was found in the fraction of around 50,000 M.W. where protein and uronic acid were contained. The molecular weight of this protein was 42,000 by SDS electrophoresis. Uronic acid was a keratan sulphate by a two-dimensional electrophoresis. In peak C there were mainly chondroitin sulphate and a small amount of heparan sulfate which had high inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)

[Isolation of urinary heparan sulfate and chondroitin sulfate and their inhibitory activity of calcium oxalate crystal growth].

Urinary heparan sulfate (U-HS) and chondroitin sulfate (U-ChS) were isolated and partially purified using two steps of anion-exchange chromatography (DE-52 and Dowex 1 x 2). Uronic acid content of these urinary glycosaminoglycans (GAGs) and commercial heparan sulfate (C-HS) and chondroitin-6-sulfate (C-C6S) was as follows; U-HS: 22.4%, U-ChS: 32.0%, C-HS: 41.6%, C-C6S: 36.0%. The inhibitory activity of these GAGs on calcium oxalate crystal growth was examined using the crystal-seed system described by Koide et al. The influence of uric acid on the inhibitory activity of the GAGs was also studied. U-HS and U-ChS exhibited nearly equal inhibitory activity to their respective commercial preparations. The inhibitory activity of U-HS was stronger than that of U-ChS. Uric acid suppressed the inhibitory activity of both, and the degree of suppression was stronger in U-ChS than in U-HS. The molecular weight of the urinary GAGs was compared to that of the commercial chondroitin-6-sulfate (C6S; m.w. between 40,000 and 80,000) by gel-filtration using Sephacryl S-300HR. C6S was eluted slightly behind the void volume. U-ChS was eluted much behind C6S. Therefore, the molecular weight of U-ChS was thought to be much smaller than that of C6S. U-HS was eluted into two peaks; the first one at the void volume and the second one between the peaks of C6S and U-ChS. These findings indicate the possibility that part of U-HS exists in the form of proteoglycan.

[A comparative clinical trial of UFT medication and intravesical BCG in the recurrence of superficial bladder cancer. Study Group of UFT and BCG adjuvant therapy for bladder cancer].

In sixty-four patients who had TUR-bt for superficial bladder cancer, the effect of intravesical BCG and UFT medication were compared. Group A (n = 20) were treated with BCG, group B (n = 22) were treated with UFT and group C (n = 22) were treated with both agents. The patients were followed up for more than 12 months by cystoscopy at a interval of 3 months, urinary cytology and bladder cold cup biopsy, if necessary. Thirteen of the 20 patients in group A and 13 of the 22 patients in group B were free of tumors, compared to 5 of the 22 patients in group C. Although the recurrence-free-rate in each group does not differ significantly, the combination therapy of intravesical BCG and UFT medication seems to decrease the rate of tumor recurrence for superficial bladder cancer. Side effects of BCG and UFT were tolerated well.

Cell line-dependent changes in sensitivity to mitomycin C by 1-methyl-3-isobutylxanthine is due to an altered intracellular dose of the anticancer drug and/or to changes in DT-diaphorase activity.

Sensitization by 1-methyl-3-isobutylxanthine (MIX), a potent inhibitor of cAMP phosphodiesterase, of cellular sensitivity to mitomycin C (MC) was tested using various cell lines. Sensitization was seen with CHO cells and Balb/c 3T3 cells, but a decrease in sensitivity was seen with HeLa, K-balb and other cell lines. To measure the extent of crosslinks produced by MC, we used an alkaline elution assay. The extent of crosslinks was increased by MIX in CHO cells and decreased in K-balb cells. These changes in extent are well reflected by changes in sensitivity to MC in both cell lines. In CHO cells, an intracellular dose of MC was increased by MIX with no change in the rate of uptake or efflux of MC. There was a slight decrease in the dose in HeLa or K-balb cells. Among the enzymes which engage in the reductive activation of MC, we tested DT-diaphorase and NADPH-cytochrome P450 reductase using CHO, HeLa and K-balb cells. MIX had almost no effect on the activity of NADPH-cytochrome P450 in each cell line, whereas it suppressed the activity of DT-diaphorase, significantly in HeLa and K-balb cells, and slightly in CHO cells. All these facts indicate that MIX caused an increase in the extent of crosslinks via an increase in the intracellular dose of MC in CHO cells, and that these increases may lead to the sensitization. On the other hand, the decrease in the sensitivity shown in HeLa and K-balb cells may be due to the suppression of DT-diaphorase activity and/or to a decrease in MC dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Neocarzinostatin-induced mutations at the hprt locus in exponentially growing CHO cells, compared with spontaneous mutations.

Spontaneous mutations and neocarzinostatin-induced mutations were investigated in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in exponentially growing Chinese hamster ovary cells. Neocarzinostatin (NCS) treatment caused an 4.5-fold increase in mutation frequency. Analysis by multiplex polymerase chain reaction and sequencing of hprt cDNA revealed that spontaneous mutations in this system were characterized by a relatively high rate of large deletions and double-base substitutions, and a low rate of splice mutations compared with data reported in fibroblastic cell lines. NCS hardly affected this spectrum of spontaneous mutations, which indicates the rare incidence of NCS-specific change in the exponential growth phase. This is in contrast to aprt gene mutations reported in plateau phase cells in which base substitutions occur preferentially at sites affected by NCS. These results suggest that differences in the loci assayed or in the processes involved in mammalian mutagenesis in the exponential growth phase and the plateau phase may be the source of the different results.

Immuno-localization of H+/peptide cotransporter in rat digestive tract.

In the mammalian digestive tract, small peptides are absorbed by a H+-coupled peptide transport system. Using an antibody against the rat H+/peptide cotransporter (PepT1), we examined the localization of PepT1 immunohistochemically along the rat digestive tract. PepT1 was detected in the small intestine (duodenum, jejunum, and ileum), but not in the esophagus, stomach, colon, or rectum. PepT1 was especially enriched in the villi, where it was localized in the brush border of the absorptive epithelial cells. PepT1 was not detected in the mucus-secreting goblet cells or less-differentiated epithelial cells in the crypts. These observations show that PepT1 is specific to the brush border of the differentiated absorptive epithelial cells and suggest that H+-coupled uptake of small peptides and peptide-like drugs occurs at the apical membrane of these cells in the small intestine.