PubMed Health⌕ Search

Biomedical subjects

B Shane

Publications and source records attributed to B Shane.

At least 55 records · Page 3Linked to original sources
PubMed1992

Characterization of the coexisting multiple mechanisms of methotrexate resistance in mouse 3T6 R50 fibroblasts.

We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary B11 0.5 cells that overexpress comparable levels of dihydrofolate reductase, yet exhibit a 100-fold difference in MTX resistance while maintaining similar sensitivity to the lipophilic antifolates trimetrexate and piritrexim. These data suggested that R50 cells may possess additional mechanism(s) of antifolate resistance, such as MTX transport alteration. Flow cytometric analysis using fluorescein methotrexate revealed comparable levels of fluorescein MTX displacement with lipophilic antifolates in viable R50 and B11 0.5 cells, but marked insensitivity of R50 cells to MTX competition, thus suggesting a poor uptake of MTX into R50 cells. Analysis of the kinetic parameters of dihydrofolate reductase from R50 cells neither showed alterations in enzyme affinities for various antifolates nor in the Michaelis constant for folic acid and NADPH nor a change in the pH activity optimum. R50 cell-free extracts contained wild-type levels of folylpoly-gamma-glutamyl synthetase activity. However, following metabolic labeling with [3H]MTX, no MTX polyglutamates could be detected in R50 cells. We conclude that the high level of MTX resistance in R50 cells is multifactorial, including overexpression of dihydrofolate reductase, reduced MTX transport, and possibly altered formation of MTX polyglutamates. The potential interactions between the different modalities of MTX resistance in R50 cells are being discussed.

Animals↗
PubMed1992

Enhancement of biological activity by conjugation reactions.

Conjugation reactions, such as the formation of sulfates, glucuronides and amino acid derivatives of biologically active compounds, are classically considered as detoxication processes. In essence, common nonpolar compounds are rendered less active by the formation of water-soluble metabolites, which then can readily be excreted in the urine. In the past decade, however, it has become clear that some natural metabolites become more active rather than less so by the formation of such conjugates. Several compounds in this special group will be considered in this article; namely, glucuronide and hydroxyphenylamide conjugates of retinoic acid, glutathione and cysteine conjugates of halogenated hydrocarbons, glucuronide conjugates of various xenobiotics and polyglutamates of folic acid and its inhibitors. The metabolism, biological activity and toxicity of the parent molecule is profoundly affected in complex ways by such reactions. Thus, the historical concept of conjugation reactions as general detoxication processes is no longer tenable.

Animals↗
PubMed1991

In vitro metabolism of 5,8-dideazafolates and 5,8-dideazaisofolates by mammalian folylpoly-gamma-glutamate synthetase.

The in vitro metabolism of a variety of 5,8-dideazafolate and 5,8-dideazaisofolate analogues by pig liver folylpolyglutamate synthetase and the specificity of the enzyme for some polyglutamate derivatives of these analogues have been investigated. All 4-oxo-quinazoline analogues were metabolized to long chain polyglutamate derivatives, primarily the pentaglutamate, whereas 4-amino-quinazolines were metabolized to a lesser extent, with the accumulation of di- and triglutamate derivatives. This pattern of metabolism was consistent with the large drop in Vmax/Km and Vmax values for folylpolyglutamate synthetase observed with diglutamate derivatives of 4-aminofolate analogues. The extent of metabolism of the various analogues did not correlate with the relative substrate effectiveness of their parent monoglutamate derivatives. The 5-chloro and 5-methyl substitutions of quinazolines enhanced the addition of glutamate residues to 4-amino derivatives but markedly impaired the metabolism of 4-oxo derivatives.

Aminopterin↗
PubMed1991

Studies on the mechanism of antitumor action of 2-desamino-2-methyl-5,8-dideazaisofolic acid.

