67 research outputs found

    MDR-1 gene expression is a minor factor in determining the multidrug resistance phenotype of MCF7/ADR and KB-V1 cells

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    AbstractThe relevance of MDR-1 gene expression to the multidrug resistance phenotype was investigated. Drug-resistant cells, KB-V1 and MCF7/ADR, constantly expressed mRNA of the MDR-1 gene and were more resistant to vinblastine and adriamycin than drug-sensitive cells, KB-3–1 and MCF7. The drug efflux rate of KB-V1 was the same as KB-3–1 although the MDR-1 gene was expressed in only the resistant cell. The higher intracellular drug concentration of KB-3–1 than KB-V1 was due to the large drug influx. In the case of MCF7 and MCF7/ADR, the influx and efflux of the drug had nearly the same pattern and drug efflux was not affected by verapamil. The amount of ATP, cofactor of drug pumping activity of P-glycoprotein, was not changed by the resistance. These observations suggested that drug efflux mediated by MDR-1 gene expression was not a major determining factor of drug resistance in the present cell systems, and that the drug resistance could be derived from the change in drug uptake and other mechanisms

    Isolation of p-hydroxycinnamaldehyde as an antibacterial substance from the saw fly, Acantholyda parki S.

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    AbstractWe purified an antibacterial substance from larvae of the saw fly, Acantholyda parki S., and identified its molecular structure as p-hydroxycinnamaldehyde. We then synthesized it by reduction of p-hydroxycinnamic acid. The antibacterial activity of the synthetic p-hydroxycinnamaldehyde was equal to that of the authentic substance. This molecule was found to have a broad antibacterial spectrum against not only Gram-negative, but also Gram-positive bacteria. Furthermore, it showed antifungal activity against Candida albicans. We suggest that this substance may play a role in the defense system of this insect. This is the first report of p-hydroxycinnamaldehyde of animal origin

    Broad spectrum thiopeptide recognition specificity of the Streptomyces lividans TipAL protein and its role in regulating gene expression.

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    Microbial metabolites isolated in screening programs for their ability to activate transcription of the tipA promoter (ptipA) in Streptomyces lividans define a class of cyclic thiopeptide antibiotics having dehydroalanine side chains ("tails"). Here we show that such compounds of heterogeneous primary structure (representatives tested: thiostrepton, nosiheptide, berninamycin, promothiocin) are all recognized by TipAS and TipAL, two in-frame translation products of the tipA gene. The N-terminal helix-turn-helix DNA binding motif of TipAL is homologous to the MerR family of transcriptional activators, while the C terminus forms a novel ligand-binding domain. ptipA inducers formed irreversible complexes in vitro and in vivo (presumably covalent) with TipAS by reacting with the second of the two C-terminal cysteine residues. Promothiocin and thiostrepton derivatives in which the dehydroalanine side chains were removed lost the ability to modify TipAS. They were able to induce expression of ptipA as well as the tipA gene, although with reduced activity. Thus, TipA required the thiopeptide ring structure for recognition, while the tail served either as a dispensable part of the recognition domain and/or locked thiopeptides onto TipA proteins, thus leading to an irreversible transcriptional activation. Construction and analysis of a disruption mutant showed that tipA was autogenously regulated and conferred thiopeptide resistance. Thiostrepton induced the synthesis of other proteins, some of which did not require tipA

    Adenovirus-mediated p53 tumor suppressor gene therapy against subcutaneous HuH7 hepatoma cell line nodule of nude mice.

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    Mutations of the tumor-suppressor gene p53 have been found in 30-50% cases of hepatocellular carcinoma (HCC). In this study, E1-negative adenoviral vector encoding wild-type p53 under the control of the human cytomegalovirus promoter (AdCMV-p53w) was constructed to evaluate its therapeutic efficacy against tumor nodules developing after injection of HuH7 cell lines in ten nude mice. When each nodule had reached 10 mm in perpendicular diameter, 1.5 x 10(8) pfu of AdCMV-p53w per session was injected intratumorally as follows: In group I (n=3), five sessions were injected every other day. In group II (n=3), only one session. Group III (n=4) as negative controls. The mice were sacrificed at 28 days post AdCMV-p53w injection. Tumor growth was significantly suppressed and delayed in group I and II compared to group III as compared by tumor volume at the end of observation. These results suggest that AdCMV-p53w may not only be effective in treating HCCs expressing mutant p53, but also useful as a local injectable gene therapy

    A kinetic model and simulation of starch saccharification and simultaneous ethanol fermentation by amyloglucosidase and Zymomonas mobilis

