Population Genetics of an Expanding Family of Mobile Genetic Elements

A model of an expanding family of dispersed repetitive DNA was studied. Based on the previous result of the model of duplicative transposition, an approximate solution to give allelism and identify coefficients as functions of time was obtained, and theoretical predictions were verified by Monte Carlo experiments. The results show that, even if the copy number per genome increases very rapidly, allelism and identity coefficients may take a long time to reach equilibrium. The changes of allelism and allelic identity are similar to that of homozygosity at an ordinary single locus, whereas that of nonallelic identity can be much slower, particularly when the copy number per genome is large. Thus, many existing families of highly repetitive sequences may represent nonequilibrium states for nonallelic identity. The present model may be extended to include other evolutionary forces such as gene conversion or the recurrent insertion from normal gene copies.     

A Close Relationship between Increases in Galactosyltransferase Activity and the Accumulation of Galactolipids during Plastid Development in Cucumber Seedlings

Changes in the activity of UDP-galactose:diacylglycerol galactosyltransferase(UDGT), a key enzyme in galactolipid biosynthesis, during germinationwere investigated in cucumber (Cucumis sativus L. cv. Aonagajibai)seedlings. After germination, UDGT activity increased duringgrowth in darkness for 4 days, reaching 10 times the activityin ungerminated seeds. Illumination of 4-day-old dark-grownseedlings strongly stimulated the activity. By contrast, inseedlings grown continuously in darkness, the increase in UDGTactivity ceased after 4 days and the activity remained constantthereafter. A similar increase in the specific activity of UDGTwas observed i n the envelope fraction from seedlings, indicatingthat the increase in the enzymatic activity preceded synthesisof other proteins in the envelope membrane. Coincident withthe change in the enzymatic activity, here was an increase inlevels of monogalactosyl diacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), two major constituents of chloroplastmembrane lipids, in the germinated seedlings. Cycloheximideinhibited the light-mediated increase in the enzymatic activityby illumination of 4-day-old dark-grown seedlings, and, as aconsequence, it inhibited the accumulation of MGDG and DGDG.It was clear, therefore, that protein synthesis was necessaryduring this activation. Addition of a cytokinin, benzyladenine(BA), stimulated the increase in the UDGT activity. The increasein the UDGT activity caused by BA was accompanied by the accumulationof galactolipids, as in the case of the activation by light.These results suggest that activation of the final reactionin the synthesis of MGDG, which is catalyzed by the galactosyl-transferase,contributes to the accumulation of galactolipids during thedevelopment of the chloroplast membrane. (Received December 3, 1994; Accepted July 3, 1995)     

Endothelin-3 stimulates production of endothelium-derived nitric oxide via phosphoinositide breakdown.

Cultured bovine endothelial cells (EC) have specific receptors for endothelin (ET)-3 functionally coupled to phosphoinositide breakdown. We studied whether ET-3 stimulates synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that activates soluble guanylate cyclase in EC, and whether the ET-3-induced NO formation involves G-proteins. ET-3 dose-dependently stimulated production of intracellular cGMP in EC, of which effects were abolished by pretreatment with NG-monomethyl L-arginine, an inhibitor of NO synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The stimulatory effects of ET-3 on cGMP production, inositol trisphosphate formation and increase in cytosolic free Ca2+ concentration were similarly blocked by pretreatment with pertussis toxin (PTX). These data suggest that ET-3 induces synthesis of NO mediated by phosphoinositide breakdown via PTX-sensitive G-protein in EC.     

Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.     

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.     

Relation between insulin resistance and fast-migrating LDL subfraction as characterized by capillary isotachophoresis

The proportion of the electronegative low density lipoprotein [LDL(-)] subfraction, which is atherogenic, is increased in type 2 diabetes but is not reduced by glycemic control. Therefore, we evaluated the ability of a new technique, capillary isotachophoresis (cITP), to quantify charge-based LDL subfractions and examined the relation between insulin resistance and the cITP fast-migrating (f) LDL levels. Seventy-five 10-year-old boys were included. The two cITP LDL subfractions, fLDL and major LDL subfractions, were proportional to the LDL protein content within the range of 0.1-0.8 mg/ml LDL protein. Levels of cITP fLDL were positively correlated with triglyceride (TG) levels and negatively correlated with LDL size. Insulin resistance as assessed by the homeostasis model assessment (HOMA-IR) was positively correlated (P < 0.01) with cITP fLDL levels (r = 0.41). The relation between HOMA-IR and cITP fLDL levels depended on TG levels but was independent of body mass index and LDL size. cITP lipoprotein analysis is an accurate and sensitive method for quantifying charge-based LDL subfractions in human plasma, and insulin resistance is related to cITP fLDL independent of LDL size.     

Identification of MK-1925: A selective,orally active and brain-penetrable opioid receptor-like 1 (ORL1) antagonist

Structure–activity relationship studies directed toward improving the metabolic stability of compound 1 resulted in the identification of 3-[5-(3,5-difluorophenyl)-3-({[(1S,3R)-3-fluorocyclopentyl]amino}methyl)-4-methyl-1H-pyrazol-1-yl]propanenitrile 39 (MK-1925) as a selective, orally available and brain-penetrable opioid receptor-like 1 (ORL1) antagonist. The compound also showed in vivo efficacy after oral dosing. Therefore, compound 39 was selected to undergo further studies as a clinical candidate.     

Discovery and biological profile of isoindolinone derivatives as novel metabotropic glutamate receptor 1 antagonists: A potential treatment for psychotic disorders

We describe here the discovery and biological profile of a series of isoindolinone derivatives as developed mGluR1 antagonists. Our combined strategy of rapid parallel synthesis and conventional medicinal optimization successfully led to N-cyclopropyl 22 and N-isopropyl isoindolinone analogs 21 and 23 with improved in vivo DMPK profiles. Moreover the most advanced analog 23 showed an oral antipsychotic-like effect at a dose of 1 mg/kg in an animal model.     

Structural interactions in chondroitin 4-sulfate mediated adherence of Plasmodium falciparum infected erythrocytes in human placenta during pregnancy-associated malaria

Infection with Plasmodium falciparum during pregnancy results in the adherence of infected red blood cells (IRBCs) in placenta, causing pregnancy-associated malaria with severe health complications in mothers and fetuses. The chondroitin 4-sulfate (C4S) chains of very low sulfated chondroitin sulfate proteoglycans (CSPGs) in placenta mediate the IRBC adherence. While it is known that partially sulfated but not fully sulfated C4S effectively binds IRBCs, structural interactions involved remain unclear and are incompletely understood. In this study, structurally defined C4S oligosaccharides of varying sulfate contents and sizes were evaluated for their ability to inhibit the binding of IRBCs from different P. falciparum strains to CSPG purified from placenta. The results clearly show that, with all parasite strains studied, dodecasaccharide is the minimal chain length required for the efficient adherence of IRBCs to CSPG and two 4-sulfated disaccharides within this minimal structural motif are sufficient for maximal binding. Together, these data demonstrate for the first time that the C4S structural requirement for IRBC adherence is parasite strain-independent. We also show that the carboxyl group on nonreducing end glucuronic acid in dodecasaccharide motif is important for IRBC binding. Thus, in oligosaccharides containing terminal 4,5-unsaturated glucuronic acid, the nonreducing end disaccharide moiety does not interact with IRBCs due to the altered spatial orientation of carboxyl group. In such C4S oligosaccharides, 14-mer but not 12-mer constitutes the minimal motif for inhibition of IRBC binding to placental CSPG. These data have important implications for the development and evaluation of therapeutics and vaccine for placental malaria.