The new folate analogue, 2-desamino-2-methyl-5,8-dideazaisofolic acid, 2c, was synthesized and evaluated using a variety of biochemical and antitumor assays. For purposes of comparison, its 2-desamino, 2b, and 2-amino, 2a, counterparts, as well as N10-propargly-5,8-dideazafolic acid, 1a, and the corresponding 2-desamino, 1b, and 2-desamino-2-methyl, 1c, modifications were included in these studies. Compound 2c was found to be a potent inhibitor of the growth of L1210 and MCF-7 cells in culture, being only 2-fold and 5-fold less effective than 1c, respectively. However, although analogue 2c was 189-fold less inhibitory toward L1210 thymidylate synthase (TS) than 1c, its cytotoxicity was reversed completely by thymidine alone which suggests that the compound behaves as a TS inhibitor in cells. Enzymatically synthesized polyglutamates of 2c were substantially more inhibitory toward human TS than the parent compound. Compound 2c was the most efficient substrate for mammalian folyl-polyglutamate synthetase of the compounds studied having a Vmax/Km nearly 12-fold larger than 1c. Both 1c and 2c were effective inhibitors of the uptake of [3H]methotrexate into MOLT-4 cells, implying that each is efficiently transported into tumor cells. These results suggest that a weak inhibitor of TS in vitro can be a potent cytotoxic agent if it can readily gain entry into target cells and be converted to polyglutamated metabolites.

Animals↗
PubMed1990

Impaired milk folate secretion is not corrected by supplemental folate during iron deficiency in rats.

Decreased milk folate secretion in iron-deficient rat dams contributes to the impairment of folate metabolism in nursing pups. The present study was designed to assess whether impaired milk folate secretion secondary to iron deficiency is due to a decrease in the supply of folate to the mammary gland or to an inability of the mammary gland to effectively use folate. Rats were fed diets containing 0.5, 2.0 or 7.0 mg folate/kg and 8 (-Fe) or 250 (+Fe) mg Fe/kg throughout gestation and until d 17 of lactation. Regardless of dietary Fe content, maternal plasma, red blood cell, liver and kidney folate concentrations correlated with dietary folate content (r = 0.75-0.85, p less than 0.0001). With the exception of plasma folate level, which was 46% lower for -Fe than +Fe dams fed 0.5 mg folate/kg, no other differences in indices of folate status were noted between +Fe and -Fe dams. Dietary folate content had a direct impact on milk folate content in +Fe dams but not in -Fe dams. Mammary tissue methionine synthase and folylpolyglutamate synthetase activities were not depressed in Fe deficiency; rather, mean activities were elevated among -Fe dams fed 0.5 mg folate/kg. In conclusion, the reduction in milk folate secretion during Fe deficiency is not due to a decrease in the amount of folate supplied to the mammary gland; rather, the defect causing this reduction is specific to the mammary gland.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1989

Folylpolyglutamate synthesis and role in the regulation of one-carbon metabolism.

The physiological importance of folylpolyglutamates is now well established. These derivatives are the intracellular substrates and regulators of one-carbon metabolism, and their synthesis is required for normal folate retention by tissues. Over the last few years, a considerable amount of information has been obtained on the mechanism by which these compounds are synthesized, on how this synthesis is regulated, and on the effects of the polyglutamate chain on the interaction of folate substrates and inhibitors with folate-dependent enzymes. Many regulatory implications have been suggested by these studies, but the physiological relevance of some of these observations remains to be explored. Folates in mammalian tissues are metabolized to polyglutamates of chain lengths considerably longer than that required for folate retention, but the metabolic advantages of this are not entirely clear. Several in vivo model systems have been developed to explore the functioning of specific folylpolyglutamate chain lengths in metabolic cycles of one-carbon metabolism, and these are likely to shed further light on this point. The role of folate-binding proteins in folate transport, the metabolic role of glutamylhydrolases, and the role of folylpolyglutamates in putative multifunctional protein complexes are also areas that are being actively pursued at present and are likely to produce new insights in the future. Recent studies on the retention of antifolates by cells and on their substrate efficacy for folylpolyglutamate synthetases have also suggested mechanisms for the differential cytotoxicity of these agents for different tissues.