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    A mathematical model is described for the simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and Zymomonas mobilis. By introducing the degree of polymerization (DP) of oligosaccharides produced from sago starch treated with α -amylase, a series of Michaelis-Menten equations were obtained. After determining kinetic parameters from the results of simple experiments carried out at various substrate and enzyme concentrations and from the subsite mapping theory, this model was adapted to simulate the SSF process. The results of simulation for SSF are in good agreement with experimental results.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47811/1/449_2004_Article_BF00369488.pd

    Two Tetranucleotide Repeats within the Xq21.3/Yp11.2 Human Specific Region of Homology and Their Conservation in Primate Evolution

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    We locate two tetranucleotide repeat sequences (AT3 and T2C2) between the markers sY44 and sY46 within the Xq21.3/Yp block of homology that has been created since the separation of the chimpanzee and human lineages, and trace their origin in primate evolution. The T2C2 repeat is present only in hominoid primates. The sequence AT3 is present in Old World monkeys but not in New World monkeys, and has been lost in some gibbon species. In the bonobo, the AT3 repeat is the site of a new Alu insertion. These findings and their relationship to the conservation of other markers in this region cast light on the structure of a genomic region that has been subject to change in the course of primate evolution and may include one or more sites of instability

    On the rocks: a follow-up study of crack users in London.

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    Contains fulltext : 16821.pdf (publisher's version ) (Open Access

    Homofermentative Production of d- or l-Lactate in Metabolically Engineered Escherichia coli RR1

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    We investigated metabolic engineering of fermentation pathways in Escherichia coli for production of optically pure d- or l-lactate. Several pta mutant strains were examined, and a pta mutant of E. coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to d-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation in ppc made the mutant produce d-lactate like a homofermentative lactic acid bacterium. When the pta ppc double mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of d-lactate production, more than 62.2 g of d-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenous l-lactate pathway, an l-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain which lacked phosphotransacetylase and d-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to l-lactate as the major fermentation product, and up to 45 g of l-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of E. coli can be reoriented to the production of d-lactate, an indigenous fermentation product, or to the production of l-lactate, a nonindigenous fermentation product

    Role of Lys335 in the metastability and function of inhibitory serpins.

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    The native form of inhibitory serpins (serine protease inhibitors) is not in the thermodynamically most stable state but in a metastable state, which is critical to inhibitory functions. To understand structural basis and functional roles of the native metastability of inhibitory serpins, we have been characterizing stabilizing mutations of human alpha1-antitrypsin, a prototype inhibitory serpin. One of the sites that has been shown to be critical in stability and inhibitory activity of alpha1-antitrypsin is Lys335. In the present study, detailed roles of this lysine were analyzed by assessing the effects of 13 different amino acid substitutions. Results suggest that size and architect of the side chains at the 335 site determine the metastability of alpha1-antitrypsin. Moreover, factors such as polarity and flexibility of the side chain at this site, in addition to the metastability, seem to be critical for the inhibitory activity. Substitutions of the lysine at equivalent positions in two other inhibitory serpins, human alpha1-antichymotrypsin and human antithrombin III, also increased stability and decreased inhibitory activity toward alpha-chymotrypsin and thrombin, respectively. These results and characteristics of lysine side chain, such as flexibility, polarity, and the energetic cost upon burial, suggest that this lysine is one of the structural designs in regulating metastability and function of inhibitory serpins in general

    Phylogenetic analysis of genus Sporobolomyces based on partial sequences of 26S rDNA

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    The sequences of the D1/D2 region of 26S rDNA from seven Sporobolomyces species, Bensingtonia subrosea, and Rhodosporidium toruloides were determined and compared with those from representatives of the genera Leucosporidium, Rhodosporidium, Rhodotorula, and Sporidiobolus. The five species of Sporobolomyces analyzed were distantly related to a monophyletic clade consisting of species of Sporidiobolaceae and Sporobolomycetaceae. Sporobolomyces falcatus was found to be closely related to Tremella exigua. The members of Sporidiobolaceae and Sporobolomycetaceae were divided into four groups. Group 1 was composed of Leucosporidium scottii and two Rhodotorula species, and group 2 contained three Rhodotorula species. Group 3 was designated as the Sporobolomyces/Sporidiobolus core group, as it contained Sporidiobolus johnsonii, the type species of Sporidiobolus and the teleomorphic state of Sporobolomyces salmonicolor (the type species of Sporobolomyces). Group 4, named the Rhodotorula/Rhodosporidium core group, included Rhodosporidium toruloides and Rhodotorula glutinis, the type species of the genera Rhodosporidium and Rhodotorula, respectively. The four groups were differentiated on the basis of their physiological characteristics including the assimilation of D-glucosamine, glucuronate, 2-keto-gluconate, Larabinitol, raffinose, methyl-alpha-glucoside, and starch. The taxonomy of the genera Leucosporidium, Rhodosporidium, Rhodotorula, Sporidiobolus, and Sporobolomyces will require a major revision when more data becomes available.
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