Circadian phenotypes of Drosophila fragile x mutants in alternative genetic backgrounds

Drosophila FMR1 mutants are models of human fragile X syndrome. They show a loss of locomotor activity rhythm and severe degradation of eclosion timing. We analyzed the circadian behavior of FMR1 mutants (dfmr1(B55)) in two genetic backgrounds, yellow white (yw) and Canton S (CS). The arrhythmic phenotype of circadian locomotor activity in constant darkness (DD) did not significantly change in either genetic background. Surprisingly, eclosion timing was completely restored by backcrossing dfmr1(B55) with yw or CS flies. Morphological analysis of the small ventrally located lateral neurons of FMR1 mutants revealed that the dorsal-projection area was significantly larger in arrhythmic than rhythmic flies. In addition, dfmr1(B55) mutants in both genetic backgrounds had a significantly lower evening peak in the light-dark (LD) cycle. These results indicate that lack of FMR1 does not affect eclosion timing, but alters locomotor activity patterns in both LD and DD conditions by affecting the arborization of small ventrally located lateral neurons. Thus, the FMR1 gene may regulate the circadian-related locomotor activity of Drosophila.     

Enantioseparation using urea- and imide-bearing chitosan phenylcarbamate derivatives as chiral stationary phases for high-performance liquid chromatography

Completely deacetylated chitosan was prepared by the treatment of commercial chitosan with 50% aqueous NaOH, and then derivatized into several new chitosan phenylcarbamate derivatives having a urea and an imide moiety at the 2-position of the glucosamine ring by the reaction with isocyanate and phthalic anhydride/isocyanate, respectively. The chitosan derivatives were coated on macroporous silica gel and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. The chiral recognition ability of the chitosan derivative was improved using the completely deacetylated chitosan. Among the novel chitosan derivatives, the 3,5-dimethyl-, 3,5-dichloro-, and 3,4-dichlorophenylcarbamate derivatives were found to possess relatively high chiral resolution abilities. The CSPs based on the chitosan phenylcarbamate-urea and -imide derivatives were stable in the presence of chloroform and ethyl acetate as a component of the eluents, and some racemates were better resolved by such eluents. The dichlorophenylcarbamate-imide derivatives showed a high chiral recognition for metal acetylacetonate complexes. The enantiomerization of Al(acac)3 was performed on the chitosan 3,5-dichlorophenylcarbamate-imide derivative CSP and the resulting chromatogram showed a 26% (+)-isomer enrichment.     

A preliminary report on the vegetation zonation of palsas in the Arctic National Wildlife Refuge,northern Alaska,USA

We measured vegetation patterns on palsas with reference to topographic characteristics on the Arctic National Wildlife Refuge, northern Alaska, to obtain benchmark data because of the changes expected from global warming. Vegetation was examined in 60 plots of area 50 cm × 50 cm by five environmental factors: water content in the peat and duff layers, groundwater level, slope angle, depth to frozen surface, and presence of pellets and feces. Three palsas were selected for the survey, and the heights were fewer than 50 cm from the groundwater surface. Based on TWINSPAN and canonical correspondence analysis, we confirmed that clear patterns of vegetation zonation had developed within a 60-cm difference in water level. Vaccinium vitis-idaea occurred well on the top areas of palsas, while Carex aquatilis was established on the bottom areas. Sphagnum spp. were established on intermediate locations between V. vitis-idaea and C. aquatilis. The prime determinant of the vegetation zonation seems to be water content in peat and duff layers rather than water level, although the five factors that we examined interact intricately with each other.     

