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Biomedical subjects

A K Smith

Publications and source records attributed to A K Smith.

At least 55 records · Page 3Linked to original sources
PubMed1993

5,10-Dideazatetrahydrofolic acid (DDATHF) transport in CCRF-CEM and MA104 cell lines.

5,10-Dideazatetrahydrolic acid (DDATHF) is representative of a new class of antifolates acting through inhibition of de novo purine synthesis. We report here the transport characteristics of the diastereomers of DDATHF, which differ in configuration at C6, and comparison studies with other folate and antifolate analogs. (6R)-DDATHF showed high affinity for the influx system of CCRF-CEM cells with a Km of 1.07 microM and an influx Vmax of 4.04 pmol/min/10(7) cells. Comparative studies with methotrexate yielded an influx Km of 4.98 microM and a Vmax of 6.64 pmol/min/10(7) cells, and with 5-formyltetrahydrofolate an influx Km of 2.18 microM and a Vmax of 6.84 pmol/min/10(7) cells. Uptake of (6R)-DDATHF was competitively inhibited by (6S)-DDATHF, methotrexate (MTX), and 5-formyltetrahydrofolate, all with Ki values similar to their influx Km. The (6S)-DDATHF diastereomer had an influx Km of 1.04 microM, similar to that of (6R)-DDATHF; however, the Vmax of 1.72 pmol/min/10(7) cells was 2.3-fold lower than for (6R)-DDATHF. The transport properties of DDATHF were also studied in a mutant cell line (CEM/MTX), resistant to MTX based on impaired drug transport. In this system (6R)-DDATHF showed an influx Km of 1.49 microM and a decreased influx Vmax of 0.60 pmol/min/10(7) cells. A similar effect was shown for MTX (Km of 7.48 microM, Vmax of 1.02 pmol/min/10(7) cells). The number of binding sites in CCRF-CEM cells was similar for (6R)-DDATHF, (6S)-DDATHF, and MTX, 0.74, 0.71, and 0.76 pmol/10(7) cells, respectively. These values were slightly higher in the CEM/MTX cell line (1.07 and 1.09 pmol/10(7) cells for (6R)-DDATHF and MTX, respectively). Treatment of CCRF-CEM cells with either the N-hydroxysuccinimide ester of MTX or the corresponding N-hydroxysuccinimide ester of (6R)-DDATHF caused substantial inhibition (> 90%) of the influx of (6R)-[3H]DDATHF and [3H]MTX, respectively. These results suggest strongly that DDATHF and MTX share a common influx mechanism through the reduced folate transport system. The internalization of DDATHF by monkey kidney epithelial MA104 cells, which express a high affinity folate receptor, was also studied. Competitive binding studies using purified folate receptor and radiolabeled 5-methyltetrahydrofolate showed that (6S)- and (6R)-DDATHF both had I50 values lower than 5-methyltetrahydrofolate (12 nM). Further studies indicate that both DDATHF isomers are actively intracellularly concentrated through this route and are also rapidly converted to high chain length polyglutamates. Transport via this system was inhibited in folate-depleted cells by 10 nM folic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Transport↗
PubMed1993

Accumulation of 5-methyltetrahydrofolic acid and folylpolyglutamate synthetase expression by mitogen stimulated human lymphocytes.

The accumulation of 5-methyl[3H]tetrahydrofolic acid (5CH3[3H]FH4) by phytohaemagglutinin stimulated lymphocytes (PHA-L) cultured in folate free media was investigated to determine the mechanism of uptake of 5CH3FH4 and the requirement of the cells for this vitamin as assessed by monitoring de novo thymidine synthesis. When grown in 20 nM 5CH3[3H]FH4 PHA-L accumulate radiolabel at a rate of 0.04 pmol/h/10(6) cells. This doubles the endogenous folate pool of unstimulated cells (0.6 +/- 0.16 pmol/10(6) cells) in about 15 h. Uptake proceeded via a saturable process, independent of a high affinity folate receptor as assessed by ligand binding and by Northern and Western blot analysis. However, transport was blocked by probenecid, which is consistent with an anion carrier mechanism. Unstimulated cells lacked folypolyglutamate synthetase (FPGS) activity and did not express significant amounts of FPGS mRNA. After 48 h of mitogen stimulation there was a 4-10-fold increase in FPGS mRNA and folypolyglutamate formation (Glu > or = 5) was essentially simultaneous with 5CH3[3H]FH4 transport. Increasing extracellular folate to 2 microM only increased intracellular folate 8-fold, but the length of the folylpolyglutamates decreased. The increased folate did not increase de novo thymidine synthesis compared to cells grown in physiological folate. We conclude that mitogen stimulation activates the process(es) for folate accumulation, especially FPGS, and that physiological uptake (0.04 pmol/h/10(6) cells) is adequate for meeting the cells' need for the vitamin.

Cells, Cultured↗
PubMed1991

The folate receptor works in tandem with a probenecid-sensitive carrier in MA104 cells in vitro.

Previous studies have defined a novel route of internalization for the essential vitamin 5-methyltetrahydrofolate in MA104 cells that begins with binding of the vitamin to the membrane receptor for folate. One of the critical steps in the pathway is the passage of 5-methyltetrahydrofolate through the membrane into the cytoplasm. Utilizing both probenecid and low temperature as selective inhibitors, we have successfully blocked transmembrane movement of the vitamin into the cytoplasm without affecting binding to the receptor or the internalization of the vitamin-receptor complex, which suggests that passage is through an anion carrier. This anion carrier, which mediates inward movement of 5-methyltetrahydrofolate after it dissociates from the receptor, also appears to mediate the efflux of folylmonoglutamate, but not folylpolyglutamate, when the concentration of the former in the cytoplasm is sufficiently high. Since we also found that the synthesis of folylpolyglutamates is regulated in these cells, most likely the intracellular concentration of the vitamin is controlled by regulating the flux of folylmonoglutamate through this carrier.

