Target site search and effective inhibition of leukaemic cell growth by a covalently closed multiple anti-sense oligonucleotide to c-myb

Systematic secondary structure simulation of a target mRNA sequence is shown to be effective for locating a good anti-sense target site. Multiple selected anti-sense sequences were placed in a single molecule. The anti-sense oligonucleotide (oligo) was covalently closed to avoid exonuclease activities and was designated CMAS (covalently closed multiple anti-sense)-oligo. CMAS-oligo was found to be stable, largely preserving its structural integrity after 24 h of incubation in the presence of either exonuclease III or serum. When human c-myb mRNA was targeted by the c-myb CMAS-oligo, expression of the gene was completely abolished. Further, tumour cell growth was inhibited by 82 ± 3% as determined by an MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and by 90 ± 1% by [3H]thymidine incorporation. When a leukaemic cell line K562 was treated with CMAS-oligo, colony formation on soft agarose was also decreased by 93%. In contrast, treatment with a scrambled control oligo did not significantly inhibit leukaemic cell growth. These results suggest that a rational target site search is possible for an anti-sense oligo and that CMAS-oligo can be employed as an effective anti-sense agent with enhanced stability.ope

Molecular cloning and characterization of the mouse Peroxiredoxin V gene

We have cloned two cDNA isoforms as well as genomic sequences of the mouse Prx V gene and characterized their molecular genetic features. Two isoforms of the mouse Prx V cDNA were identified from liver and testis. The testis-originated long transcripts had extra 1164-bp 5'-UTR sequences compared to the liver-originated short transcripts. Primer extension and sequence analyses revealed that the two isoforms were presumably transcribed at the same gene locus. The gene was composed of six exons spanning 3.2 kb. The short transcript was abundantly expressed in the kidney, liver, and heart of the adult mouse tissues and in the extra-membrane of the 10.5 dpc embryos. The long transcript of 1985 bp was abundantly detected in testis with trace amounts in other tissues. Interestingly, in testis and fetus, only mRNA expression of the long form was identified. However, the protein expression was not found in testis, implying that the long form could not properly direct the protein expression. The long Prx V cDNA has eight uORFs in the extra 5'-UTR, which proceed the major ORF. The inability of protein expression for the long-form cDNA in testis suggests that the uORFs might inhibit translation of the major ORF and thereby confer the tissue-specific regulation of the mouse Prx V gene.ope

Characterization of glk, a gene coding for glucose kinase of Corynebacterium glutamicum

The glk gene from Corynebacterium glutamicum was isolated by complementation using Escherichia coli ZSC113 (ptsG ptsM glk). We sequenced a total of 3072 bp containing the 969-bp open reading frame encoding glucose kinase (Glk). The glk gene has a deduced molecular mass of 34.2 kDa and contains a typical ATP binding site. Comparison with protein sequences revealed homologies to Glk from Streptomyces coelicolor (43%) and Bacillus megaterium (35%). The glk gene in C. glutamicum was inactivated on the chromosome via single crossover homologous recombination and the resulting glk mutant was characterized. Interestingly, the C. glutamicum glk mutant showed poor growth on rich medium such as LB medium or brain heart infusion medium in the presence or absence of glucose, fructose, maltose or sucrose as the sole carbon source. Growth yield was reduced significantly when maltose was used as the sole carbon source using minimal medium. The growth defect of glk mutant on rich medium was complemented by a plasmid-encoded glk gene. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression, and it was found that glk was expressed constitutively under all tested conditions with different carbon sources.ope

Phylogenetic analysis of HERV-K LTR family in human chromosome Xq26 and new world monkeys

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Lipid peroxidation inhibitory activity of some constituents isolated from the stem bark of Eucalyptus globulus

