Biomedical subjects

C M Lin

Publications and source records attributed to C M Lin.

At least 55 records · Page 3Linked to original sources

PubMed1996

Interactions of 2-methoxyestradiol, an endogenous mammalian metabolite, with unpolymerized tubulin and with tubulin polymers.

2-Methoxyestradiol (2ME) is an endogenous mammalian catabolite of estradiol with antimitotic activity. Although it is a competitive inhibitor of the binding of colchicine to tubulin, it has unusual effects on glutamate-induced tubulin polymerization. Polymer that was little changed in morphology assembled at a reduced rate and was relatively cold stable. We have now examined interactions of [4-3H]-2ME with unpolymerized tubulin and polymer. The [3H]2ME binds avidly to tubulin even on ice, and it is readily displaced by other colchicine site drugs. An association rate constant on ice of 1.9 x 10(2) M-1s-1 was obtained. Scatchard analysis indicated a single class of binding site and an association equilibrium constant of 5.7 x 10(5) M-1. These values lead to a calculated dissociation rate constant of 3.3 x 10(-4) s-1. In glutamate-induced tubulin assembly, a reaction that requires GTP and leads to the formation of sheets of parallel protofilaments, increasing amounts of [3H]2ME were incorporated into polymer, reaching near-stoichiometry with tubulin at 100 microM 2ME. Equivalent binding of [3H]2ME occurred when the drug was added to preformed polymer, but binding of [3H]2ME to polymer was not readily inhibited by colchicine site drugs. Significant amounts of [3H]2ME were also incorporated into microtubule polymer formed with microtubule-associated proteins, glycerol, or 4-morpholineethanesulfonate buffer, but the stoichiometry was substantially lower than that in the sheet polymer induced by either glutamate or 1,4-piperazineethanesulfonate buffer. The structural differences between the microtubule and sheet polymers leading to these differences in apparent affinity for 2ME are unknown, but presumably interaction of the estrogen metabolite with cellular microtubules has functional significance related to the antimitotic properties of the compound.

2-Methoxyestradiol↗

PubMed1996

Antitumor 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone derivatives. Interactions with tubulin.

A series of derivatives of 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone (DHQZ) with known antitumor activity was re-evaluated in the National Cancer Institute cancer cell line screen. Analysis by the COMPARE algorithm suggested that their cytotoxicity derived from interactions with tubulin. Significant inhibition of tubulin assembly and of the binding of radiolabeled colchicine to tubulin was demonstrated with several of the compounds, particularly NSC 145669, 175635, and 175636. The DHQZ derivatives are structurally analogous to a number of antimitotic agents, flavonols and derivatives of 2-styrylquinazolin-4(3H)-one and of 2-phenyl-4-quinolone. Structure-activity analogies between these agents, the combretastatins, and the colchicinoids were analyzed and summarized.

Animals↗

PubMed1996

Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than taxol.

Computer-assisted structure analysis indicated (+)-discodermolide, a polyhydroxylated alkatetraene lactone marine natural product, was an antimitotic compound, and we confirmed this prediction. Previous work had shown an accumulation of discodermolide-treated cells in the G2/M portion of the cell cycle, and we have now found that discodermolide arrests Burkitt lymphoma cells in mitosis. Discodermolide-treated breast carcinoma cells displayed spectacular rearrangement of the microtubule cytoskeleton, including extensive microtubule bundling. Microtubule rearrangement that occurred with 10 nM discodermolide required 1 microM taxol. Discodermolide had equally impressive effects on tubulin assembly in vitro. Near-total polymerization occurred at 0 degree C with tubulin plus microtubule-associated proteins (MAPs) under conditions in which taxol at an identical concentration was inactive. Without MAPs and/or without GTP, tubulin assembly was also more vigorous with discodermolide than with taxol under every reaction condition examined. Discodermolide-induced polymer differed from taxol-induced polymer in that it was completely stable at 0 degree C in the presence of high concentrations of Ca2+. In a quantitative assay designed to select for agents more effective than taxol in inducing assembly, discodermolide had an EC50 value of 3.2 microM versus 23 microM for taxol.

Alkanes↗

PubMed1996

A convenient tubulin-based quantitative assay for paclitaxel (Taxol) derivatives more effective in inducing assembly than the parent compound.

