Nucleotide Sequence of a 28-kb Mouse Genomic Region Comprising the Imprinted Igf2 Gene

The mouse insulin-like growth factor II gene (Igf2) is physicallylinked to the insulin II gene (Ins2) and both are subject totissue-specific genomic imprinting. The paternal-specific expressionof Igf2 has been associated with hypermethylation of some CpGsites in the 5' flanking region and in the body of the gene.As a first step in analyzing the structural features of thisimprinted locus, we here report the complete nucleotide sequenceof Igf2, including all introns and the intergenic region adjacentto Ins2. This 28-kb segment of mouse chromosome 7 exhibits 80%overall identity with the corresponding rat sequence and hasa high GC content of 52%. In addition to the known CpG islandwithin the second Igf2 promoter, another island was identifiedapproximately 2 kb 5' to the first exon. Other features of thislocus include a 35-fold tandem repeat of an 11-bp sequence thatoverlaps Igf2 pseudo-exon 2, and a B2 repeat element in theintergenic region between Ins2 and Igf2. The GC-richness andthe presence of CpG islands associated with tandem repeats arecommon features of imprinted genes and thus may play a rolein the imprinting mechanism.     

Design,synthesis, and biological evaluation of a novel series of quercetin diacylglucosides as potent anti-MRSA and anti-VRE agents

A series of novel quercetin diacylglucosides were designed and first synthesized by Steglich esterification on the basis of MRSA strains inhibiting natural compound A. The in vitro inhibition of different multi-drug resistant bacterial strains and Escherichia coli DNA gyrase B was investigated. In the series, compound 10h was up to 128-fold more potent against vancomycin-resistant enterococci and more effective than A, which represents a promising new candidate as a potent anti-MRSA and anti-VRE agent.     

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific,toxin-producing fungus Alternaria alternata Japanese pear pathotype

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant–pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.     

Campest-5-en-3-one, an oxidized derivative of campesterol, activates PPARalpha, promotes energy consumption and reduces visceral fat deposition in rats

Dietary campest-5-en-3-one (campestenone), an oxidized derivative of campesterol, significantly reduced visceral fat weight and the concentration of triacylglycerol in serum and liver of rats. Dietary campestenone dramatically increased the activities and the mRNA expressions of mitochondrial and peroxisomal enzymes involved in beta-oxidation in the liver. Campestenone activated human peroxisome proliferator-activated receptor (PPAR) alpha as determined using the novel GAL4 ligand-binding domain chimera assay system with coactivator coexpression. In contrast, dietary campestenone reduced the activities and the mRNA expressions of enzymes involved in fatty acid synthesis, except for the malic enzyme. Dietary campestenone decreased the sterol regulatory element binding protein-1 (SREBP-1) mRNA level. Energy expenditure was significantly higher in the feeding of campestenone in rats. Dietary campestenone reduced hepatic cholesterol concentration and increased fecal excretion of neutral steroids originated from cholesterol. Lymphatic absorption of cholesterol was reduced by the coadministration of campestenone in rats cannulated in the thoracic duct. These observations suggest a possibility that campestenone has an ability to prevent coronary heart disease by improving obesity and abnormality of lipid metabolism.     

Isomer-specific anti-obese and hypolipidemic properties of conjugated linoleic acid in obese OLETF rats

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has attracted considerable attention because of its potentially beneficial biologic effects both in vitro and in vivo. Our results clearly show the specific action of the 10trans,12cis-CLA isomer against hyperlipidemia and obesity in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks of feeding with 10t,12c-CLA, but not 9cis,11trans-CLA, abdominal adipose tissue weight and serum and hepatic lipid levels in OLETF rats were lower than those in linoleic acid-fed rats. These effects were attributable to suppressed fatty acid synthesis and enhanced fatty acid beta oxidation in the liver on a 10t,12c-CLA diet. Additionally, we showed that mRNA expression of fatty acid synthase, carnitine palmitoyltransferase, leptin, and sterol regulatory element binding protein-1 was also regulated by 10t,12c-CLA. We suppose that 10t,12c-CLA reveals hypolipidemic and anti-obese activity through the alteration of mRNA expressions in the liver and white adipose tissue.     

Identification of genes affecting lipid content using transposon mutagenesis in Saccharomyces cerevisiae

Genes involved in lipid accumulation were identified in Saccharomyces cerevisiae using transposon insertion mutagenesis. Five ORFs, such as SNF2, IRA2, PRE9, PHO90, and SPT21 were found from the analysis of the insertion sites in transposon insertion mutants with higher lipid content. Since these ORFs are not directly involved in storage lipid biosynthesis, we speculate that they are involved in carbon fluxes into storage lipids in response to nutrient conditions. Lipid analysis of disruptants of these ORFs indicated that the Deltasnf2, and Deltaira2 disruptants had significantly higher lipid content. Cultivation in a nitrogen-limited medium increased the lipid content in all disruptants, among which the Deltapre9 disruptant was the most sensitive to nitrogen limitation. We then focused on the Deltasnf2 disruptant due to its higher lipid content and its function as a regulator of phospholipid synthesis. Lipid class analysis indicated that triacylglycerol and free fatty acids contributed to the increase in total lipids of the Deltasnf2 disruptant. The addition of exogenous fatty acids was not so effective at increasing the lipid content in the Deltasnf2 disruptant as it was in the wild type. It should be noticed that exogenous free linoleic acid was much higher in the Deltasnf2 disruptant than in the wild type, as in the case of endogenous free fatty acids. In addition, the incorporation of exogenous fatty acids into cells increased in the disruptant, suggesting that fatty acid transporters were regulated by SNF2. The results suggest that metabolic fluxes into storage lipids, which are activated in the Deltasnf2 disruptant, is repressed by the incorporation of exogenous fatty acids. They provide new insight into the biosynthesis of storage lipids in yeast.     

2,4-Disubstituted piperidines as selective CC chemokine receptor 3 (CCR3) antagonists: synthesis and selectivity

Linear unselective CCR3 antagonist leads with IC(50) values in the 200 nM range were converted into low nM binding compounds selective at CCR3 by moving the piperidine nitrogen substituent to the carbon at the 2-position of the ring. Substitution of the piperidine nitrogen with simple alkyl and acyl groups was found to improve the selectivity of this new compound class. In particular, N-{3-[(2S, 4R)-1-(propyl)-4-(4-fluorobenzyl)piperidinyl]propyl}-N'-(3-acetylphenyl)urea exhibited single digit nanomolar IC(50) values for CCR3 with >100-fold selectivity against an extensive counter screen panel.