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W M Watkins

Publications and source records attributed to W M Watkins.

At least 55 records · Page 3Linked to original sources
PubMed1995

Native and asialo-Tamm-Horsfall glycoproteins as important ligands for the detection of GalNAc beta 1-->and Gal beta 1-->4GlcNAc active lectins.

The binding properties of human Tamm-Horsfall Sd(a+) urinary glycoprotein(THGP) and asialo-THGP with various applied lectins was investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins completely precipitated Abrus precatorius agglutinin(APA). They also reacted well with Wistaria floribunda (WFA), Glycine max (soybean, SBA), and Ricinus communis agglutinins and precipitated over 78% of the lectin nitrogen added, but reacted poorly or weakly with all alpha-anomeric GalNAc specific lectins, such as Helix pomatia (HPA), Phaseolus lunatus (lima bean, LBL), and Maclura pomifera (MPL) lectins. The glycoprotein-lectin interaction was inhibited by GalNAc beta 1-->, Gal beta 1-->4GlcNAc, or by both. The findings suggest that Sd (a+) THGP and asialo-THGP are among the best water-soluble glycoprotein ligands for GalNAc beta 1-->and Gal beta 1-->4GlcNAc active lectins.

Asialoglycoproteins↗
PubMed1995

Purification, properties and possible gene assignment of an alpha 1,3-fucosyltransferase expressed in human liver.

alpha 1,3-Fucosyltransferase solubilized from human liver has been purified 40,000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with alpha 1,3-fucosyltransferase activity and had a specific activity of approximately 5-6 mumol min-1 mg-1 and an M(r) approximately 44,000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal beta 1-4GlcNAc, NeuAc alpha 2-3Gal beta 1-4GlcNAc and Fuc alpha 1-2Gal beta 1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc alpha 1-2Gal beta 1-4Glc and the Type 1 compound Gal beta 1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc alpha 2-6Gal beta 1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated alpha 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with alpha 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with the Fuc-TVI cDNA, suggests a provisional identification of Fuc-TVI as the major alpha 1,3-fucosyltransferase gene expressed in human liver.

Ammonium Sulfate↗
PubMed1995

Halofantrine pharmacokinetics in Kenyan children with non-severe and severe malaria.

1. Kenyan children with uncomplicated malaria given oral halofantrine (HF; non-micronised suspension; 8 mg base kg-1 body weight 6 hourly for three doses) showed wide variation in the disposition of HF and desbutylhalofantrine (HFm). 2. Eight Kenyan children with severe (prostrate) falciparum malaria who were receiving intravenous quinine, were given the same HF regimen by nasogastric tube. One patient had undetectable HF and two had undetectable HFm at all times after drug administration. 3. The mean AUC(0,24 h) of HF in prostrate children was half (7.54 compared with 13.10 micrograms ml-1 h) (P = 0.06), and that for HFm one-third (0.84 compared with 2.51 micrograms ml-1 h) (P < 0.05) of the value in children with uncomplicated malaria. 4. Oral HF may be appropriate for some cases of uncomplicated falciparum malaria in Africa, but in patients with severe malaria, the bioavailability of HF and HFm may be inadequate.

Administration, Oral↗
PubMed1995

Potent antimalarial activity of the alkaloid nitidine, isolated from a Kenyan herbal remedy.

Bioassay-guided fractionation of extracts of Toddalia asiatica, a plant used by the Pokot tribe of Kenya to treat fevers, has yielded the alkaloid nitidine as the major antimalarial component. Fractions containing nitidine have in vitro 50% inhibitory concentrations against Plasmodium falciparum in the range of 9 to 108 ng/ml for a range of chloroquine-susceptible and -resistant strains. The results show a lack of cross-resistance between chloroquine and nitidine.

Animals↗
PubMed1995

In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs.