Acute effects on the lung and the liver of oral administration of cerium chloride on adult, neonatal and fetal mice

We evaluated tissue changes associated with cerium chloride administration via gavage to adult mice, via milk to neonatal mice and transplacentally to fetal mice. Change in adults consisted of extensive pulmonary hemorrhage, pulmonary venous congestion, thickened alveolar septae, hepatic necrosis and neutrophil infiltrations. Those in fetal mice consisted of pulmonary and hepatic congestion. These results indicate that gavage cerium administration elicited subtle tissue changes, though oral toxicity is rather low. These changes were less severe in neonatal and fetal mice. When cerium was injected into adult mice through the tail vein, cerium was distributed mainly to the liver, spleen and lung dose-dependently with the cerium concentration gradually decreasing after 3 days. A study of cerium anticoagulation in mouse plasma showed that clotting time was significantly prolonged when cerium was added to plasma. These results suggest that cerium may disturb blood coagulation and cause pulmonary and hepatic vascular congestion.     

Antiviral activities against herpes simplex virus type 1 by HPH derivatives and their structure-activity relationships

The compound named Histidine-pyridine-histidine (HPH) is an oxygen-activating ligand derived from the structure of bleomycin. We synthesized HPH derivatives, namely HPH-1 to -8, and investigated their antiviral activities against herpes simplex virus type 1. HPH-8 showed potent antiviral activity with an EC50 of 15 microM, and relatively high cytotoxicity with a CC50 of 37 microM. In contrast, HPH-4 indicated a weaker antiviral activity with an EC50 of 79 microM, but exhibited a far more less cytotoxicity (CC50 500 microM). Other HPH derivatives showed no effects against antiviral activities and cytotoxicities.     

Antiproliferative and apoptosis-inducing activities of alkyl gallate and gallamide derivatives related to (-)-epigallocatechin gallate

Green tea and (-)-epigallocatechin gallate (EGCG: one of the main components of green tea) are reported to have cancer-preventive activity in humans. A previous SAR study of EGCG and derivatives indicated that a galloyl group is essential for the activity. To test this hypothesis, we synthesized various alkyl gallate and gallamide derivatives and evaluated their antiproliferative effects on human leukemia HL-60 cells. Dodecyl 3,4,5-trihydroxybenzoate (6c) showed the most potent activity, being more potent than EGCG. To clarify the molecular mechanism of the antiproliferative action, we investigated the effects of 6c on various factors. Compound 6c was found to induce apoptosis mediated by endoplasmic reticulum (ER)-stress-related caspase-12. Upregulation of gadd-153, an ER-stress marker protein, was also observed. These results indicate that 6c induced apoptosis via the ER-stress-related pathway.     

Phylogeny, recombination, and mechanisms of stepwise mitochondrial genome reorganization in mantellid frogs from Madagascar

In Malagasy frogs of the family Mantellidae, the genus Mantellais known to possess highly reorganized mitochondrial (mt) genomeswith the following characteristics: 1) some rearranged genepositions, 2) 2 distinct genes and a pseudogene correspondingto the transfer RNA gene for methionine (trnM), and 3) 2 controlregions (CRs) with almost identical nucleotide sequences. Theseunique genomic features were observed concentrated between theduplicated CRs surrounding cytochrome b (cob) and nicotinamideadenine dinucleotide dehydrogenase subunit 2 (cnad2) genes.To elucidate the mechanisms and evolutionary pathway that yieldedthe derived genome condition, we surveyed the reorganized genomicportion for all 12 mantellid genera. Our results show that themt genomes of 7 genera retain the ancestral condition. In contrast,adding to Mantella, 4 genera of the subfamily Mantellinae, Blommersia,Guibemantis, Wakea, and Spinomantis, share several derived genomiccharacters. Furthermore, mt genomes of these mantellines showedadditional structural divergences, resulting in different genomeconditions between them. The high frequency of genomic reorganizationdoes not correlate with nucleotide substitution rate. The encounteredmt genomic conditions also suggest the occurrences of stepwisegene duplication and deletion events during the evolution ofmantellines. Simultaneously, the majority of duplication eventsseems to be mediated by general (homologous) or illegitimaterecombination, and general recombination also plays a role inconcerted sequence evolution between multiple CRs. Consideringour observations and recent conditional evidences, the followingoutlines can be expected for recombination processes in mt genomereorganization. 1) The CR is the "hot spot" of recombination;2) highly frequent recombination between CRs may be mediatedby a replication fork barrier lying in the CR; 3) general recombinationhas a potential to cause gene rearrangement in upstream regionsof multiple CRs as the results of gene conversion and unequalcrossing over processes. Our results also suggest that recombinationactivity is not a direct cause of convergent gene rearrangement;rather, homoplasious gene rearrangement seems to be mediatedby persistence of a copied genomic condition through severallineage splits and subsequent parallel deletions.     