A room temperature biochemical assay, based on centrifugal removal of tubulin polymer, was developed to permit ready detection of paclitaxel analogs more active than the parent compound and to permit reliable quantification of differences in activity relative to paclitaxel in terms of drug concentration. The assay was validated by comparing paclitaxel to two compounds (docetaxel and 2-debenzoyl-2-meta-azidobenzoylpaclitaxel) known to be more active under multiple reaction conditions. The assay was designed to yield a relatively high EC50 (23 microM) for paclitaxel. This was possible because paclitaxel only weakly induced tubulin assembly at room temperature in 0.4 M glutamate without exogenous GTP. Under these same reaction conditions 50% assembly occurred with 4.7 microM 2-debenzoyl-2-meta-azidobenzoylpaclitaxel and 11 microM docetaxel. These biochemical EC50 values were in agreement with the relative cytotoxicity of the three compounds for human Burkitt lymphoma CA46 cells (IC50 values for paclitaxel, docetaxel, and 2-debenzoyl-2-meta-azidobenzoylpaclitaxel were 40, 10, and 3 nM, respectively).

Animals↗

PubMed1996

Echocardiographic predictors of survival after surgery for mitral regurgitation in the age of valve repair.

The objective of this study was to identify echocardiographic and clinical predictors of survival after mitral valve surgery when mitral repair is an option. In 132 patients undergoing mitral valve repair or replacement for the diagnosis of mitral regurgitation, preoperative echocardiograms were analyzed quantitatively and reviewed by two independent observers for structural abnormalities of the mitral valve. In Cox regression analysis, clinical factors such as age (mortality rate ratio [MRR] 1.7/decade, 95% confidence intervals [CI] 1.1, 2.4), and New York Heart Association class IV (MRR 3.1, 95% CI 1.4, 6.7) and echocardiographic factors including morphologic evidence of endocarditis or myxomatous disease (MRR 0.3, 95% CI 0.1, 0.7) were significant predictors of overall survival, although valve repair itself was not. End-systolic dimensions and volumes were not, likely related to the small number of patients with markedly increased end-systolic dimensions or volumes (5 patients [4%] with end-systolic dimension > 5.5 cm, 12 patients [9%] with end-systolic volume index > 60 ml/m2). New York Heart Association class IV (MRR 2.9, 95% CI 1.3, 6.4), age (MRR 1.7/decade, 95% CI 1.2, 2.6), and the presence of calcification (MRR 4.6, 95% CI 1.3, 16.2) were independent predictors of survival in multivariate analysis. In this contemporary cohort of patients undergoing repair or replacement for mitral regurgitation, factors such as echocardiographically determined cause of disease and presence of calcification predicted survival; traditional measurements such as end-systolic dimensions and volumes were less predictive, most likely because patients underwent surgery before their ventricles became markedly enlarged. Clinical factors such as age and functional status remained the most potent predictors of survival after surgery for mitral regurgitation.

Age Factors↗

PubMed1996

Transgenic fish and its application in basic and applied research.

Since 1985, transgenic fish have been successfully produced by microinjecting or electroporating desired foreign DNA into unfertilized or newly fertilized eggs using many different fish species. More recently, transgenic fish have also been produced by infecting newly fertilized eggs with pantropic, defective retroviral vectors carrying desired foreign DNA. These transgenic fish can serve as excellent experimental models for basic scientific investigations as well as in biotechnological applications. In this paper, we will review the current status of the transgenic fish research and its potential application in basic and applied research.

Animals↗

PubMed1995

Interactions of tubulin with guanine nucleotides that have paclitaxel-like effects on tubulin assembly: 2',3'-dideoxyguanosine 5'-[alpha,beta-methylene]triphosphate, guanosine 5'-[alpha,beta-methylene]triphosphate, and 2',3'-dideoxyguanosine 5'-triphosphate.

Despite reduced affinity for the exchangeable nucleotide binding site of tubulin relative to GTP, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) and guanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pG] are highly active in promoting tubulin assembly. Like the antimitotic drug paclitaxel, which interacts with the same part of the beta-tubulin molecule as exchangeable-site GTP, both analogs enhance nucleation reactions and promote formation hyperstable polymers. These observations led us to synthesize the doubly modified analog 2',3'-dideoxyguanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pddG]. We compared the effects of pp(CH2)pddG to those of ddGTP, pp(CH2)pG, and the three-cognate diphosphates in their interactions with tubulin. We found that pp(CH2)pddG was as active as ddGTP and pp(CH2)pG in supporting formation of polymer of increased stability, but that its affinity for the exchangeable site was lower than that of both singly modified analogs [relative affinities for the exchangeable site for pp(CH2)pddG:ddGTP:pp(CH2)-pG:GTP were 1:2.8:10:273]. There were significant differences in interactions of each of the three analogs with tubulin, and the behavior of pp(CH2)pddG was intermediate between that of ddGTP and that of pp(CH2)pG. Most importantly, under the reaction conditions studied, with heat-treated microtubule-associated proteins (MAPs) ddGTP-induced polymer consisted of short microtubules, while polymer formed with both pp(CH2)pddG and pp(CH2)pG consisted of short sheets. On the other hand, assembly without MAPs had a fivefold lower critical concentration for tubulin with ddGTP and pp(CH2)pddG (0.5 mg/ml) than with pp(CH2)pG (2.5 mg/ml). De novo assembly, which occurs readily with 2',3'-dideoxyguanosine 5'-diphosphate, was not observed with either alpha, beta-methylenediphosphate GDP analog.

