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At least 685 records · Page 38Linked to original sources
PubMed2002

The spinocerebellar ataxias: order emerges from chaos.

In the past decade, the genetic etiologies accounting for most cases of adult-onset dominant cerebellar ataxia have been discovered. This group of disorders, generally referred to as the spinocerebellar ataxias (SCAs), can now be classified by a simple genetic nosology, essentially a sequential list in which each new SCA is given a number. However, recent advances in the elucidation of SCA pathogenesis provide the opportunity to subclassify the disorders into three discrete groups based on pathogenesis: 1) the polyglutamine disorders, SCAs 1, 2, 3, 7, and 17, which result from proteins with toxic stretches of polyglutamine; 2) the channelopathies, SCA6 and episodic ataxia types 1 and 2 (EA1 and EA2), which result from disruption of calcium or potassium channel function; and 3) the gene expression disorders, SCAs 8, 10, and 12, which result from repeat expansions outside of coding regions that may quantitatively alter gene expression. SCAs 4, 5, 9, 11, 13-16, 19, 21, and 22 are of unknown etiology, and may or may not fit into one of these three groups. At present, most diagnostic and therapeutic strategies apply equally to all of the SCAs. Therapy specific for individual diseases or types of diseases is a realistic goal in the foreseeable future.

Adult↗
PubMed1985

Fluorometric high-performance liquid chromatographic analysis of 10-deazaaminopterin, 10-ethyl-10-deazaaminopterin, and known metabolites.

The antifolate compounds 10-deazaaminopterin (10-dAM) and 10-ethyl-10-deazaaminopterin (10-EdAM) are therapeutically superior to methotrexate in transplanted murine tumor systems and in human tumor xenografts growing in immunodeficient "nude" mice. The increased therapeutic index of these analogs correlates with their selective uptake, retention, and polyglutamation within neoplastic cells. We have developed a fluorescence high-performance liquid chromatographic assay applicable to 10-dAM, 10-EdAM, their polyglutamate anabolites, and their 7-hydroxy (7-OH) and deglutamate catabolites. The assay is based upon the high native fluorescence of pteridine-containing compounds which contain carbon in the 10 position. The assay employs a reverse-phase C-18 column and an ascending acetonitrile gradient in 50 mM phosphate, pH 7.0. The compounds are extracted from plasma and urine with 95 +/- 7% and 98 +/- 2% recoveries, respectively, using C-18 Sep-Paks. The linear range of the assay is, for 10-dAM, 2-100 nM, and for 10-EdAM, 1-100 nM. Polyglutamated metabolites of [3H]10-EdAM isolated from L1210 cells have been separated by HPLC with identification of five derivatives (Glu 1-5) confirmed by enzymatic peak shift using serum conjugase and by quantitative correlation of fluorescence intensity, radioactivity, and titration inhibition of dihydrofolate reductase. The assay has been used successfully in pharmacokinetic analyses of plasma and urine samples from patients receiving 10-dAM and 10-EdAM. In patients who had received 10-EdAM, 7-OH-10-EdAM, and the deglutamate catabolite were also detected. This HPLC fluorescence assay is superior to the dihydrofolate reductase inhibition and binding assays with regard to specificity and precision; moreover, it can provide a means for simultaneous assay of the physiologically important anabolites and catabolites of these new antifolates.

Aminopterin↗
PubMed1983

Regulatory aspects of the glutamylation of methotrexate in cultured hepatoma cells.