Animals↗
PubMed1989

Inhibition of mammalian folylpolyglutamate synthetase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin bearing a terminal L-ornithine.

Six new 5,8-dideaza analogues of folic acid and aminopterin containing a terminal L-ornithine residue were prepared by using multistep synthetic sequences. Each was evaluated as an inhibitor of hog liver folylpolyglutamate synthetase and human dihydrofolate reductase. Structural modifications at positions 2, 4, 5, and 10 were included to help define structure-activity relationships for compounds of this type. The compound N alpha-(4-amino-4-deoxy-5-chloro-5,8-dideazapteroyl)-L-ornithine (3f) was identified as the most potent inhibitor of mammalian folylpolyglutamate synthetase reported thus far (Ki congruent to 2 nM). Its 4-oxy counterpart, N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, was only 5-fold less inhibitory than 3f toward folylpolyglutamate synthetase but was found to be a much weaker inhibitor of dihydrofolate reductase than 3f.

Aminopterin↗
PubMed1989

Survey report. Jamaica.

Explore the source record for details and available documents.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Identification of 10-formyltetrahydrofolate dehydrogenase-hydrolase as a major folate binding protein in liver cytosol.

10-Formyltetrahydrofolate dehydrogenase (10-formyltetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.6) purified from pig liver contained bound tetrahydropteroylhexa-gamma-glutamate, a potent product inhibitor. Dehydrogenase purified from rat liver had chromatographic properties indistinguishable from those of a previously described major cytosolic folate binding protein of unknown function (Zamierowski, M.M. and Wagner, C. (1977) J. Biol. Chem. 252, 933-938; Cook, R.J. and Wagner, C. (1982) Biochemistry 21, 4427-4434). The dehydrogenase catalyzes the oxidative deformylation of 10-formyltetrahydrofolate to carbon dioxide and tetrahydrofolate. The tight binding of product to the enzyme suggests that oxidation of one-carbon moieties is regulated by the ratio of formyltetrahydrofolate to tetrahydrofolate in liver.

Animals↗
PubMed1988

Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates.

The transfer of 17O and/or 18O from (COOH-17O or -18O) enriched substrates to inorganic phosphate (Pi) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-18O-labeled folate, methotrexate, and dihydropteroate, in addition to [17O]-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. Pi was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for 17O and 18O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate 18O-enriched carboxyl group to Pi occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of Pi obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that 18O transfer had occurred.

Animals↗
PubMed1988

Substrate specificity of mammalian folylpoly-gamma-glutamate synthetase for 5,8-dideazafolates and 5,8-dideaza analogues of aminopterin.

The substrate specificity of pig liver folylpolyglutamate synthetase (tetrahydrofolate:L-glutamate gamma-ligase (ADP-forming), EC 6.3.2.17) for classical 5,8-dideaza analogues of folic acid, isofolic acid aminopterin and isoaminopterin has been investigated. 5,8-Dideazafolate and 5,8-dideazaaminopterin are very effective substrates with activities approaching those of the best reduced folate substrates. The analogous isofolate analogues are less effective substrates, but still better than folic acid. The 5-chloro substituent is the only modification that consistently increases the on rate, with 5-chloro-5,8-dideazaaminopterin being the most effective substrate found, thus far, for the enzyme. Methylation at positions 9 or 10 generally decreases binding, while 5-methylation increases the binding of 4-oxoquinazolines, but decreases the binding of their 4-amino counterparts. The presence of a formyl group at N9 or N10 has the opposite effect, decreasing the binding of 4-oxo analogues while increasing the rate for 4-amino derivatives. Increases in on rate with methyl, formyl or 4-amino substitutions are only significant when the parent compound is a poor substrate, suggesting that these groups do not interact directly with the enzyme but cause conformational changes in the structure of the substrate that influence binding to the enzyme.

Adenosine Triphosphate↗
PubMed1988

Folate bioavailability in humans: effects of wheat bran and beans.