Antineoplastic Agents↗

PubMed1995

Transmembrane signaling by the aspartate receptor: engineered disulfides reveal static regions of the subunit interface.

Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18-75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays.

Cell Membrane↗

PubMed1995

Synthesis, antitubulin and antimitotic activity, and cytotoxicity of analogs of 2-methoxyestradiol, an endogenous mammalian metabolite of estradiol that inhibits tubulin polymerization by binding to the colchicine binding site.

In order to define the structural parameters associated with the antitubulin activity and cytotoxicity of 2-methoxyestradiol, a mammalian metabolite of estradiol, an array of analogs was synthesized and evaluated. The potencies of the new congeners as inhibitors of tubulin polymerization and colchicine binding were determined using tubulin purified from bovine brain, and the cytotoxicities of the new compounds were studied in a variety of cancer cell cultures. Maximum antitubulin activity was observed in estradiols having unbranched chain substituents at the 2-position with three non-hydrogen atoms. 2-Ethoxyestradiol and 2-((E)-1-propenyl)-estradiol were substantially more potent than 2-methoxyestradiol itself. The tubulin polymerization inhibitors in this series displayed significantly higher cytotoxicities in the MDA-MB-435 breast cancer cell line than in the other cell lines studied. The potencies of the analogs as cytotoxic and antimitotic agents in cancer cell cultures correlated with their potencies as inhibitors of tubulin polymerization, supporting the hypothesis that inhibition of tubulin polymerization is the mechanism of the cytotoxic action of 2-methoxyestradiol and its congeners. Several of the more potent analogs were tested in an estrogen receptor binding assay, and their affinities relative to estradiol were found to be very low.

2-Methoxyestradiol↗

PubMed1995

Production of a biologically active recombinant teleostean growth hormone in E. coli cells.

We have isolated and characterized several recombinant lambda phage clones carrying growth hormone (GH) cDNA of striped bass (Morone saxatilis). Nucleotide sequence and the predicted amino acid sequence of sbGH was determined from a recombinant clone carrying the longest cDNA insert. The sbGH cDNA encodes a pre-hormone of 204 amino acid residues. Comparison of the predicted amino acid sequence of sbGH with those of other vertebrates revealed different degrees of sequence identity: approximately 98% with European sea bass; 90% with bluefin tuna; bonito and red seabream; 71% with winter flounder; 64% with salmonids; 55% with carp; and 38% with human. Expression of the mature sbGH cDNA (without the signal peptide sequence) in E. coli cells under regulation of the lambda phage PL promoter produced a polypeptide of 20 kDa. Following renaturation, this recombinant hormone was shown to be biologically active in a radioreceptor competition binding assay and in the induction of hepatic insulin-like growth factor I (IGF-I) mRNA synthesis in vivo.

Amino Acid Sequence↗

PubMed1995

Effects of alcohol moderation on blood pressure and intracellular cations in mild essential hypertension.

It is known that moderation of alcohol intake reduces blood pressure, although the exact mechanism has not yet been established. To clarify the hypotensive mechanism of alcohol reduction, we evaluated the change in cellular magnesium and sodium metabolism during alcohol reduction in mild hypertensive patients. We measured intraerythrocyte sodium and magnesium, intraplatelet free magnesium concentrations, and erythrocyte ouabain-sensitive 22Na efflux rate constant (Kos) in 17 mild essential hypertensive patients regularly consuming more than 40 g/day of alcohol, before and after 4 weeks of alcohol reduction, and 12 age-matched nondrinking hypertensives. Intraerythrocyte magnesium (P < .01) and intraplatelet free magnesium (P < .05) concentrations were significantly lower in drinkers than in nondrinkers. In drinkers, advice to reduce alcohol intake for 4 weeks resulted in a reduction in self-reported alcohol consumption from 461.7 to 71.6 g/week, a significant fall in both supine systolic blood pressure (136.3 +/- 10.8 to 130.8 +/- 11.3 mm Hg, P < .001) and supine diastolic blood pressure (85.1 +/- 8.6 to 82.6 +/- 8.7 mm Hg, P < .05). The fall in mean blood pressure correlated positively with the reduction in weekly alcohol consumption. Intraerythrocyte magnesium and Kos were increased (P < .05, P < .01, respectively), while intraerythrocyte sodium was decreased (P < .01). The increase in intraerythrocyte magnesium correlated negatively with the fall in mean blood pressure and positively with the increase in Kos, which correlated negatively with the decrease in intraerythrocyte sodium.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

Antitumor agents, 154. Cytotoxic and antimitotic flavonols from Polanisia dodecandra.