Animals↗
PubMed1988

Effect of dietary supplementation with n-3 fatty acids on clinical manifestations of psoriasis.

In a double-blind, block randomized study we investigated the effect of dietary supplementation with eicosapentaenoic acid in patients with psoriasis. The experimental group received 10 g of fish oil daily containing approximately 1.8 g eicosapentaenoic acid, while the controls were given an isoenergetic amount of olive oil. We found no significant change in the clinical manifestations of psoriasis in either group after 8 weeks of treatment. In the experimental group, the amount of n-3 fatty acids in serum phospholipids was significantly increased at the end of trial as compared to pre-treatment values, whereas the level of n-6 fatty acids was decreased.

Adolescent↗
PubMed1982

A novel iron protein from Desulfovibrio gigas.

The isolation, purification, and partial characterization of a novel iron-containing protein from the sulfate-reducing anaerobic bacterium, Desulfovibrio gigas, is described. The highly insoluble protein was isolated from the cell debris following osmotic shock of the bacteria. The insoluble fraction consistently contained about 90% of the cell-associated iron. Elemental analysis of a crude protein preparation gave 5.3% iron, 2.9% sulfur and 11.9% nitrogen. An independent colorimetric iron analysis showed 6.4% iron. The iron could be dissociated from the protein by treatment with 5% SDS. The iron-free protein was purified by a combination of organic extraction and DEAE-cellulose chromatography. The purified protein showed only one major band, Mr 14000, by SDS-polyacrylamide gel electrophoresis. The protein could be reconstituted upon treatment with an appropriate mixture of FeS and beta-mercaptoethanol. The reconstituted protein had the same physical and chemical properties as the native protein. The amino acid composition was not unusual except for the high isoleucine content.

Amino Acids↗
PubMed1974

Heat urticaria.

Explore the source record for details and available documents.

Adult↗
PubMedDate not supplied

Characterization of binding of simian rotavirus SA-11 to cultured epithelial cells.

Rotavirus causes enteritis in both man and animals. The identity of the rotavirus receptor is not known. The nature of the binding interaction and the relationship between virus binding and internalization have not previously been reported. We studied the binding of [5,6-3H]uridine-labeled rotavirus SA11 to confluent monolayers of MA104 cells. We found approximately 13,000 receptor units per cell. The binding was sodium-dependent, pH-insensitive between 5.5 and 8, independent of added calcium, and dependent on sialic acid residues in the membrane. It could be inhibited by mucin. These features may provide clues to the identity of the receptor. Virus was not internalized to significant extent at 4 degrees C. After warming to 37 degrees C, virus was internalized over about 60 min. All binding sites appeared to be equally internalizable. The techniques developed to distinguish between binding and internalization may help to elucidate the mechanism of internalization.

Animals↗
PubMedDate not supplied

Delayed methotrexate clearance in a patient with sickle cell anemia and osteosarcoma.

A 15-year-old girl with homozygous sickle cell anemia (HbSS) and osteosarcoma is described. Delayed clearance of methotrexate (MTX) after the second course of high-dose MTX (HDMTX) led to the development of renal and hepatic toxicities. Rescue was accomplished with high-dose leucovorin, intravenous carboxypeptidase G2, and thymidine. Although the renal and hepatic abnormalities resolved, focal tonic-clonic seizures developed, accompanied by abnormal brain imaging. Four weeks after this episode, all clinical and biochemical abnormalities resolved. Preexistent end-organ damage associated with HbSS may compromise the ability to deliver high-dose chemotherapy with curative intent in patients with malignant disease.

Adolescent↗
PubMedDate not supplied

Ratio of methotrexate to folate uptake by lymphoblasts in children with B-lineage acute lymphoblastic leukemia: a pilot study.

PURPOSE: Methotrexate (MTX) remains one of the most effective drugs for the treatment of children with acute lymphoblastic leukemia (ALL). Because MTX and 5-methyltetrahydrofolate (5CH3THF) share uptake and metabolic pathways, the efficacy of MTX is likely to depend not only on its metabolism but also on how well folate is accumulated by lymphoblasts. The authors' goal was to compare in vitro folate and antifolate uptake in B-lineage lymphoblasts from patients who remained in continuous complete remission (CCR) and those in whom relapse occurred. PATIENTS AND METHODS: Twenty-four children with B-lineage ALL were studied at diagnosis (n = 20) or relapse (n = 4). Lymphoblasts obtained by bone marrow aspiration were incubated for 24 hours in vitro with 0.05 microM 5CH3[3H]THF or 1 microM [3H]MTX. RESULTS: As of July 1999, 16 patients studied at diagnosis remained in CCR at a median follow-up of 45 months after achieving remission. Two of the patients studied at relapse are in second CCR; the remaining two died from progressive disease. The median uptake of neither [3H]MTX nor 5CH3[3H]THF differed significantly between the 16 patients in first CCR studied at diagnosis and the 4 patients studied at relapse. However, the median ratio of [3H]MTX:5CH3[3H]THF uptake differed significantly for patients who remained in first CCR versus patients studied at relapse. CONCLUSIONS: The uptake of [3H]MTX in relation to 5CH3[3H]THF by leukemic lymphoblasts in vitro may correlate positively with treatment outcome in children with B-lineage ALL. A larger study of homogeneously treated patients is necessary to confirm these results.

Adolescent↗