The membrane-spanning domain of CD98 heavy chain promotes alpha(v)beta3 integrin signals in human extravillous trophoblasts

CD98 heavy chain (CD98hc) is expressed highly in developing human placental trophoblast. CD98hc is an amino acid transporter and is thought to function in cell fusion, adhesion, and invasion by interacting with integrins. In invasive extravillous trophoblast, alpha(v)beta(3) integrin is expressed in a temporally and spatially specific manner, which prompted us to investigate the potential role of CD98hc in signal transduction of alpha(v)beta(3) integrin. Immunocytochemistry of extravillous trophoblast derived from human placenta revealed that CD98hc colocalized with alpha(v)beta(3) integrin and with alpha(v)beta(3)-associated cytoplasmic proteins including paxillin, vinculin, and focal adhesion kinase. Coimmunoprecipitation of CD98hc and its mutants revealed that the transmembrane domain of CD98hc is necessary for the association of CD98hc with alpha(v)beta(3) integrin. When CD98hc negative liver cells (FLC4) were stably transfected with CD98hc and the extracellular domain of CD98hc was cross-linked by anti-CD98 antibody, FLC4 cells binding affinity to fibronectin and cell motility increased. The anti-CD98 antibody cross-linking promoted actin stress fiber formation and activation of signal transduction downstream of RhoA GTPase, and elevated the phosphorylation of focal adhesion kinase, paxillin, and protein kinase B. Pretreatment of transfected FLC4 cells with specific inhibitors for alpha(v)beta(3)integrin, phosphatidylinositol 3-kinase, and RhoA diminished these effects caused by anti-CD98 antibody cross-linking. These results suggest that notoriously invasive activity of extravillous trophoblast is mediated by CD98hc, which promotes alpha(v)beta(3) integrin-dependent signals.     

Mercaptoacetate inhibition of fatty acid beta-oxidation attenuates the oral acceptance of fat in BALB/c mice

We investigated the effect of beta-oxidation inhibition on the fat ingestive behavior of BALB/c mice. Intraperitoneal administration to mice of mercaptoacetate, an inhibitor of fatty acid oxidation, significantly suppressed intake of corn oil but not intake of sucrose solution or laboratory chow. To further examine the effect of mercaptoacetate on the acceptability of corn oil in the oral cavity, we examined short-term licking behavior. Mercaptoacetate significantly and specifically decreased the number of licks of corn oil within a 60-s period but did not affect those of a sucrose solution, a monosodium glutamate solution, or mineral oil. In contrast, the administration of 2-deoxyglucose, an inhibitor of glucose metabolism, did not affect the intake or short-term licking counts of any of the tasted solutions. These findings suggest that fat metabolism is involved in the mechanism underlying the oral acceptance of fat as an energy source.     

Role of Cu,Zn-SOD in the synthesis of endogenous vasodilator hydrogen peroxide during reactive hyperemia in mouse mesenteric microcirculation in vivo

We have recently demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous H2O2 in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous H2O2 during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-SOD(-/-) mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, N(G)-monomethyl-L-arginine (L-NMMA) alone, and L-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or L-NMMA alone and was almost completely inhibited by L-NMMA + catalase in wild-type mice, whereas it was inhibited by L-NMMA and L-NMMA + catalase in the Cu,Zn-SOD(-/-) mice. RH-induced increase in blood flow after L-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-SOD(-/-) mice. In mesenteric arterioles of the Cu,Zn-SOD(-/-) mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular H(2)O(2) production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-SOD(-/-) mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous H(2)O(2) that contributes to RH in mouse mesenteric smaller arterioles.