Three flavonols, 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone [1], 5,4'-dihydroxy-3,6,7,8,3'-pentamethoxyflavone [2], and quercetin 3-O-beta-D-glucopyranosyl-7-O-alpha-L-rhamnopyranoside [3], were isolated from Polanisia dodecandra. Compound 1 showed remarkable cytotoxicity in vitro against panels of central nervous system cancer (SF-268, SF-539, SNB-75, U-251), non-small cell lung cancer (HOP-62, NCI-H266, NCI-H460, NCI-H522), small cell lung cancer (DMS-114), ovarian cancer (OVCAR-3, SK-OV-3), colon cancer (HCT-116), renal cancer (UO-31), a melanoma cell line (SK-MEL-5), and two leukemia cell lines (HL-60 [TB], SR), with GI50 values in the low micromolar to nanomolar concentration range. This substance also inhibited rubulin polymerization (IC50 = 0.83 +/- 0.2 microM) and the binding of radiolabeled colchicine to tubulin with 59% inhibition when present in equimolar concentrations with colchicine. Compound 2 also showed cytotoxicity against medulloblastoma (TE-671) tumor cells with an ED50 value of 0.98 microgram/ml. Compound 1 appears to be the first example of a flavonol to exhibit potent inhibition of tubulin polymerization and, therefore, warrants further investigation as an antimitotic agent.

Antineoplastic Agents↗

PubMed1994

Antitumor agents. 155. Synthesis and biological evaluation of 3',6,7-substituted 2-phenyl-4-quinolones as antimicrotubule agents.

A series of 3',6,7-substituted 2-phenyl-4-quinolones were designed and synthesized as antimitotic antitumor agents. All compounds showed cytotoxic effects (log GI50 < or = -4.0; log drug molar concentration required to cause 50% inhibition) against the growth of a variety of human tumor cell lines, including those derived from solid tumors such as non-small cell lung, colon, central nervous system, ovary, prostate, and breast cancers, when evaluated in the National Cancer Institute's 60 human tumor cell line in vitro screen. The most potent compound (26) demonstrated strong cytotoxic effects with GI50 values in the nanomolar or subnanomolar range in almost all the tumor cell lines. Compound 26 was also a potent inhibitor of tubulin polymerization and radiolabeled colchicine binding to tubulin, with activity comparable to those of the potent antimitotic natural products colchicine, podophyllotoxin, and combretastatin A-4.

Animals↗

PubMed1994

2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.

A metabolite of estradiol, 2-methoxyestradiol (2ME), inhibits angiogenesis in the chicken embryo chorioallantoic membrane assay. Since 2ME causes mitotic perturbations, we examined its interactions with tubulin. In our standard 1.0 M glutamate system (plus 1.0 mM MgCl2 at 37 degrees C), superstoichiometric concentrations (relative to tubulin) of 2ME inhibited the nucleation and propagation phases of tubulin assembly but did not affect the reaction extent. Although polymer formed in the presence of 2ME was more cold-stable than control polymer, morphology was little changed. Under suboptimal reaction conditions (0.8 M glutamate/no MgCl2 at 26 degrees C), substoichiometric 2ME totally inhibited polymerization. No other estrogenic compound was as effective as 2ME as an inhibitor of polymerization or of the binding of colchicine to tubulin. Inhibition of colchicine binding was competitive (Ki, 22 microM). Thus, a mammalian metabolite of estradiol binds to the colchicine site of tubulin and, depending on reaction conditions, either inhibits assembly or seems to be incorporated into a polymer with altered stability properties.

2-Methoxyestradiol↗

PubMed1994

Antitumor agents. 150. 2',3',4',5',5,6,7-substituted 2-phenyl-4-quinolones and related compounds: their synthesis, cytotoxicity, and inhibition of tubulin polymerization.