The glutamylation of methotrexate has been evaluated in H35 hepatoma cells in vitro as a function of the conditions of culture. Glutamylation yields methotrexate polyglutamate with two to five additional glutamate residues and is a saturable process. The rate of glutamylation increases little above 10 microM extracellular methotrexate which corresponds to an intracellular concentration of approximately 4 microM. The rate of glutamylation measured over a 6-h period was stimulated by a reduction in cellular folates and prior incubation of the cells with insulin. Glutamylation was also more rapid in dividing cultures than in confluent cells. The combination of insulin inclusion and folate reduction, which was additive, caused approximately a fourfold increase in the rate of glutamylation over control cells under the conditions tested. The maximal rate of methotrexate glutamylation, which was 100 nmol/g/h, occurred in folate-depleted, insulin-supplemented cells. Supplementing folate-depleted cells with reduced folate coenzymes caused the glutamylation to be reduced by more than 90%. The turnover of methotrexate polyglutamates in cells saturated with these derivatives occurred at approximately one-half the rate of net synthesis and was stimulated to nearly the same extent by folate depletion and insulin. In addition to showing that folates can modify the rates of methotrexate polyglutamate formation, data are presented suggesting that methotrexate polyglutamates can regulate their own synthesis. The consequences of the formation of these retained forms of methotrexate in H35 hepatoma cells (M. Balinska, J. Galivan, and J.K. Coward (1981) Cancer Res. 41,2751-2756) and the effects of potential regulators of this process are discussed in terms of the glutamylation of folates in the cells and the chemotherapeutic effects of antifolates.

Animals↗
PubMed1986

Modulation by basic polypeptides of ATP-induced activation of tyrosine hydroxylase prepared from bovine adrenal medulla.

The effects of basic polypeptides on the activation of adrenal tyrosine hydroxylase by ATP were investigated to show a possible involvement of macromolecular cell components in the regulation of the enzyme activity. Basic polypeptides caused an enhancement of the activation of tyrosine hydroxylase by low concentrations of ATP, and the potentiating effects of these polypeptides were observed to be dependent on their concentrations. Kinetic studies showed that basic polypeptides caused an increase in the Vmax of the ATP-activated enzyme for the cofactor without any change in the Km. These results suggest that basic polypeptides convert the enzyme from a nonsusceptible form to a form susceptible to ATP, thus resulting in the potentiation of the ATP-induced activation. Furthermore, the activation by ATP of tyrosine hydroxylase was not observed after treatment of the enzyme preparation with CM-cellulose, and the responsiveness of the enzyme treated with CM-cellulose to ATP was partially restored by addition of basic polypeptides. These observations suggest the possibility that macromolecular cell components, presumably basic proteins, may be involved in the regulation of the activity of tyrosine hydroxylase through their modulating effects on the sensitivity of the enzyme to ATP within the cell.

Adenosine Triphosphate↗
PubMed1987

The kinetics of methotrexate polyglutamation in human breast cancer cells.

The polyglutamation kinetics of methotrexate (MTX) in MCF-7 human breast cancer cells have been formulated mathematically. The model takes account of glutamation and hydrolysis kinetics up through the pentaglutamate level, increased synthesis of dihydrofolate reductase following exposure to drug, reversible tight-binding to reductase, and membrane transport of polyglutamates. The glutamation, hydrolysis, and efflux parameters have been determined from fits to experimental MTX polyglutamate uptake and efflux data. The preferred substrate for folypolyglutamyl synthase in the intact cell appears to be MTX diglutamate, on average being two to three times as reactive as either the parent drug or the triglutamate. Hydrolysis rate constants range from 0.03 to 0.19 h-1, but no clear trend with chain length is observable given the large uncertainty of each parameter estimate. However, the efflux of MTX polyglutamates from MCF-7 cells does show a trend with chain length decreasing with increasing length as expected. The best characteristic time of MTX diglutamate efflux is 4.1 h, about one-third that of the higher polyglutamate species, in agreement with observations on the MDA.MB.436 breast cancer cell line. The model shows quantitative agreement with the fraction of MTX polyglutamates found still to be bound to reductase in MCF-7 cells following 24 h of efflux, and qualitative agreement with the time dependence of bound MTX-polyglutamate concentration profiles obtained on the ZR-75 breast cancer line.

Breast Neoplasms↗
PubMed1984

Enzymatic synthesis of polyglutamate derivatives of 7-hydroxymethotrexate.

