Differential regulation of GLUT-1 and GLUT-4 glucose transporter mRNA levels in 3T3-L1 adipocytes.
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Adipose Tissue↗
Biomedical subjects
D T Chu
Publications and source records attributed to D T Chu.
Structure-activity relationship of quinolone antibacterial agents: the effects of C-2 substitution.
Very little is known about the structure-activity relationship of quinolone antibacterials at the 2-position. Because of the loss of biological activity with 2-methyl and 2-hydroxyl substitution, modifications at C-2 were generally considered to be unfavourable. Quinolone derivatives having a ring between positions 1 and 2 were recently shown to have biological activity. The sulfur-bridged analogs such as the benzothiazolo[3,2-a]quinolone, KB-5246 and NAD-394 have been reported to be highly active in vitro. The authors have synthesized 2-methylthiociprofloxacin, 2-methylofloxacin, the 5-oxopyrrolo[1,2-a]quinoline and isothiazolonaphthyridine to assess the importance of the sulfur atom on biological activity as well as the effect of C-2 substituent on the spatial arrangements of N-1 or the 3-carboxylic group. It was found that the planarity between the 4-keto and 3-carboxylic acid groups of quinoline molecules is the most important criterion for biological activity. The syntheses of the above four compounds are also described.
Anti-Infective Agents↗
Synthesis and biological properties of A-71497: a prodrug of tosufloxacin.
Tosufloxacin (5, A-61827 tosylate or T-3262) is currently under product development by both Abbott Laboratories and Toyama Chemical Co. Its registration as antibacterial agent has been approved in Japan. It has been found to be extremely effective in treating several bacterial infections. However, due to its inherent low water solubility, the development of an intravenous formulation will be extremely difficult and may preclude its parenteral use. In search of a more water-soluble analog of tosufloxacin for parenteral use, the 3-formyl derivative of tosulfoxacin, A-71497 (13), was synthesized for evaluation. It was found to produce high plasma levels of tosufloxacin upon both oral and subcutaneous administration to mice. High plasma levels of tosufloxacin were also obtained when 13 was administered both orally and intravenously to dogs. It possesses increased water solubility and makes the development of intravenous formulation possible. The chemical synthesis as well as biological properties of A-71497 (13) are described.
4-Quinolones↗
Rat gene 33: analysis of its structure, messenger RNA and basal promoter activity.
Several overlapping cDNA and genomic DNA clones corresponding to mRNA g33 and gene 33, respectively, were isolated and characterized. The mRNA g33 molecule is 2971 nt in length, exclusive of a poly(A+) tail, and encodes a putative 459 amino acid protein (49,946 daltons). The 13.2 kbp gene consists of four exons, three introns, and has two initiation sites located 27 and 30 bp downstream from a TATA box. Transfection of H4IIE cells with a fusion gene 33 (-1900 to +32)/luciferase construct (pSL330A) gave rise to readily detectable luciferase activity. In addition, primer extension analysis of the gene 33/luciferase mRNA transcribed in these experiments showed that transcription initiates in the gene 33 DNA at two sites consistent with those found in vivo. Analysis of the effect of 5' deletions on basal promoter activity showed that, in relation to the promoter activity of pSL330A, approximately 40% of the activity is lost between -1643 and -1050, another approximately 40% between -550 and -475, and deletion to -55 causes a total loss of detectable luciferase activity.
Amino Acid Sequence↗
The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus.
Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.
Acetylation↗
Structure-activity relationships of the fluoroquinolones.
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4-Quinolones↗
[Immune restoration of local xenogeneic graft-versus-host reaction in cancer patients in vitro and reversal of cyclophosphamide-induced immune suppression in the rat in vivo by fractionated Astragalus membranaceus].