Twelve compounds with lipid peroxidation inhibitory activity were isolated from the stem bark of E. globulus. Their structures were assigned as a new aromatic monoterpene (1) and eleven known compounds, pinoresinol (2), vomifoliol (3), 3,4,5-trimethoxyphenol 1-O-beta-D-(6'-O-galloyl)glucopyranoside (4), methyl gallate (5), rhamnazin (6), rhamnetin (7), eriodictyol (8), quercetin (9), taxifolin (10), engelitin (11), and catechin (12) on the basis of UV, mass, and NMR spectroscopic analyses. These compounds except vomifoliol significantly inhibited lipid peroxidation in rat liver microsome.ope

안면읍, 구미시 유래 야생마우스의 형태유전학적 조사

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규산질다공체(CellCaSi)에 의한 미세조류 제어

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Cis-fumagillin, a new methionine aminopeptidase(yp 2) inhibitor produced by Penicillium sp. F2757

Selective inhibition against the yeast MetAP2 (methionine aminopeptidase type 2) was detected in the fermentation broth of a fungus F2757 that was later identified as Penicillium janczewskii. A new compound cis-fumagillin methyl ester (1) was isolated from the diazomethane treated fermentation extracts together with the known compound fumagillin methyl ester (2). The cis-fumagillin methyl ester, a stereoisomer of fumagillin methyl ester at the C2'-C3' position of the aliphatic side chain, selectively inhibited growth of the map 1 mutant yeast strain (MetAP1 deletion strain) at a concentration as low as 1 ng. However, the wild type yeast w303 and the mutant map2 (MetAP2 deleted) strains were resistant up to 10 μg of the compound. In enzyme experiments, compound 1 inhibited the MetAP2 with an IC50 value of 6.3 nM, but it did not inhibit the MetAP1 (IC50 >200 μM). Compound 2 also inhibited the MetAP2 with an IC50 value of 9.2 nM and 105 μM against MetAP1.ope

Glucosamine-mediated detoxification of p-benzoquinone and its removal by chitosan

p-Benzoquinone non-enzymatically reacted with D-glucosamine at physiological pH and moderate temperature. The reaction of p benzoquinone with glucosamine was signaled by changes in the UV and visible spectra. The reactivity proceeded fastest at pH values above 7, with a sharp drop from pH 6.5 to 7.0, and the reaction was negligible in acidic conditions. The order of reactivity of amino sugars was D-mannosamine > D-glucosamine > D- galactosamine. From the reaction mixture, four conversion products were isolated and none was toxic to Escherichia coli even at 500-700 μg ml-1, while p-benzoquinone was cytotoxic to E. coli at 20 μg ml-1. Chitosan could react with p-benzoquinone efficiently and remove this toxicant in aqueous solution.ope

Reduced glutathione oxidation ratio and 8 ohdG accumulation by mild ischemic pretreatment

A critical role of oxidative stress has been implicated in ischemic brain damage. Mild ischemic pretreatment and/or synthesis of heat shock proteins (HSPs) has been suggested to protect against oxidative brain damage. However, experimental support of this suggestion have proven to be difficult partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. In this study, we have attempted to establish biochemical assay systems to quantitate oxidative brain damage following ischemia. We produced experimental brain ischemia in the Mongolian gerbil (Meriones unguiculatus) and examined the hippocampus for ischemic brain damage. The results obtained from ischemic gerbil hippocampus demonstrated that oxidative brain damage can be quantitated by determining glutathione oxidation ratio together with the accumulation of the oxidative DNA damage product, 8-hydroxy-2'-deoxyguanosine (8 ohdG). Our results also demonstrated a role for mild ischemic pretreatment and synthesis of HSPs against oxidative brain damage. We showed that mild 2-min ischemic pretreatment reduced the degree of both glutathione oxidation ratio and 8 ohdG accumulation in gerbil hippocampus subsequent to 10 min ischemic challenge. We also showed that the accumulation of HSP70 was closely associated with the reduction of oxidative brain damage. To our knowledge, this is the first report to investigate glutathione redox states and oxidative DNA damage levels to evaluate a protective role of mild ischemic pretreatment and HSP synthesis following brain ischemia. Our data validate the previous suggestions and provide new additional data that argue for the protective role of mild ischemic pretreatment and HSP70 synthesis against oxidative brain damage.ope