7-Hydroxymethotrexate, an important metabolite of methotrexate, is a substrate for folylpolyglutamate synthetase (FPGS) isolated from rat liver and several human leukemia cell lines. The substrate activity it displays over a wide range of concentrations (0-200 microM) is nearly equivalent to that of methotrexate. The 7-hydroxy derivative of dichloromethotrexate is also a substrate for FPGS. The pattern of polyglutamate products synthesized by rat liver FPGS was nearly identical with both 7-hydroxymethotrexate and methotrexate. In addition, conversion of MTX polyglutamates to the corresponding 7-hydroxy compounds was demonstrated using partially purified rabbit liver aldehyde oxidase. The rate of conversion was concentration dependent, and the relative rate decreased as the MTX polyglutamate chain length increased. We propose that 7-hydroxymethotrexate polyglutamates may be formed by initial hydroxylation of methotrexate and subsequent polyglutamate formation or by direct hydroxylation of methotrexate polyglutamates. It was further shown that the relative substrate activity of folate analogs for folylpolyglutamate synthetase is dependent on the source of the enzyme.

Aldehyde Oxidase↗
PubMed1984

Detection by high-performance liquid chromatography of methotrexate and its metabolites in tumor tissue from osteosarcoma patients treated with high-dose methotrexate/leucovorin rescue.

Methotrexate (MTX) polyglutamates were detected in osteogenic sarcoma tumor samples obtained from patients 24 or 48 h after receiving high-dose MTX/leucovorin rescue therapy. Tumor samples were assayed by high-performance liquid chromatography, and polyglutamyl metabolites, along with MTX, were quantitated using both direct u.v. absorption at 313 nm and an enzyme titration assay. Good agreement between these two methods was found although the more sensitive enzyme assay detected peaks in some samples not detected by u.v. absorbance. A wide variation in MTX:MTX polyglutamate levels (1:1 to 25:1) was found among the six clinical samples studied. Also, no correlation between the extent of polyglutamate formation and plasma levels (determined at the time of tumor sampling) was observed. High intracellular levels of a derivative which appears to be the 7-hydroxy metabolite of MTX were also detected in four of six samples. This material coeluted with authentic standard, showed spectral properties like standard 7-OH-MTX, and did not inhibit dihydrofolate reductase.

Chromatography, High Pressure Liquid↗
PubMed1985

The effect of leucovorin on the synthesis of methotrexate poly-gamma-glutamates in the MCF-7 human breast cancer cell line.

The modulating effects of leucovorin on the synthesis of methotrexate (MTX) polyglutamates in the MCF-7 human breast cancer cell line have been investigated using a paired-ion high performance liquid chromatography (HPLC) system. Leucovorin decreased the intracellular level of MTX and profoundly affected polyglutamate synthesis irrespective of whether it was administered with or after MTX. Inhibition of MTX polyglutamate synthesis was also observed when concentrations of leucovorin too low to affect intracellular levels of MTX were employed. Leucovorin did not promote efflux of MTX from the MCF-7 cells and did not affect the distribution of the retained drug amongst the various polyglutamate forms.

Breast Neoplasms↗
PubMed1985

The effect of the rate of cell proliferation on the synthesis of methotrexate poly-gamma-glutamates in two human breast cancer cell lines.

The synthesis of methotrexate poly-gamma-glutamates by the MDA-MB-436 and MCF-7 human breast cancer cell lines is highly dependent on the rate of cell growth. Slowly proliferating cells accumulate methotrexate to the same extent as rapidly proliferating cells but convert a lower percentage of the drug to polyglutamate forms. The longest polyglutamate-derivatives of methotrexate are generally only synthesized when the cells are doubling rapidly. The MDA-MB-436 cells exhibit a biphasic response of doubling time and polyglutamation to increasing initial cell number. Extremes of cell density are associated with long doubling times and reduced polyglutamate synthesis. MCF-7 cells show increasing doubling time and decreasing polyglutamate synthesis in response to increasing initial cell number.

Breast Neoplasms↗