The effect of wheat bran or California small white beans in the diet on absorption of monoglutamyl (PteGlu) and heptaglutamyl folic acid (PteGlu7) was studied in six men confined to a metabolic unit. Relative folate absorption was determined by measuring 24-h urinary folate excretion and serum folate levels at 0, 1, and 2 h after ingestion of a formula meal containing 1.13 mumol PteGlu or PteGlu7 (500 micrograms PteGlu equivalent). Serum data showed PteGlu absorption was more rapid than PteGlu7 absorption. Urinary excretion of PteGlu7 was 63% (50 less than or equal to mean less than or equal to 76%) of PteGlu excretion. Addition of 30 g wheat bran to the formula meal accelerated PteGlu absorption whereas PteGlu7 absorption was not significantly affected by either food. Effects of the two foods were qualitatively different. Wheat bran increased the absorption of PteGlu relative to PteGlu7 whereas beans minimized the difference between PteGlu and PteGlu7 serum areas.

Adult↗
PubMed1987

Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene.

The nucleotide sequence of the gene for folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase (folC) has been determined. The deduced amino acid sequence codes for a protein of Mr 45,380 and contains regions with homology to the A and B regions of ATP-binding sites. The folC gene is adjacent to a gene located 70 base pairs upstream of the initiation codon for folC. The nucleotide sequence of this upstream gene was also determined. Deletion of the upstream gene sequences from recombinant plasmids containing the folC gene results in about a 100-fold decrease in plasmid-dependent folylpolyglutamate synthetase activity, suggesting that the major promoter for folC is 5' to the upstream gene. The upstream gene codes for a protein of Mr 33,346, which is expressed in maxicells and amplified in cells containing the upstream gene in recombinant pUC8 plasmids. Expression of the upstream gene in maxicells was greater than that of folC, as determined by the intensity of 35S-labeled proteins after sodium dodecyl sulfate-gel electrophoresis. A region of dyad symmetry exists between the coding sequences of the two genes which may code for a transcription termination signal and be responsible for the attenuation of the expression of the folC gene relative to the upstream gene. The folC gene is located about 1 kilobase upstream of the purF gene region at 50 min on the Escherichia coli map. The function of the upstream gene product is unknown. It contains sequences with homology to metal-binding domains in nucleic acid-binding proteins. A new purification procedure for obtaining large quantities of folylpolyglutamate synthetase-dihydrofolate synthetase is described.

Amino Acid Sequence↗
PubMed1987

Folylpolyglutamates in Leishmania major.

The intracellular folates of the protozoan parasite Leishmania major have been examined. About 95% of the exogenous [3H]folate accumulated by the protozoan is metabolized to polyglutamate conjugates within 65 hr, and the intracellular folates are about forty-fold concentrated over the folate in the medium. The predominant metabolite of folic acid is the pentaglutamate conjugate (85%), with lessor amounts of the tetraglutamate (approximately 9%) and hexaglutamate (approximately 3%), and trace (less than 2.5%) amounts of di-, tri- and hepta-glutamate conjugates. Chromatographic properties of the products indicate that the conjugates are linked through the gamma-carboxyl groups. The folylpolyglutamate distribution in Leishmania is similar to that found in mammalian tissues.

Animals↗
PubMed1987

Mammalian folylpoly-gamma-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme.

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of Mr 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K+ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the Km value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.

Animals↗
PubMed1987

Mammalian folylpoly-gamma-glutamate synthetase. 2. Substrate specificity and kinetic properties.

The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis (kcat = 2.5 s-1) while 5- and 10-position substitutions of the folate molecule impair catalysis. kcat values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. The on rates for most pteroyldiglutamates are similar to the rates for their respective monoglutamate derivatives, but further extension of the glutamate chain results in a progressive decrease in on rates. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The Km for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and beta,gamma-methylene-ATP, beta,gamma-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P1,P5-di(adenosine-5') pentaphosphate, and free ATP4- are potent inhibitors of the reaction.

Animals↗