Novel DNA polymorphism in the mouse tumor necrosis factor receptors type 1 and type 2

The introduction of the polymerase chain reaction (PCR) provides an entirely new means of analyzing DNA polymorphism and makes practical the analysis of length variation in simple-sequence tandem repeats of dinucleotides. In the process of cloning and sequencing the mouse genomic DNA for tumor necrosis factor (TNF) receptors type 1 and type 2, we identified two simple dinucleotide repeats within the noncoding regions of TNF receptor type 1 and three such sequences within TNF receptor type 2. PCR analysis of these sequences, using genomic DNA from 21 different inbred and wild mouse strains, as demonstrated by running the amplified products on sequencing gels, showed that the repeats are highly polymorphic. We identified seven alleles of TNF receptor type 2 and five alleles of TNF receptor type 1. Using these polymorphic markers in two sets of recombinant inbred strains of mice, the chromosomal localization of Tnfr-1 was mapped to mouse chromosome 6 and Tnfr-2 was located to the distal portion of mouse chromosome 4.     

Characteristics of the energy-transducing NADH-quinone oxidoreductase ofParacoccus denitrificans as revealed by biochemical,biophysical, and molecular biological approaches

A comparison of the mitochondrial NADH-ubiquinone oxidoreductase and the energy-transducing NADH-quinone oxidoreductase (NDH-1) ofParacoccus denitrificans revealed that both systems have similar electron-transfer and energy-transduction pathways. In addition, both complexes are sensitive to the same inhibitors and contain similar electron carriers, suggesting that theParacoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding theParacoccus NDH-1 has been cloned and sequenced. It is composed of 18,106 base pairs and contains 14 structural genes and six unidentified reading frames (URFs). The structural genes, URFs, and their polypeptides have been characterized. We also discuss nucleotide sequences which are believed to play a role in the regulation of the NDH-1 gene cluster andParacoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.     

Determination of non-protein-bound iron in human synovial fluid by high-performance liquid chromatography with electrochemical detection

Non-protein-bound iron in human synovial fluid was determined using high-performance liquid chromatography with electrochemical detection. The procedure was based on the separation of the iron—diethylenetriaminepentaacetic acid (DPTA) complex formed directly on a chromatographic column containing an anion-exchange resin followed by electrochemical detection. The method enabled more than 0.1 μM Fe(III) to be determined with an injection volume of 10 μl. A mixture of synovial fluid, 20 μM DTPA and acetate buffer was incubated in the presence and absence of superoxide (O⁻₂) generated by a xanthine—xanthine oxidase system and was ultrafiltered through a 30 000 molecular mass cut-off filter. No iron was detected in the ultrafiltrate at physiological pH. However, the presence of iron was observed in the ultrafiltrate at low pH, and O⁻₂ and decreased pH, iron may be released into the synovial fluid.     

Changes in the Content of Two Glutamate Synthase Proteins in Spikelets of Rice (Oryza sativa) Plants during Ripening

Nitrogen accumulation in the apical spikelets on the primary branches of the main stem of rice plants have been studied during the ripening process (0-35 d after flowering). The level of NADH-dependent glutamate synthase (GOGAT) protein and activity increased 4- and 6-fold, respectively, in the first 15 d after flowering. Maximum levels of NADH-GOGAT were found at that time when the spikelets had just begun to increase in dry weight and to accumulate storage proteins. Subsequently, both the level of NADH-GOGAT protein and its activity in spikelets declined rapidly. Although changes in ferredoxin (Fd)-dependent GOGAT paralleled changes in NADH-GOGAT, the relative abundance of NADH-GOGAT protein in the spikelets was about 3 times higher than that of Fd-GOGAT from 5 to 15 d after flowering. When the chaff (lemma and palea) was separated from the spikelets 10 d after the flowering, 16% of the NADH-GOGAT protein was found in the chaff and 84% in the young grain tissues (endosperm, testae, aleurone tissues, and embryo). On the other hand, Fd-GOGAT protein was distributed 52% in the chaff and 48% in the young grain tissues in spikelets of the same age. Activity of NADP-isocitrate dehydrogenase, which may generate the 2-oxoglutarate required for the GOGAT reactions, was much higher than that of total GOGAT activities on a spikelet basis during the ripening process. These results suggest that in rice plants NADH-GOGAT is responsible for the synthesis of glutamate from the glutamine that is transported from senescing tissues to the spikelets.     

Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in transgenic rice.

A previously isolated rice (Oryza sativa) rbcS gene was further characterized. This analysis revealed specific sequences in the 5' regulatory region of the rice rbcS gene that are conserved in rbcS genes of other monocotyledonous species. In transgenic rice plants, we examined the expression of the beta-glucuronidase (gusA) reporter gene directed by the 2.8-kb promoter region of the rice rbcS gene. To examine differences in the regulation of monocotyledonous and dicotyledonous rbcS promoters, the activity of a tomato rbcS promoter was also investigated in transgenic rice plants. Our results indicated that both rice and tomato rbcS promoters confer mesophyll-specific expression of the gusA reporter gene in transgenic rice plants and that this expression is induced by light. However, the expression level of the rice rbcS-gusA gene was higher than that of the tomato rbcS-gusA gene, suggesting the presence of quantitative differences in the activity of these particular monocotyledonous and dicotyledonous rbcS promoters in transgenic rice. Histochemical analysis of rbcS-gusA gene expression showed that the observed light induction was only found in mesophyll cells. Furthermore, it was demonstrated that the light regulation of rice rbcS-gusA gene expression was primarily at the level of mRNA accumulation. We show that the rice rbcS gene promoter should be useful for expression of agronomically important genes for genetic engineering of monocotyledonous species.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

Biosynthesis of brassinolide from castasterone in cultured cells of Catharanthus roseus

Feeding experiments with tritium- and deuterium-labeled castasterone (CS) were conducted with three cell lines of Catharanthus roseus, including crown gall cells and nontransformed cells. In all three cell lines, the conversion of CS to brassinolide (BL) was observed and unequivocally confirmed by gas chromatography/mass spectrometry (GC/MS). This is the first conclusive evidence that CS is the biosynthetic precursor of BL.Biosynthesis of brassinosteroids in Catharanthus roseus. Part II. Part I of this series: Yokota et al. (1990a).     

The Chromosomal Organization of the Human Endogenous Retrovirus-like Sequence HERV-H: Clustering of the HERV-H Sequences in a 300-kb Region Close to the GRPR Locus on the X Chromosome

Within the haploid genome there are approximately 1,000 copiesof the human endogenous retroviruslike sequence, HERV-H. Althoughthese sequences are scattered throughout the entire genome,in situ hybridization experiments revealed that there are discreteclusters positioned on chromosomes 1p and 7q. In this study,we have located three HERV-H sequences which were unexpectedlyclustered within a 300-kilobase region close to the GRPR locuson the X chromosome. In previous studies, no clusteringof thissequence has been reported at this locus. Our finding demonstratesthat, like other repetitive sequences, clustering of HERV-Hoccurs in the human genome, although these sequences may notalways be detected by in situ hybridization methods.