Through the process of fractionation, purification by gel filtration chromatography and thereafter the screening with an in vitro local xenogeneic graft-versus-host reaction (XGVHR) model, a fraction was identified as a potent immunorestorative agent and was designated "Fraction 3" (F3). Using the XGVHR in vitro as a model assay for T cell function again, F3 was studied on mononuclear cells (MNC) from 13 cancer patients and exhibited significant immunorestorative activity, with an increase in local XGVHR (compared to untreated cells) of 151.34 +/- 46.02 mm3 vs 57.80 +/- 16.44 mm3, P less than 0.001. The in vitro augmented immune reactions induced by F3 in cancer patients also significantly exceeded the local XGVHR observed in the untreated MNC derived from 9 normal donor controls (94.15 +/- 9.16 mm3, P less than 0.005). In a newly developed in vivo XGVHR animal model, pretreatment of rats with F3 resulted in a significant abrogation of the local XGVHR with a reversal of the immunosuppressive effect of cyclophosphamide from 99.42 +/- 9.2 mm3 (positive control) to 39.78 +/- 8.3 mm3 (P less than 0.001). This reversal was complete as the volume of the abrogated local XGVHR was comparable to that of the negative control (no cyclophosphamide-priming, saline injection only) 34.79 +/- 5.69 mm3 (P greater than 0.1). These results suggest that F3 retained the immunopotentiating activity of the original crude extract and form the rational basis for the use of Astragalus in immunotherapy.
Adjuvants, Immunologic↗
Reciprocal regulation of gene transcription by insulin. Inhibition of the phosphoenolpyruvate carboxykinase gene and stimulation of gene 33 in a single cell type.
Two H4IIE hepatoma cell genes, phosphoenolpyruvate carboxykinase (PEPCK) and gene 33 (g33), are reciprocally regulated by insulin. Quantitation of mRNAPEPCK and mRNAg33 in total RNA isolated from cells treated with insulin showed a 7-fold increase in mRNAg33 amount and a 3-fold decrease of mRNAPEPCK. The cAMP analog 8-(4-chlorophenylthio)-cAMP induced mRNAPEPCK but had no effect on mRNAg33. The responses to various insulins and related molecules showed that the insulin receptor mediates the effects of physiologic concentrations of insulin on each of these genes. This inverse pattern of regulation by insulin was further characterized by determining the transcription rates of both genes in nuclei isolated at various times after the addition of insulin and 8-(4-chlorophenylthio)-cAMP to H4IIE cells. Insulin increased the rate of synthesis of mRNAg33 from 35 to 354 ppm and decreased the synthesis of mRNAPEPCK from 1175 to 109 ppm. These effects of insulin occurred rapidly and reached their maxima by 60 min. In both cases, greater effects were observed as insulin concentrations were increased from 10(-12) to 10(-8) M. Although the effects of insulin were concentration-dependent for both genes, the PEPCK gene was significantly more sensitive to low concentrations of insulin than was gene 33. The reciprocal effects of insulin on the synthesis of mRNAPEPCK and mRNAg33 in H4IIE cells provide a means of investigating how a hormone can exert opposing effects on two genes in the same cell.
Animals↗
Asymmetric synthesis and properties of the enantiomers of the antibacterial agent 7-(3-aminopyrrolidin-1-yl)-1-(2,4-difluorophenyl)-1,4-dihydro-6- fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acid hydrochloride.
Compound 1 [7-(3-aminopyrrolidin-1-yl)-1-(2,4-difluorophenyl)-1,4-dihydro-6-f luoro-4-oxo-1,8-naphthyridine-3-carboxylic acid hydrochloride] is a potent member of the quinolonecarboxylic acid class of antibacterial agents and is currently undergoing clinical evaluation. We have developed efficient asymmetric syntheses of the enantiomers of this agent. The S-(+) enantiomer 1a is 1-2 log2 dilutions more active than the R-(-) enantiomer 1b against aerobic bacteria and 1-2 or more log2 dilutions more active against anaerobic bacteria in vitro. The enantiomer 1a shows significantly better in vivo activity in a Pseudomonas aeruginosa mouse protection model compared to racemic 1. Coupled with the improved solubility profile of 1a relative to racemic material, these features may be of practical significance from a clinical standpoint.
Anti-Bacterial Agents↗
Design, synthesis, and properties of (4S)-7-(4-amino-2-substituted-pyrrolidin-1-yl)quinolone-3-carboxylic acids.
