Nuclear and chloroplast DNA differentiation in Andean potatoes.

Over 3500 accessions of Andean landraces have been known in potato, classified into 7 cultivated species ranging from 2x to 5x (Hawkes 1990). Chloroplast DNA (ctDNA), distinguished into T, W, C, S, and A types, showed extensive overlaps in their frequencies among cultivated species and between cultivated and putative ancestral wild species. In this study, 76 accessions of cultivated and 19 accessions of wild species were evaluated for ctDNA types and examined by ctDNA high-resolution markers (ctDNA microsatellites and H3 marker) and nuclear DNA restriction fragment length polymorphisms (RFLPs). ctDNA high-resolution markers identified 25 different ctDNA haplotypes. The S- and A-type ctDNAs were discriminated as unique haplotypes from 12 haplotypes having C-type ctDNA and T-type ctDNA from 10 haplotypes having W-type ctDNA. Differences among ctDNA types were strongly correlated with those of ctDNA high-resolution markers (r = 0.822). Differentiation between W-type ctDNA and C-, S-, and A-type ctDNAs was supported by nDNA RFLPs in most species except for those of recent or immediate hybrid origin. However, differentiation among C-, S-, and A-type ctDNAs was not clearly supported by nDNA RFLPs, suggesting that frequent genetic exchange occurred among them and (or) they shared the same gene pool owing to common ancestry.     

Early embryonic death-associated changes in genome-wide gene expression profiles in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo

Successful somatic nuclear transfer-derived cloning has been reported in cattle; however, the cloned embryo is highly susceptible to death around day 60 of gestation leading to early embryonic loss. The early embryonic death is postulated to possibly arise in part from an atypical placentation. We have performed cDNA macroarray analysis using 3,353 of the previously cataloged 4,165 genes, in order to characterize the early embryonic death-associated changes in genome-wide gene expression profiles in the fetal placenta of the cow carrying somatic nuclear transfer-derived cloned embryo. A more marked difference in the expression profiles was observed between the fetal placentas of the cows with the cloned immotile embryo (CD) and with the cloned motile embryo (CL) or artificial insemination-derived motile embryo (AI), as compared to between the CL and AI placentas, suggesting an aberration of the expression profile in the CD placenta among the three placentas. Further, 291 and 77 genes showed more than twofold elevation and less than 50% reduction, respectively, in either or both of two CD (CD1 and CD2) placentas in comparison with the CL placenta, but no differential expression between the CL and AI placentas. The expression patterns of six genes in the AI, CL, and CD placentas were confirmed in an experiment with an additional sample for each of the three placentas. Among the placental genes showing the early embryonic death-associated changes of expression in the cow with the cloned embryo, IGF2 (elevated gene), and HBA1, HBA2, SPTB, and SPTBN1 genes (reduced gene) are intriguing in that the changes of expression in these genes were observed in an additional sample of CD placenta as well as the CD1 and CD2 placentas, and in that overexpression (for IGF2) and dysfunction or deficiency (for HBA1, HBA2, SPTB, and SPTBN1) result in embryonic lethality.     

Bead-like passage of chloride ions through ClC chloride channels

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H+-Cl- exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open-close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H+, Na+, K+, or H3O+ in the intracellular region to elucidate the open-close mechanism, and to clarify the role of H+ ion an H+-Cl- exchange transporter. Our simulations revealed that H+ and Na+ caused channel opening and the passage of Cl- ions. Na+ induced a bead-like string of Cl- -Na+-Cl--Na+-Cl- ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.     

Tti1 and Tel2 Are Critical Factors in Mammalian Target of Rapamycin Complex Assembly

Mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family and is a major regulator of translation, cell growth, and autophagy. mTOR exists in two distinct complexes, mTORC1 and mTORC2, that differ in their subunit composition. In this study, we identified KIAA0406 as a novel mTOR-interacting protein. Because it has sequence homology with Schizosaccharomyces pombe Tti1, we named it mammalian Tti1. Tti1 constitutively interacts with mTOR in both mTORC1 and mTORC2. Knockdown of Tti1 suppresses phosphorylation of both mTORC1 substrates (S6K1 and 4E-BP1) and an mTORC2 substrate (Akt) and also induces autophagy. S. pombe Tti1 binds to Tel2, a protein whose mammalian homolog was recently reported to regulate the stability of PIKKs. We confirmed that Tti1 binds to Tel2 also in mammalian cells, and Tti1 interacts with and stabilizes all six members of the PIKK family of proteins (mTOR, ATM, ATR, DNA-PKcs, SMG-1, and TRRAP). Furthermore, using immunoprecipitation and size-exclusion chromatography analyses, we found that knockdown of either Tti1 or Tel2 causes disassembly of mTORC1 and mTORC2. These results indicate that Tti1 and Tel2 are important not only for mTOR stability but also for assembly of the mTOR complexes to maintain their activities.     

Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the ∆snf2 disruptant produces a significant increase in its enzyme activity

We previously found that overexpression of DGA1 encoding diacylglycerol acyltransferase (DGAT) in the ∆snf2 disruptant of Saccharomyces cerevisiae caused a significant increase in lipid accumulation and DGAT activity. The present study was conducted to investigate how Dga1p is activated in the ∆snf2 disruptant. To analyze the expression of Dga1p in wild type and the ∆snf2 disruptant, we overexpressed Dga1p with a 6x His tag at the N-terminus and a FLAG tag at the C-terminus. Immunoblotting using anti-6x His and anti-FLAG antibodies revealed that, in addition to full-length protein, Dga1p lacking the N-terminus was produced only in the ∆snf2 disruptant. Full-length Dga1p and N-terminally truncated Dga1p were separated and purified from the lipid body fraction by using anti-FLAG M2 agarose and TALON metal affinity resin. Major DGAT activity was recovered in the purified fraction of N-terminally truncated Dga1p, indicating that proteolytic cleavage at the N-terminal region is involved in DGAT activation in the ∆snf2 disruptant. Analysis of the cleavage site of N-terminally truncated Dga1p revealed a major site between Lys-29 and Ser-30. We then overexpressed truncated Dga1p variants that lacked different N-terminal amino acids and had a FLAG tag at the C-terminus. The homogenate and lipid body fraction of the ∆snf2 disruptant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1∆N2p) had higher DGAT activity than that overexpressing Dga1p, indicating that Dga1∆N2p is activated Dga1p. Dga1∆N2p-FLAG(C-terminus) was purified to near homogeneity by anti-FLAG M2 agarose chromatography and maintained significant DGAT activity. These results provide a new strategy to engineer expression of DGAT.     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.     

Neurosteroids in the Purkinje Cell: Biosynthesis, Mode of Action and Functional Significance

Neurosteroids are synthesized de novo from cholesterol in the brain. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently the Purkinje cell, an important cerebellar neuron, has been identified as a major site for neurosteroid formation in vertebrates. This is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. Since this discovery, organizing actions of neurosteroids are becoming clear by the studies using the Purkinje cell as an excellent cellular model. In mammals, the Purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life. Both progesterone and estradiol promote dendritic growth, spinogenesis, and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such organizing actions that may be mediated by neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), contribute to the formation of cerebellar neuronal circuit during neonatal life. Allopregnanolone, a progesterone metabolite, is also synthesized in the cerebellum and acts on Purkinje cell survival in the neonate. This review summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of neurosteroids in the Purkinje cell.     

Natural killer cell lytic activity and CD56(dim) and CD56(bright) cell distributions during and after intensive training.

The purpose of this study was to examine the impact of intensive training for competitive sports on natural killer (NK) cell lytic activity and subset distribution. Eight female college-level volleyball players undertook 1 mo of heavy preseason training. Volleyball drills were performed 5 h/day, 6 days/wk. Morning resting blood samples were collected before training (Pre), on the 10th day of training (During), 1 day before the end of training (End), and 1 wk after intensive training had ceased (Post). CD3(-)CD16(bright)CD56(dim) (CD56(dim) NK), CD3(-)CD16(dim/-)CD56(bright) NK (CD56(bright) NK), and CD3(+)CD16(-)CD56(dim) (CD56(dim) T) cells in peripheral blood were determined by flow cytometry. The circulating count of CD56(dim) NK cells (the predominant population, with a high cytotoxicity) did not change, nor did the counts for other leukocyte subsets. However, counts for CD56(bright) NK and CD56(dim) T cells (subsets with a lower cytotoxicity) increased significantly (P < 0.01) in response to the heavy training. Overall NK cell cytotoxicity decreased from Pre to End (P = 0.002), with a return to initial values at Post. Lytic units per NK cell followed a similar pattern (P = 0.008). Circulating levels of interleukin-6, interferon-gamma, and tumor necrosis factor-alpha remained unchanged. These results suggest that heavy training can decrease total NK cell cytotoxicity as well as lytic units per NK cell. Such effects may reflect in part an increase in the proportion of circulating NK cells with a low cytotoxicity.     

Synthesis and biological evaluation of alkoxycoumarins as novel nematicidal constituents

We synthesized all of the monomethoxycoumarins, 5-alkoxycoumarins and their derivatives, and investigated their nematicidal activity against the phytopathogenic nematode, Bursaphelenchus xylophilus. Among the compounds, 5-ethoxycoumarin showed the highest nematicidal activity. Furthermore, 5-ethoxycoumarin was comparatively harmless against both the brine shrimps, Artemia salina, and the Japanese killifish, Oryzias latipes.