As part of our continuing search for potential anticancer drug candidates in the 2-phenyl-4-quinolone series, we have synthesized a series of 6,7-methylenedioxy-substituted and unsubstituted 2-phenyl-4-quinolones, as well as related compounds. Their in vitro inhibition of human tumor cell lines and tubulin polymerization is reported. In general, a good correlation was found between cytotoxicity and inhibition of tubulin polymerization. Compounds 7, 9, 13, 16, 22, 23, 36, and 37 showed potent inhibitory effects in both assays. All rigid analogs (47-49) and trimethoxy-substituted compounds showed little or no activity. Substitution at the 4'-position also resulted in compounds with little or no activity, except for hydroxyl or methyl groups at this position. Further investigation is underway to determine if substitution at the 3'-position will result in compounds with increased activity.

Animals↗

PubMed1994

Calcium supplementation inhibits the expression of parathyroid hypertensive factor in DOCA-salt hypertensive rats.

To elucidate the antihypertensive mechanism of oral calcium supplementation in salt-dependent hypertension, we investigated the hypertensive activity of plasma from DOCA-salt hypertensive rats fed with (DS-Ca) or without (DS) a high calcium diet. Four weeks of calcium supplementation (4% CaCl2) attenuated the blood pressure increase in DS rats. Intravenous bolus injection of dialyzed plasma (1.0-kDa cutoff) from DS rats to normotensive rats resulted in a sustained elevation in blood pressure, whereas that from DS-Ca rats did not. As the endothelin concentration was not different between the two groups, the circulating hypertensive substance in DS rats may be identical to parathyroid hypertensive factor (PHF) and the inhibition of its expression by calcium may be involved in the hypotensive mechanism of high calcium diets in salt-dependent hypertension.

Animals↗

PubMed1993

Synthesis and evaluation of a series of benzylaniline hydrochlorides as potential cytotoxic and antimitotic agents acting by inhibition of tubulin polymerization.

Although certain substituted cis-stilbenes have displayed potent tubulin polymerization inhibitory activity and significant cytotoxicities in cancer cell cultures, these compounds have limited aqueous solubility and are therefore difficult to formulate for in vivo evaluation. A series of water-soluble N-(3,4,5-trimethoxybenzyl)aniline salts has therefore been synthesized in which the olefinic bridge of the stilbenes is replaced by an aminomethylene hydrochloride moiety. A relationship was found between the size of the substituent in the 4-position of the aniline ring and both antitubulin activity and cytotoxicity, such that the smaller the substituent, the greater the potency. The most promising of the newly synthesized compounds was 4-methyl-N-(3,4,5-trimethoxybenzyl)aniline hydrochloride, with an IC50 value of 3.5 microM for inhibition of tubulin polymerization and cytotoxicity for a wide variety of cancer cell lines. The cytotoxicities of the benzylaniline hydrochlorides correlated remarkably well with their antitubulin activities.

Aniline Compounds↗

PubMed1993

Antitumor agents. 141. Synthesis and biological evaluation of novel thiocolchicine analogs: N-acyl-, N-aroyl-, and N-(substituted benzyl)deacetylthiocolchicines as potent cytotoxic and antimitotic compounds.

Three series of novel thiocolchicine analogs, N-acyl-, N-aroyl-, and N-(substituted benzyl)-deacetylthiocolchicinoids, have been synthesized and evaluated for their cytotoxicity against various tumor cell lines, especially solid tumor cell lines, and for their inhibitory effects on tubulin polymerization in vitro. Most of these compounds showed strong inhibitory effects on tubulin polymerization comparable to that obtained with thiocolchicine and greater than that obtained with colchicine. Only compounds with a long side chain at the C(7) position, such as 22-24, did not inhibit tubulin polymerization. Several of the active N-aroyldeacetylthiocolchicine analogs had positive optical rotations, in contrast to the negative optical rotation observed with most colchicinoids. This property might be attributed to a reversal of biaryl configuration from the normal aS to aR. Therefore, the N-aroyl analogs were further evaluated by circular dichroism, which readily distinguishes between the aS and aR biaryl configurations. This latter technique demonstrated that the active N-aroyl analogs do have an aS configuration despite their positive optical rotations. However, comparison of 1H NMR and UV spectral data of N-(substituted benzyl)-deacetylthiocolchicines with those of corresponding N-aroyldeacetylthiocolchicines suggested a different biaryl dihedral angle [even though these compounds have the same aS biaryl configuration]. The similar tubulin binding properties of these compounds suggest that a biaryl dihedral angle of 53 degrees is not essential for colchicinoid-tubulin interaction. The increased cytotoxicity of N-(substituted benzyl)deacetylthiocolchicines compared to the N-aroyldeacetylthiocolchicines may be attributed to different lipophilicity, drug uptake, or drug metabolism in the tumor cells. The side chain at the C(7) position affects inhibition of tubulin polymerization and the cytotoxic activity of colchicinoids as a function of its size and its contribution to lipophilicity.

Antineoplastic Agents↗