The quinolonecarboxylic acids constitute a class of extremely potent and orally active broad-spectrum antibacterial agents. These compounds have been shown to inhibit DNA gyrase, a key enzyme in bacterial DNA replication. The 7-(3-aminopyrrolidinyl)quinolone A-60969 (1) is a particularly potent member of this class and is currently undergoing clinical evaluation. We have studied a series of enantiomerically homogeneous (4S)-7-(4-amino-2-substituted-pyrrolidinyl)quinolones in an effort to utilize the 2-position of the pyrrolidine moiety to improve upon the solubility and pharmacokinetic properties of this class of compounds while still maintaining potent antibacterial activity. We have found that the absolute stereochemistry at the 2-position of the pyrrolidine ring is critical to the maintenance of such activity. In this paper, we report the full details of the asymmetric synthesis and the in vitro and in vivo structure-activity relationships of this series of compounds as well as the physiochemical properties, such as water solubility and log P, associated with the structural modifications. We also discuss the pharmacokinetic properties of several of these compounds in mice and the pharmacokinetics of 59, which has the best overall properties of agents in this study, in dog.
Animals↗
A-61827 (A-60969), a new fluoronaphthyridine with activity against both aerobic and anaerobic bacteria.
A-61827 (A-60969 is the hydrochloric salt of A-61827) is a new aryl-fluoronaphthyridine which is active against aerobic and anaerobic bacteria. The MICs of A-61827 for 90% of strains (MIC90) of staphylococci and streptococci were less than or equal to 1 microgram/ml and were generally 1 to 4 twofold dilutions less than those of ciprofloxacin for these bacteria. The MIC90S of A-61827 for members of the family Enterobacteriaceae and Pseudomonas aeruginosa were also less than or equal to 1 microgram/ml. Ciprofloxacin was 1 to 3 twofold dilutions more active than A-61827 against these gram-negative bacteria. Neisseria gonorrhoeae, Campylobacter jejuni, and Haemophilus influenzae were susceptible to less than 0.06 microgram of A-61827 per ml. The MIC90 of A-61827 for Legionella pneumophila was 0.25 microgram/ml. A-61827 was as potent or 1 to 2 twofold dilutions more potent than ciprofloxacin against these organisms. The MIC90 of A-61827 for all anaerobic bacteria was less than or equal to 4 micrograms/ml compared with less than or equal to 32 micrograms/ml for ciprofloxacin. In mouse protection tests, A-61827 was as active as ciprofloxacin against Escherichia coli, P. aeruginosa, and Salmonella typhimurium and 5 to 10 times more active than ciprofloxacin against Staphylococcus aureus and Streptococcus pyogenes. A-61827 was as active as ciprofloxacin against P. aeruginosa in a mouse pyelonephritis model and more active than ciprofloxacin and metronidazole in a mouse Bacteroides fragilis abscess model. After oral administration of 100 mg/kg to mice, the peak concentrations of A-61827 and ciprofloxacin in serum were 2.3 and 2.4 micrograms/ml and the half-lives in serum were 3.9 and 1.2 h, respectively.
Abscess↗
Structure-activity relationships in quinolone antibacterials: design, synthesis and biological activities of novel isothiazoloquinolones.
Over the past years it was found that modification of the 3-carboxylic acid group of quinolones generally produced compounds with a substantial decrease in antibacterial activity. The 3-carboxylic acid moiety together with the 4-carbonyl function are believed to be the most structurally critical sites for this class of compounds to DNA gyrase. The authors have designed and synthesized a series of quinolone analogues in which the 3-carboxylic acid group has been modified. These compounds, 2,3,4,9-tetrahydroisothiazolo[5,4-b]quinoline-3,4-diones, possess biological activities far superior to their parent counterparts. For example, the MICs (microgram/ml) for A-62824 (ciprofloxacin 3-carboxylic acid modified analogue) and ciprofloxacin against some organisms are as follows: S. aureus ATCC 6538P (0.02, 0.2); S. epidermidis 3519 (0.05, 0.2); E. coli Juhl (0.005, 0.01); P. aeruginosa A5007 (0.05, 0.1) and Acinetobacter sp. CMX 699 (0.05, 0.78). This investigation has produced the first successful modification of the 3-carboxylic acid group of quinolones resulting in a series of extremely potent antibacterials. The design and synthesis as well as the biological activities of these new derivatives are described.
