Transcriptional activities of nuclear SREBP-1a, -1c,and -2 to different target promoters of lipogenic and cholesterogenic genes

Recent studies on the in vivo roles of the sterol regulatory element binding protein (SREBP) family indicate that SREBP-2 is more specific to cholesterogenic gene expression whereas SREBP-1 targets lipogenic genes. To define the molecular mechanism involved in this differential regulation, luciferase-reporter gene assays were performed in HepG2 cells to compare the transactivities of nuclear SREBP-1a, -1c, and -2 on a battery of SREBP-target promoters containing sterol regulatory element (SRE), SRE-like, or E-box sequences. The results show first that cholesterogenic genes containing classic SREs in their promoters are strongly and efficiently activated by both SREBP-1a and SREBP-2, but not by SREBP-1c. Second, an E-box containing reporter gene is much less efficiently activated by SREBP-1a and -1c, and SREBP-2 was inactive in spite of its ability to bind to the E-box. Third, promoters of lipogenic enzymes containing variations of SRE (SRE-like sequences) are strongly activated by SREBP-1a, and only modestly and equally by both SREBP-1c and -2. Finally, substitution of the unique tyrosine residue within the basic helix-loop-helix (bHLH) portion of nuclear SREBPs with arginine, the conserved residue found in all other bHLH proteins, abolishes the transactivity of all SREBPs for SRE, and conversely results in markedly increased activity of SREBP-1 but not activity of SREBP-2 for E-boxes. These data demonstrate the different specificity and affinity of nuclear SREBP-1 and -2 for different target DNAs, explaining a part of the mechanism behind the differential in vivo regulation of cholesterogenic and lipogenic enzymes by SREBP-1 and -2, respectively.     

Construction of prediction models for growth traits of soybean cultivars based on phenotyping in diverse genotype and environment combinations

As soybean cultivars are adapted to a relatively narrow range of latitude, the effects of climate changes are estimated to be severe. To address this issue, it is important to improve our understanding of the effects of climate change by applying the simulation model including both genetic and environmental factors with their interactions (G×E). To achieve this goal, we conducted the field experiments for soybean core collections using multiple sowing times in multi-latitudinal fields. Sowing time shifts altered the flowering time (FT) and growth phenotypes, and resulted in increasing the combinations of genotypes and environments. Genome-wide association studies for the obtained phenotypes revealed the effects of field and sowing time to the significance of detected alleles, indicating the presence of G×E. By using accumulated phenotypic and environmental data in 2018 and 2019, we constructed multiple regression models for FT and growth pattern. Applicability of the constructed models was evaluated by the field experiments in 2020 including a novel field, and high correlation between the predicted and measured values was observed, suggesting the robustness of the models. The models presented here would allow us to predict the phenotype of the core collections in a given environment.     

Cytogenetic demonstration of out-of-phase DNA synthesis in endoreduplicated CHO cells: evidence for partial endoreduplication

Out-of-phase DNA synthesis, which is demonstrated cytogenetically as premature chromosome condensation (PCC), was analyzed in endoreduplicated Chinese hamster ovary (CHO) cells induced by colchicine or vincristine. Like conventional polyploid cells, endoreduplicated cells exhibited PCC in either S or G2. The former was more frequently observed in drug-treated cultures. In addition to these two types of PCC, other mitotic figures showing out-of-phase DNA synthesis were found. Such cells contained both conventional chromosomes (monochromosomes) and diplochromosomes. Differential FPG staining of chromatids in these cells showed that diplochromosomes incorporated BrdU twice while monochromosomes did so once, indicating the occurrence of partial endoreduplication in one of the sister nuclei of multinucleate cells. The possible mechanisms underlying induction of out-of-phase DNA synthesis and production of partial endoreduplication are discussed.     

Association of a cytoplasmic dynein-like protein recognizable by anti-MAP 1C with the mammalian mitotic spindle

A rabbit antibody to bovine brain MAP 1C was prepared. The antibody stained the mitotic spindle of PtK2 cells by immunofluorescence. On immunoblots of PtK2 cell extract the antibody reacted with polypeptides of molecular weights greater than 350 and 80 KD that resemble the subunit proteins of bovine brain MAP 1C. An additional 135 KD polypeptide in the extract was also stained. These results indicate that a cytoplasmic dynein recognizable by the anti-MAP 1C antibody is localized in the mitotic spindle.     

