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Biomedical subjects

F Ahmed

Publications and source records attributed to F Ahmed.

At least 145 records · Page 8Linked to original sources
PubMed1986

Evidence for the existence of covalent nucleotide-thymidylate synthase complexes, identification of site of attachment, and enhancement by folates.

The formation of covalent binary complexes of thymidylate synthase and its nucleotide substrate dUMP, product dTMP, and inhibitor, 5-fluorodeoxyuridylate (FdUMP) was investigated using the trichloroacetic acid precipitation method. It was observed that, in addition to FdUMP, both dUMP and dTMP were capable of covalent interactions with the enzyme in the absence of added folates. The presence of folate, dihydrofolate, or tetrahydrofolate (H4folate) was found to produce substantial enhancements in the covalent binding of both FdUMP and dUMP to the enzyme with H4folate being the most effective agent. Further, covalent binary complexes of the enzyme with the three radiolabeled nucleotides were isolated by trichloroacetic acid precipitation and subjected to CNBr cleavage. The active-site CNBr peptide was isolated by reverse phase high performance liquid chromatography, and the first five N-terminal amino acid residues were sequenced by the dansyl-Edman procedure. Each active site peptide obtained from the covalent binary complexes as well as that from the covalent inhibitory ternary complex formed from enzyme, FdUMP, and 5,10-methylene-H4folate exhibited an identical sequence of Ala-Leu-Pro-Pro-(X)-, and the 5th amino acid was found to be associated with radiolabeled nucleotide ligand. Dansyl-Edman sequence analysis of the active site CNBr peptide, derived from enzyme which had been treated with iodoacetic acid, gave a sequence of Ala-Leu-Pro-Pro-CmCys (where CmCys is carboxymethylcysteine), thus confirming the fact that the fifth residue from the N terminus is Cys-198. In all the cases, the active site Cys-198 residue was found to be covalently linked to the nucleotides. These results provide unequivocal proof that the covalent binary complexes of enzyme with dUMP and dTMP predicted in the catalytic reaction mechanism actually exist.

Deoxyuracil Nucleotides↗
PubMed1986

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthase.

The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Radioisotopes↗
PubMed1985

A nitrocellulose-filter assay for the binary complex of 5-fluorodeoxyuridylate and Lactobacillus casei thymidylate synthetase.

The interaction of 5-fluorodeoxyuridylate (FdUMP) with thymidylate synthetase to form a binary complex has been widely reported, yet previous attempts to detect this complex by nitrocellulose filtration have failed. In contrast, a nitrocellulose-filter-binding assay utilizing [6-3H]FdUMP which measures the interaction of the enzyme with the nucleotide is reported. Extensive washing of the nitrocellulose-filtered complex between FdUMP and the enzyme resulted in no loss of bound ligand. Following denaturation with trichloroacetic acid, intact complex was detected by nitrocellulose filtration. No binding was observed between 5-fluorodeoxyuridine and the enzyme or between FdUMP and the N-ethylmaleimide-modified enzyme. As measured by the nitrocellulose filtration method, at least a 600-fold excess of FdUMP to enzyme was required to achieve saturation. The stoichiometry of FdUMP bound to the enzyme detected at saturation was 0.5-0.6 for native samples. When identical samples were subjected to denaturation prior to filtration, the stoichiometry of nucleotide binding was 0.3-0.4.

Chemical Phenomena↗
PubMed1985

Some effects of nifedipine in guinea-pig isolated trachealis.

In trachealis depolarized by a K+-rich medium, nifedipine (0.001-1 mumol 1(-1) caused concentration-dependent antagonism of CaCl2-induced increase in tension, moving the CaCl2 log concentration-effect curve to the right and depressing the maximal response. In trachealis in normal Krebs solution, similar concentrations of nifedipine had marked antispasmogenic activity against the responses to potassium chloride (KCl) and tetraethylammonium (TEA). However, nifedipine had little, if any, antispasmogenic activity against the responses to acetylcholine or histamine. Nifedipine 1 mumol 1(-1) was tested for spasmolytic activity in tissues generating tension in response to the EC50 of acetylcholine, KCl or CaCl2. In producing spasmolysis nifedipine was most effective against CaCl2 and least effective against acetylcholine. Nifedipine (0.01-1 mumol-1) had little or no effect on the tone of trachealis in normal Krebs solution. Intracellular electrophysiological recording showed that nifedipine 1 mumol 1(-1) could abolish spontaneous slow wave activity. This was associated with very minor depolarization and little or no loss of mechanical tone. In tissues treated with TEA (8 mmol 1(-1) nifedipine abolished spike and slow wave discharge and reduced mechanical activity to the pre-TEA level. It is concluded that nifedipine prevents KCl- or TEA-induced spasm by inhibition of Ca2+ influx. Spasm evoked by acetylcholine or histamine and the maintenance of spontaneous tone depend largely on mechanisms for increasing the cytoplasmic concentration of free Ca2+ which are resistant to nifedipine.