Anti-Infective Agents↗
Immunotherapy with Chinese medicinal herbs. I. Immune restoration of local xenogeneic graft-versus-host reaction in cancer patients by fractionated Astragalus membranaceus in vitro.
The in vitro immunomodulatory activity of fractions derived from Astragalus membranaceus, an herb commonly used in the practice of traditional Chinese medicine, was first screened by studying their individual effects on mononuclear cells (MNC) derived from healthy normal donors using the local xenogeneic graft-versus-host reaction (XGVHR). Sephacryl S-200 column-separated Fraction 3 (MW 20,000-25,000) along with its crude extract precursor, Fraction 7, and another crude extract derivative, Fraction 8, were equally augmentative (p less than 0.05) in their effect on MNC from normal donors. These three active fractions were further studied on MNC derived from 13 cancer patients. Using again the local XGVHR as a model assay for T-cell function, preincubation of MNC derived from cancer patients with Fraction 3 induced a significant increase in local XGVHR (compared to untreated cells) with a mean +/- SD of 151.34 +/- 46.02 mm3 vs 57.80 +/- 16.44 mm3; p less than 0.001. Fractions 7 and 8 likewise induced significant increases in local XGVHR (109.14 +/- 19.32 mm3 versus 50.91 +/- 17.39 mm3; p less than 0.001 and 119.74 +/- 18.33 mm3 versus 48.77 +/- 16.17 mm3; p less than 0.001, respectively). The augmented immune reactions which were induced by either Fraction 3 or Fraction 8 (but not by Fraction 7) in MNC derived from cancer patients, each significantly exceeded the local XGVHR observed in the untreated MNC derived from normal donor controls with a relative reference index (ratio) of 1.60 +/- 0.48 and 1.23 +/- 0.17 respectively; p less than 0.005.(ABSTRACT TRUNCATED AT 250 WORDS)
Adjuvants, Immunologic↗
Immunotherapy with Chinese medicinal herbs. II. Reversal of cyclophosphamide-induced immune suppression by administration of fractionated Astragalus membranaceus in vivo.
A partially purified fraction (F3) with an estimated molecular weight of 20,000 to 25,000 derived from the traditional Chinese medicinal herb Astragalus membranaceus, was found to possess a potent immunorestorative activity in vitro. Its capacity to aborogate the local xenogeneic graft versus host reaction (XGVHR) following injection in vivo was further studied in a newly developed animal model designed for preclinical evaluation of various biological response modifiers. F3 was injected intravenously into cyclophosphamide-primed rats at varied concentrations and schedules prior to grafting of mononuclear cells from healthy normal donors. Maximal abrogation of the local XGVHR mounted by the mononuclear cells, was observed following injection of 5.55 mg of F3 daily for eight days. This abrogation of XGVHR indicates a reversal of the immunosuppressive effect of cyclophosphamide as manifested by a significant decline in the local XGVHR volume from 99.42 +/- 9.2 mm3 (positive control) to 39.78 +/- 8.3 mm3 (p less than 0.001). This reversal of cyclophosphamide-induced immunosuppression by the administration of F3 was complete, since the volume of the abrogated local XGVHR (39.78 +/- 8.3 mm3) was comparable to 34.79 +/- 5.69 mm3 (p greater than 0.1) in the negative control group (no cyclophosphamide-priming; saline injection only). These data indicate that F3 administration markedly enhances the rats' ability to reject the xenogeneic graft and therefore possesses a strong immune potentiating activity in vivo. These preclinical data also provide the rational basis for the use of extracts of Astragalus membranaceus in phase I clinical trials among patients suffering from iatrogenic or inherent immune deficiency states.
