Replication of X chromosomes in complete moles

Summary DNA replication patterns of X chromosomes in complete hydatidiform moles were studied using cultured fibroblasts from three 46,XX moles resulting from duplication of a haploid sperm, and from a 46,XY mole originating from dispermy. Control cultures included skin fibroblasts from an adult woman and a female fetus as well as PB lymphocytes from an adult woman. Cultures were treated with 5-bromodeoxyuridine for the last 2–4h of the S phase, and the chromosome slides prepared were stained by the Hoechst 33258-Giemsa procedure. Each of the three XX moles studied revealed one early-replicating and one late-replicating X chromosomes, while the XY mole revealed one early-replicating X chromosome. DNA replication patterns of molar X chromosomes were similar to those of adult and fetal fibroblasts, but different from those in adult lymphocytes. These findings indicate that DNA replication kinetics of molar fibroblasts are tissue-specific rather than origin- or developmental-stage specific.     

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.     

Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.     

Stereoselective total synthesis of ceramide Di-, Tri- and tetrahexosides of wheat flour

The first total synthesis of glycosphingolipids isolated from wheat flour has been achieved in a regio- and stereo-controlled manner.Abbreviations THF tetrahydrofuran - DMF dimethylformamide Part 53 in the series Synthetic Studies on Cell Surface Glycans     

Role in translation of a triple tandemly repeated sequence in the 5''-untranslated region of human thymidylate synthase mRNA.

A triple tandem repeat (TTR) consisting of 90 nucleotides exists immediately upstream of the ATG initiator codon in human thymidylate synthase (TS) cDNA (pcHTS-1). To investigate the role of the TTR in the expression of the TS cDNA, we used pcHTS-1 to construct mutant cDNA clones in which part of the TTR was deleted or an additional element was inserted. The mutant cDNA plasmid was introduced into murine TS-negative mutant cells and the relative translation efficiencies of the mutant cDNAs were determined by measuring the transient expression of TS activity and the amount of TS mRNA transcribed. The translation efficiency in transient expression of the mutants was increased by deletions covering all the first two repeated elements, and the part of the third closest to the ATG initiator codon, but was not affected by deletions of only parts of the first two repeated elements at the 5' end. The translation efficiency was also not affected by insertion of an additional repeated element into the TTR. These results suggest that the first two repeated elements at the 5' end both have inhibitory effects on translation of the TS mRNA, probably due to the unique structural feature of this element.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

A disease with features of cutis laxa and Ehlers-Danlos syndrome

Summary A mother and daughter are described with light and electron microscopic, and biochemical abnormalities of their connective tissue characteristic of both cutis laxa and the Ehlers-Danlos syndrome. The mother was clinically normal, while her 8-year-old daughter exhibited loose, wrinkled skin and other clinical features of cutis laxa, and also fragility, bruisability and hyper-extensibility of the skin and poor healing of wounds, leaving cigarette paper scars, features characteristic of the Ehlers-Danlos syndrome. Light and electron microscopic studies of skin biopsy specimens and cultured skin fibroblasts from both individuals revealed reduced and distorted elastic fibres, a finding usually seen in cutis laxa. Electrophoretic studies of collagen excreted from cultured skin fibrobasts revealed in both individuals and alpha 2(I) chain with a molecular size smaller than usual. The father and elder daughter were normal by clinical, light and electron microscopic and electrophoretic studies. It was concluded from these findings that the mother and daughter represented a hitherto undescrbed disease of the connective tissue with dominant inheritance and variable expressivity.     

Clinical studies on cell-mediated immunity in patients with renal cell carcinoma: interleukin-2 and interferon-γ production of lymphocytes

Summary The authors examined interleukin-2 (IL-2) production and interferon (IFN) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, 5 cm and >5 cm. The production of IL-2 and IFN was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN production was impaired. However, in the patients with tumors 5 cm, IFN production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN production.     

A comparative study on immunosuppressive effects of cyclosporin A and FK 506 on peripheral blood lymphocytes in dogs

Immunosuppressive effects of cyclosporin A (CsA) and FK 506 (FK) on peripheral blood lymphocytes were studied in dogs in respect to mixed lymphocyte reaction, proliferative responses to recombinant interleukin-2 (rIL-2), phytohemagglutinin (PHA) and concanavalin-A (Con-A); phenotypes of OKIa1, CD3, CD8 and surface IgM; cytotoxic activity against xenogeneic tumor cells. CsA (2.0 or 5.0 mg/kg, intravenously) or FK (0.16 mg/kg, intramuscularly) was given to mongrel dogs every morning for serial 21 days. The blood concentrations of CsA, measured as trough levels by fluorescence polarization method, ranged from 37 to 350 ng/ml in dogs administered at 2.0 mg/kg and from 170 to 894 ng/ml in dogs administered at 5.0 mg/kg during treatment, respectively. In dogs treated with FK at a dose of 0.16 mg/kg, the drug concentrations in the plasma during treatment ranged from 0.16 to 1.8 ng/ml. Mixed lymphocyte reaction and proliferative responses to rIL-2, PHA and Con-A, which were declined by CsA, were not affected by FK. In contrast, the proportion of OKIa1+ cells was not affected by CsA, whereas FK decreased the proportion of OKIa1+ cells progressively during the course of treatment. Cytotoxic activity was suppressed by both CsA and FK. These results possibly indicate that CsA and FK exert their immunosuppressive effects via different mechanisms.     

A preliminary note on the intestinal parasites of wild chimpanzees in the Mahale Mountains,Tanzania

Feces of wild chimpanzees in the Mahale Mountains, Tanzania, were inspected for intestinal parasites under a compound microscope. Eggs or larvae ofOesophagostomum, Strongyloides, Trichuris, Prosthenorchis, andBertiella were found. Intestinal nematodes significantly increased in the mid-rainy season. This finding supports (or, at least, is not in conflict with) the hypothesis thatAspilia leaves which are occasionally swallowed by chimpanzees may function as a vermicide, since ingestion of such leaves also increases significantly in the mid-rainy season.