Animals↗
PubMed1984

Isolation of the covalent binary complex of 5-fluorodeoxyuridylate and thymidylate synthetase by trichloroacetic acid precipitation.

Strong chemical evidence for the existence of a covalent binary complex between 5-fluorodeoxyuridylate and thymidylate synthetase was provided by the isolation of the complex by trichloroacetic acid precipitation. This result together with that of a control experiment with N-ethymaleimide inactivated thymidylate synthetase demonstrated that only nucleotide covalently bound to the protein survived repeated washings of the precipitate. Under the conditions used, a maximum binding stoichiometry of about 0.9 was obtained for the covalent binary complex, Kd = 1.1 X 10(-5) M. Also, a binding ratio of 1.7 was obtained for the methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase ternary complex.

Deoxyuracil Nucleotides↗
PubMed1984

Preparation of (-)-5,6,7,8-tetrahydrofolate using immobilized dihydrofolate reductase.

Dihydrofolate reductase from methotrexate-resistant Lactobacillus casei was immobilized on carbodiimide-activated CH-Sepharose. The immobilized enzyme was utilized in the synthesis of (-)-5,6,7,8-tetrahydrofolate from dihydrofolate and NADPH in a batchwise reaction system. The products of the reaction, (-)-tetrahydrofolate and NADP+, were separated on a Sephadex G-10 column equilibrated with 50 mM NH4HCO3 containing beta-mercaptoethanol and ethanol. The tetrahydrofolate was then characterized by ultraviolet and circular dichroic spectra and its reactivity as a cofactor in the thymidylate synthetase reaction.

Bacterial Proteins↗
PubMed1984

Some features of the spasmogenic actions of acetylcholine and histamine in guinea-pig isolated trachealis.

Intracellular electrophysiological recording showed that acetylcholine (1 mumol l-1) and histamine (2 mumol l-1) depolarized trachealis cells and often increased the frequency of slow waves. Higher concentrations of these agents caused greater depolarization and abolition of slow waves. Marked depolarization was often associated with the appearance of electrical 'noise'. These electrical phenomena were accompanied by tonic tension development in a contiguous segment of trachea. Electrical 'noise' and tension evoked by high concentrations of acetylcholine or histamine could be dissipated by washing the agonist from the tissue. Acetylcholine-induced 'noise' was resistant to tetrodotoxin (3 mumol l-1) and to hexamethonium (1 mmol l-1). Neither acetylcholine (10-1,000 mumol l-1) nor histamine (2-200 mumol l-1) increased the lanthanum-resistant calcium fraction of muscle-containing strips of trachea. It is concluded that, while developing tension under the influence of acetylcholine or histamine, trachealis cells depolarize markedly but there is relatively little cellular influx of Ca2+.

Acetylcholine↗
PubMed1983

Circulating granulocyte and macrophage progenitor cells in primary and secondary myelofibrosis.

Patients with primary myelofibrosis (PMF) and myelofibrosis secondary to carcinoma (SMF) were compared with regard to circulating granulocyte macrophage progenitor cells (CFU-GM) using in vitro tissue culture techniques. Although increased numbers of CFU-GM had previously been well documented in PMF, few patients with the secondary variety had been studied. Our data indicate that there is an increase in circulating CFU-GM in patients with SMF but it is significantly lower than in those with PMF. It is suggested that in both conditions disruption of the marrow microvascular system results in a release of CFU-GM to the circulation. In PMF stem cell colonization of the spleen with its consequent myeloid metaplasia may be responsible for the additional increase in CFU-GM. The determination of CFU-GM numbers may provide additional data to help to distinguish PMF and SMF in atypical cases where the distinction is unclear.

Adult↗
PubMed1982

Acquired Gaucher's cells in Hodgkin's disease.

A patient with Hodgkin's disease associated with low glucocerebrosidase levels in the peripheral leukocytes, and Gaucher's cells in the bone marrow and lymph nodes, is described. After MOPP therapy, complete remission of Hodgkin's disease was accompanied by normalization of the glucocerebrosidase level and disappearance of Gaucher's cells. This observation appears unique when compared with the four cases of combined Hodgkin's and Gaucher's disease reported in earlier literature in which Gaucher's disease remained unchanged after chemotherapy. We conclude that our patient had Hodgkin's disease and acquired Gaucher's cells with diminished glucocerebrosidase levels, rather than a combination of Hodgkin's disease and Gaucher's disease.

Adult↗
PubMed1979

Study of the effects of a long acting injectable-progesterone on the ovarian and uterine histomorphology of the androgenised female rats of Long-Evans strain.

The study was aimed at determining the possible role of long acting progesterone-DMPA in effecting reversal of the effect of TP on neonatally treated female Long-Evans rats. Five day-old female litters injected with 1.25 mg. TP went into persistent estrous on attaining maturity. DMPA when given in proper dosage and time reverted the condition.

Animals↗