Adjuvants, Immunologic↗
Fractionated extract of Astragalus membranaceus, a Chinese medicinal herb, potentiates LAK cell cytotoxicity generated by a low dose of recombinant interleukin-2.
Success with rIL-2 immunotherapy of human cancer appears to depend on the administration of high doses which are frequently associated with excessive toxicity. Future use of rIL-2 will require certain modifications based on the use of lower doses of rIL-2 without significant loss of antitumor efficacy. We tested in vitro the possibility of potentiating the activity of rIL-2 in terms of LAK cell generation. We hypothesized that co-incubation of LAK cell precursors with a Chinese herbal extract (F3) of Astragalus membranaceus, (an immune modulator currently under study in our laboratory), along with a low concentration of rIL-2, would generate levels of LAK cell activity equivalent to those generated by high concentrations of rIL-2 alone. We found (1) a 10-fold potentiation of rIL-2 activity manifested by tumor cell-killing activity of 80% resulting from LAK cell generation with F3 plus 100 u/ml of rIL-2 versus 76% generated by 1,000 u/ml of rIL-2 alone; (2) a significant reduction in the number of effector LAK cells required for equicytotoxic reaction following LAK cell generation with F3 plus rIL-2 compared to rIL-2 alone. We conclude that potentiation of antitumor activity mediated by rIL-2 in low concentrations is possible by the concomitant use of another immune modulator such as Astragalus membranaceus.
Adjuvants, Immunologic↗
Abrogation of the local xenogeneic graft versus host reaction by preinjection of cimetidine to the immunosuppressed host rats: an experimental model for testing preclinical immunorestorative activity in vivo.
The local xenogeneic graft-vs-host reaction (XGVHR) is mediated by competent T lymphocytes. It was effectively used as a practical bioassay to assess the in vitro immunomodulatory effects of several drugs on cells derived from cancer patients. A modified version of the XGVHR model was recently developed primarily for preclinical evaluation of in vivo immunorestoration induced by experimental biological response modifiers (BRMs). A well-defined biological response modifier, cimetidine, was injected into Cytoxan-primed rats prior to their inoculation with xenogeneic human mononuclear cells. The cimetidine treatment induced a partial abrogation of the immunosuppressive effect of Cytoxan as manifested by a significant decline in the volume of the XGVHR from 115.23 +/- 15.72 mm3 (positive control) to 67.3 +/- 11.41 mm3 (P less than 0.01). This abrogation of Cytoxan-induced immunosuppression by cimetidine was incomplete since the XGVHR without Cytoxan (negative control, saline only) was still significantly lower (45.12 +/- 4.55 mm3; P less than 0.01). The effect of cimetidine injection in vivo appeared to be dose dependent and did not exhibit any nonspecific toxic effect to the mononuclear cell inoculum. These results indicate that the model can serve as a useful tool in the preclinical evaluation of newly developed immunorestorative biological response modifiers.
Animals↗
Synthesis and structure-activity relationship of 1-aryl-6,8-difluoroquinolone antibacterial agents.
A series of new arylfluoroquinolones has been prepared. These derivatives are characterized by having fluorine atoms at the 6- and 8-positions, substituted amino groups at the 7-position, and substituted phenyl groups at the 1-position. The in vitro antibacterial potency is greatest when the 1-substituent is 2,4-difluorophenyl and the 7-substituent is a 3-amino-1-pyrrolidinyl group. 1-(4-Fluorophenyl)-6,8-difluoro-7-piperazin-1-yl-1,4-dihydro-4-oxo quinoline-3- carboxylic acid (22) was found to possess excellent in vitro potency and in vivo efficacy.
Animals↗
Chiral DNA gyrase inhibitors. 2. Asymmetric synthesis and biological activity of the enantiomers of 9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H- pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid (ofloxacin).
A short and efficient synthesis, starting with (R)- and (S)-alaninol, of the two optical antipodes of the quinolone antimicrobial agent ofloxacin has been devised. Testing in vitro of the products against a range of bacteria and in an assay system incorporating purified DNA gyrase from different bacterial species demonstrates that the S-(-) enantiomer is substantially the more active.
Anti-Bacterial Agents↗