Identification of Cytokinins in Root Exudate of the Rice Plant

Cytokinins, cis-zeatin and cis- and (trans-ribosylzeatin, wereidentified in the root exudate of the rice plant (Oryza sativa,indica cultivar IR-24) after several chromatographic separationsand combined gas-liquid chromatography-selected ion monitoring(GC-SIM) analysis. The presence of trans-zeatin ribotide wassuggested by enzyme hydrolysis, subsequent chromatographic separationand GC-SIM. The comparatively high content of the ribotide inthe root exudate suggests the form of cytokinins to be transportedfrom roots to other parts in the rice plant. (Received July 22, 1982; Accepted November 25, 1982)     

A Tripolar Spindle Formed at Meiosis I Assures the Retention of the Original Ploidy in the Gynogenetic Triploid Crucian Carp, Ginbuna Carassius auratus langsdorfii

A triploid crucian carp, ginbuna ( Carassius auratus langsdorfii ), reproduces by gynogenesis, in which sperm of diploid ginbuna or of other species triggers the development of the triploid eggs, but a male genome makes no contribution to the zygotic genome. Gynogenesis is maintained by two mechanisms: exclusion of male genome during fertilization and retention of somatic ploidy levels during oogenesis. We examined the mechanisms responsible for producing unreduced eggs. Microfluorometry with a DNA staining dye showed that DNA content in the ginbuna oocytes was not reduced in half during meiosis I. Cytological observations revealed that a tripolar spindle was formed at meiosis I and the first polar body was not extruded, whereas an ordinary bipolar spindle was formed and the second polar body was extruded at meiosis II. Activity of histone H1 kinase (as an indicator of maturation-promoting factor) decreased transiently between meiosis I and II, strongly suggesting a "normal" meiotic cycle progression in the ginbuna oocytes. These results have indicated that in the gynogenetic ginbuna the somatic ploidy levels are maintained by inhibiting the first polar body extrusion via the formation of the tripolar spindle at meiosis I.     

Autologous Bone-Marrow Mesenchymal Stem Cell Implantation and Endothelial Function in a Rabbit Ischemic Limb Model

Background

The purpose of this study was to determine whether autologous mesenchymal stem cells (MSCs) implantation improves endothelial dysfunction in a rabbit ischemic limb model.

Methods

We evaluated the effect of MSC implantation on limb blood flow (LBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in rabbits with limb ischemia in which cultured MSCs were implanted (n = 20) or saline was injected as a control group (n = 20). LBF was measured using an electromagnetic flowmeter. A total of 10⁶ MSCs were implanted into each ischemic limb.

Results

Histological sections of ischemic muscle showed that capillary index (capillary/muscle fiber) was greater in the MSC implantation group than in the control group. Laser Doppler blood perfusion index was significantly increased in the MSC implantation group compared with that in the control group. LBF response to ACh was greater in the MSC group than in the control group. After administration of N^G-nitro-L-arginine, a nitric oxide synthase inhibitor, LBF response to ACh was similar in the MSC implantation group and control group. Vasodilatory effects of SNP in the two groups were similar.

Conclusions

These findings suggest that MSC implantation induces angiogenesis and augments endothelium-dependent vasodilation in a rabbit ischemic model through an increase in nitric oxide production.     

Fmoc-based solid-phase synthesis of GPR54-agonistic pentapeptide derivatives containing alkene- and fluoroalkene-dipeptide isosteres

Fmoc-protected Phe-Gly-type (Z)-alkene dipeptide isostere (ADI) and (E)-fluoroalkene dipeptide isostere (FADI) were synthesized and applied to Fmoc-based solid-phase peptide synthesis (SPPS). These cis-peptide bond mimetics were introduced into a bioactive pentapeptide [H-Amb-Phe-Gly-Leu-Arg-Trp-NH(2); Amb = 4-(aminomethyl) benzoic acid], which has potent GPR54 agonistic activity. The resulting pentapeptide derivatives showed low GPR54 agonistic activity, as compared with the parent peptide and (E)-ADI-containing derivative. This suggests that the trans-amide conformer of Phe-Gly peptide bond of the parent peptide would be significantly important for bioactivity. Contrary to our expectations, a (Z)-FADI-containing derivative exhibited essentially no activity, revealing the necessity of critical validation of FADI-bioisosterism.     

Synthesis and structure-activity relationships of 2-amino-8-hydroxyadenines as orally active interferon inducing agents

Recently, we have reported the 8-hydroxyadenine derivatives (2–4) as a novel class of interferon (IFN) inducing agents. In the present study, a series of 8-hydroxyadenines, which possess various amino moieties at the adenine C(2)-position, were synthesized and evaluated for their ability to induce endogenous IFN in comparison to the known active agent, Imiquimod. Among the compounds prepared, compound 9o possessing a 2-methoxyethylamino group at C(2)-position of adenine was found to exhibit potent IFN inducing activity in vivo. Compound 9o induced IFN from the dosage of 0.1 mg/kg, which was 30-fold potent than that of Imiquimod, and showed a good oral bioavailability (F=81%).     

Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.     

Bidirectional regulation of hippocampal mossy fiber filopodial motility by kainate receptors: a two-step model of synaptogenesis

The rapid motility of axonal filopodia and dendritic spines is prevalent throughout the developing CNS, although the function of this motility remains controversial. Using two-photon microscopy, we imaged hippocampal mossy fiber axons in slice cultures and discovered that filopodial extensions are highly motile. Axonal filopodial motility is actin based and is downregulated with development, although it remains in mature cultures. This motility is correlated with free extracellular space yet is inversely correlated with contact with postsynaptic targets, indicating a potential role in synaptogenesis. Filopodial motility is differentially regulated by kainate receptors: synaptic stimulation of kainate receptors enhances motility in younger slices, but it inhibits it in mature slices. We propose that neuronal activity controls filopodial motility in a developmentally regulated manner, in order to establish synaptic contacts in a two-step process. A two-step model of synaptogenesis can also explain the opposite effects of neuronal activity on the motility of dendritic protrusions.     

Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study

In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalancho? species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.     

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.     

Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems

Asymmetric (N(G),N(G))-dimethylarginine (ADMA) inhibits nitric oxide (NO) synthases (NOS). ADMA is a risk factor for endothelial dysfunction, cardiovascular mortality, and progression of chronic kidney disease. Two isoforms of dimethylarginine dimethylaminohydrolase (DDAH) metabolize ADMA. DDAH-1 is the predominant isoform in the proximal tubules of the kidney and in the liver. These organs extract ADMA from the circulation. DDAH-2 is the predominant isoform in the vasculature, where it is found in endothelial cells adjacent to the cell membrane and in intracellular vesicles and in vascular smooth muscle cells among the myofibrils and the nuclear envelope. In vivo gene silencing of DDAH-1 in the rat and DDAH +/- mice both have increased circulating ADMA, whereas gene silencing of DDAH-2 reduces vascular NO generation and endothelium-derived relaxation factor responses. DDAH-2 also is expressed in the kidney in the macula densa and distal nephron. Angiotensin type 1 receptor activation in kidneys reduces the expression of DDAH-1 but increases the expression of DDAH-2. This rapidly evolving evidence of isoform-specific distribution and regulation of DDAH expression in the kidney and blood vessels provides potential mechanisms for nephron site-specific regulation of NO production. In this review, the recent advances in the regulation and function of DDAH enzymes, their roles in the regulation of NO generation, and their possible contribution to endothelial dysfunction in patients with cardiovascular and kidney diseases are discussed.