Phospholipid‐flipping activity of P4‐ATPase drives membrane curvature

P4‐ATPases are phospholipid flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4‐ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid‐flipping activity of P4‐ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine‐flippase activity at the PM due to expression of ATP10A, a member of the P4‐ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM. This is the important evidence that changes in the transbilayer lipid composition induced by P4‐ATPases can deform biological membranes.     

Changes in Morphology, Levels of Endogenous IAA and Protein Composition in Relation to the Development of Tobacco Genetic Tumor Induced in the Dark

When normal shoots which had been regenerated from tobacco genetictumor cultured in the dark were cut, tumorous tissues were againinduced on the cut segments. Morphological examination of thesegments, 5 days and 15 days after cutting, in a comparisonwith normally regenerated shoots, revealed active cell divisionin vascular-bundle tissue and formation of primordial teratoma-likestructures in the induced tumorous tissues. Endogeneous IAAremained at a constant, low level throughout the tumorigeneticprocess. Proteins from each step of tumorigenesis were separatedby two-dimensional electrophoresis on mini-gels and visualizedby silver staining. Twenty-seven polypeptides showed qualitativeor quantitative changes during the tumorigenetic process. Theseresults are discussed in relation to the development of tobaccogenetic tumor. (Received July 20, 1990; Accepted December 6, 1990)     

Superficial intimal injury of the rabbit carotid artery induced by distilled water

The study of mechanical injury to the aortic endothelium in experimental animals is important in understanding the pathologic processes in atherogenesis. In this investigation distilled water was used to produced superficial injury to the rabbit carotid artery. Sterilized distilled water was injected into a temporarily isolated segment of rabbit carotid artery measuring 0.5 cm in length. After 4 min blood flow was reestablished by removal of the isolating ligatures. The carotid arteries were examined at time intervals of 5 min, 24 h, 48 h, 1 month, 3 months and 6 months after injury. Five min after injury, the carotid endothelial cells were almost completely removed but no medical injury was present. After 24 and 48 h, a few platelets were adherent to the denuded intimal surface. After 1 month, 3 months and 6 months the injured surface showed a slight intimal thickening consisting of modified smooth muscle cells. Our experimental findings suggested that the extent of the injured area is more important in the repair process than its depth.     

Identification of Two Nickel Ion-Induced Genes,NCI16 and PcGST1, in Paramecium caudatum

Here, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (∼16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; ∼25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H₂O₂ concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H₂O₂, implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tert-butylhydroquinone (tBHQ) but not by H₂O₂, suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an ∼0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control.     

Studies on endoreduplication. V. A three-dimensional scheme for diplo- and quadruple chromosomes and a model for DNA replication

The metaphase appearance of quadruple chromosomes in colchicine-treated CHO cells was compared between air-dried and gently squashed preparations. A marked difference in morphology between the two methods suggested that the planar alignment of quadruple chromosomes is an artifact of the spreading process and that quadruple chromosomes are organized within the nucleus in a three-dimensional configuration. By analyzing the alignment of the original and replicated strands, using BrdU incorporation, the three-dimensional orientation of the chromatids in quadruple chromosomes could be traced. This analysis led to a new model for DNA replication. According to this model, an opening of a DNA base pair which rotates about 90 degrees with respect to the deoxyribose phosphate backbone precedes DNA replication, resulting in the formation of the newly replicated strands perpendicular to the original plane of the base pair. This model, although derived from endoreduplication, may be applicable to a general scheme for DNA replication during normal chromosome duplication.     

Spawning season and size at sexual maturity of <Emphasis Type="Italic">Kyphosus bigibbus</Emphasis> (Kyphosidae) from northwest Kyushu,Japan

The reproductive characteristics of Kyphosus bigibbus were examined using individuals collected between June 2004 and February 2009 off Nagasaki Peninsula in northwest Kyushu, Japan. The spawning season and size at sexual maturity of this species were characterized based on a gonad index and histological examination of the gonads. The spawning season extends from June to October. This species is assumed to be an indeterminate, multiple-batch spawner. Females reached sexual maturity at larger size than males (fork length at 50% sexual maturity: males 284 mm, females 360 mm).