Rearrangements of the DNA in carbon ion-induced mutants of Arabidopsis thaliana

To elucidate the nature of structural alterations in plants, three carbon ion-induced mutations in Arabidopsis thaliana, gl1-3, tt4(C1), and ttg1-21, were analyzed. The gl1-3 mutation was found to be generated by an inversion of a fragment that contained GL1 and Atpk7 loci on chromosome 3. The size of the inverted fragment was a few hundred kilobase pairs. The inversion was found to accompany an insertion of a 107-bp fragment derived from chromosome 2. The tt4(C1) mutation was also found to be due to an inversion. The size of the intervening region between the breakpoints was also estimated to be a few hundred kilobase pairs. In the case of ttg1-21, it was found that a break occurred at the TTG1 locus on chromosome 5, and reciprocal translocation took place between it and chromosome 3. From the sequences flanking the breakpoints, the DNA strand breaks induced by carbon ions were found to be rejoined using, if present, only short homologous sequences. Small deletions were also observed around the breakpoints. These results suggest that the nonhomologous end-joining (NHEJ) pathway operates after plant cells are exposed to ion particles.     

WUSCHEL-RELATED HOMEOBOX4 Is Involved in Meristem Maintenance and Is Negatively Regulated by the CLE Gene FCP1 in Rice

The shoot apical meristem is the ultimate source of the cells that constitute the entire aboveground portion of the plant body. In Arabidopsis thaliana, meristem maintenance is regulated by the negative feedback loop of WUSCHEL-CLAVATA (WUS-CLV). Although CLV-like genes, such as FLORAL ORGAN NUMBER1 (FON1) and FON2, have been shown to be involved in maintenance of the reproductive meristems in rice (Oryza sativa), current understanding of meristem maintenance remains insufficient. In this article, we demonstrate that the FON2-LIKE CLE PROTEIN1 (FCP1) and FCP2 genes encoding proteins with similar CLE domains are involved in negative regulation of meristem maintenance in the vegetative phase. In addition, we found that WUSCHEL-RELATED HOMEOBOX4 (WOX4) promotes the undifferentiated state of the meristem in rice and that WOX4 function is associated with cytokinin action. Consistent with similarities in the shoot apical meristem phenotypes caused by overexpression of FCP1 and downregulation of WOX4, expression of WOX4 was negatively regulated by FCP1 (FCP2). Thus, FCP1/2 and WOX4 are likely to be involved in maintenance of the vegetative meristem in rice.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

Surgical stress during operation for gastrointestinal cancer increases plasma thioredoxin levels and decreases mitochondrial membrane potential in peripheral blood lymphocytes

Surgical stress is difficult to evaluate quantitatively. It has been reported that mitochondrial membrane potential (delta psi(m)) in the peripheral blood lymphocytes (PBLs) is decreased by surgical stress. Thioredoxin (TRX), a small protein with redox-active dithiol/disulfide in the active site, is induced by a variety of oxidative stresses and secreted from the cells. Accumulating evidence shows that plasma levels of TRX are elevated in oxidative stress-associated disorders. In the present study, we examined plasma levels of TRX in cases undergoing operations for gastrointestinal cancer. Plasma levels of TRX were significantly elevated on the first postoperative day compared with the pre-operative levels. The changes in the plasma TRX levels tended to show an inverse relationship with the changes in delta psi(m) in PBLs, which shows a significant decrease caused by surgical stress. Plasma TRX levels as well as delta psi(m) in PBLs are valuable markers to evaluate surgical stress.     

Bioconversion of organosilicon compounds by horse liver alcohol dehydrogenase: the role of the silicon atom in enzymatic reactions

Summary Bioconversion of three organosilicon compounds of different chain length between the silicon atom and the hydroxyl group (Me₃Si(CH₂)_nOH, n = 1–3) by horse liver alcohol dehydrogenase (HLADH, EC 1.1.1.1.) was studied. Furthermore, the effect of the silicon atom on the HLADH-catalysed reaction was examined in comparison with the corresponding carbon compounds. HLADH could catalyse the dehydrogenation of trimethylsilyeethanol (n = 2) and trimethylsilylpropanol (n = 3). Trimethylsilylethanol was a better substrate than both its carbon analogue, 3,3-dimethylbutanol, and ethanol. The improved activity of HLADH on trimethylsilylethanol could be accounted for by a higher affinity toward HLADH and a lower activation energy of the reaction by HLADH than those of the carbon counterpart. These are derived from physical properties of the silicon atom, that is, the lower electronegativity and the bigger radius than those of the carbon atom. In contrast, HLADH showed no activity on trimethylsilylmethanol (n = 1), whereas it catalysed the dehydrogenation of the carbon analogue, 2,2-dimethylpropanol, fairly well. The reason for the inactivity of HLADH in the case of trimethylsilylmethanol based on the electric effect of the silicon atom is also discussed. Offsprint requests to: A. Tanaka     

Microcalorimetric measurements of heat production in isolated rat brown adipocytes.

Heat production, oxygen consumption, and lipolysis in isolated interscapular brown adipocytes from the rat were investigated. Epinephrine, norepinephrine, and isoproterenol increased heat production in a concentration-dependent manner, showing, about 6-, 4-, and 5-fold higher effects than controls, respectively. The concentration of isoproterenol for threshold heat production and glycerol release were 10(-10) M and 10(-9) M, respectively. The fact that 10(-9) M isoproterenol increased heat production by about 3-fold while glycerol release had no effect at all indicates that calorimetry is more appropriate for investigation of brown adipocytes. At least the method is more sensitive than that of measuring glycerol release.     

Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2^271-279). The CTLs induced by PEPP2^271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.     

Characterization of dehydropeptidase I in the rat lung

The activity of dehydropeptidase I in rat tissues decreases in the order of lung greater than kidney greater than liver-spleen greater than other tissues, while aminopeptidase activity is high in the kidney, and lower in the lung than in other tissues. Dehydropeptidase I was solubilized from the membrane fraction of rat lung by treatment with papain and purified by DEAE-cellulose column chromatography, affinity chromatography on concanavalin-A-Sepharose and high-performance liquid chromatography gel filtration. The purified preparation was found to be homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The relative molecular mass was estimated to be 150,000 by gel filtration, comprising a homodimer of two 80,000-Mr subunits. The enzyme activity was inhibited by cilastatin, o-phenanthroline and ATP. This enzyme catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and N-ethylmaleimide-S-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to E4. Furthermore it catalyzed a hydrolytic splitting of L-Leu-L-Leu, but not S-benzyl-L-cysteine p-nitroanilide, which is a good substrate for aminopeptidase. Our enzyme preparation was immunologically identical to the rat renal dehydropeptidase I. The physiological significance of the pulmonary dehydropeptidase I on the metabolism of glutathione and its adducts is discussed.