The potency of antimalarial dihydrofolate reductase inhibitors, alone and in synergistic combination with dihydropteroate synthetase inhibitors, against the Kenyan K39 strain of Plasmodium falciparum (pyrimethamine resistant) and against normal replicating human bone marrow cells in in vitro culture has been studied. Therapeutic indices and rank order of synergistic potency were derived. Trimethoprim, pyrimethamine, and the quinazolines WR159412 and WR158122 had the smallest therapeutic indices (1.39, 4.38, 2.56, and 90.0, respectively), while the three triazines clociguanil, WR99210, and chlorcycloguanil had the largest (3,562, 3,000, and 2,000, respectively). In rank order of decreasing activity against P. falciparum, the six most potent drug combinations were WR99210-dapsone, chlorcycloguanil-dapsone, WR158122-dapsone, WR159412-dapsone, WR159412-sulfamethoxazole, and chlorcycloguanil-sulfamethoxazole; pyrimethamine-sulfadoxine was the least potent combination. These experiments form a basis for the selection of rapidly eliminated antifolate combinations for further clinical testing.

Animals↗
PubMed1995

Parasite viability during treatment of severe falciparum malaria: differential effects of artemether and quinine.

The effect of artemether (AR) and quinine (QN) on parasite viability ex vivo was compared in children being treated for severe Plasmodium falciparum malaria. Parasitized blood taken at intervals during treatment was cultured in vitro, and parasite development was assessed microscopically. Parasite viability (defined as the proportion of circulating rings developing to early schizonts) was 56.8% in the AR group (n = 7) 6 hr after the start of treatment, compared with 93.3% for QN (n = 6; P = 0.015). Even after 24 hr of QN treatment, parasite viability was not significantly reduced in five patients. These ex vivo findings, which confirm previous observations of the stage-specific effects of these drugs against P. falciparum, suggest that AR may be superior to QN in the treatment of severe malaria.

Animals↗
PubMed1995

Pharmacology and pharmacokinetics of new antimalarials.

Chloroquine-resistant Plasmodium falciparum is now widespread in Africa, requiring new drugs for the control of both non-severe and severe forms of the disease. For non-severe malaria, pyrimethamine-sulphadoxine, an antifolate combination antimalarial, is at present efficacious, single-dose and cheap; important characteristics for treatments in Africa. However, alternative combinations are being investigated which are intrinsically more active, less toxic and with shorter elimination half-lives. In theory, short half-life compounds reduce the selective pressure for resistance, which may be a major determinant of the useful therapeutic life of an antimalarial drug. The potential advantages/disadvantages of alternative antifolates is discussed. While the use of mefloquine and halofantrine in Africa is at present limited by cost, these drugs are likely replacements for the antifolates when parasite resistance arises. For severe, life-threatening falciparum malaria, quinine remains the treatment of choice. In contrast to quinine, artemether rapidly reduces the viability of circulating, ring-stage parasites, produces more rapid parasite clearance and may reduce the length of coma, but does not significantly reduce the mortality of severe malaria which remains at about 15% even with optimal management. It seems unlikely that chemotherapy, even with "new" antimalarials, will reduce this high figure. Other strategies are required.

Africa↗
PubMed1994

Differential extraction of artemether and its metabolite dihydroartemisinin from plasma and determination by high-performance liquid chromatography.

A method is described for the separation of artemether (ARM) from its metabolite dihydroartemisinin (DHA) and determination by HPLC. The basis of the separation is differential extraction of the drugs from plasma as a function of plasma pH. Hexane extracted ARM from basiffied plasma and both ARM and DHA from normal plasma. Derivatized extracts were chromatographed on a 5-microns ODS column with water-acetonitrile (40:60) as mobile phase and detected at 254 nm. The method removes the need for expensive absorption cartridges (BondElut). Chromatography has been improved and the elution time shortened in comparison with previous methods.

Antimalarials↗
PubMed1994

Sequence variation of the hydroxymethyldihydropterin pyrophosphokinase: dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine.

Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur-based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine-resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (CH2OH-H2pterinPP kinase) at its 5' proximal end. This domain is unusually large, with two long insertions relative to other CH2OH-H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high-level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide-resistant pathogenic bacteria. Copy-number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.

Amino Acid Sequence↗
PubMed1994

Ferriprotoporphyrin catalysed decomposition of artemether: analytical and pharmacological implications.

The ability of the products of erythrocytic haemolysis and ferriprotoporphyrin (FP-IX)-containing substances of facilitate the decomposition of the antimalarial drug artemether has been evaluated in vitro. The products of haemolysis accelerate the degradation of artemether to unidentified compounds which are undetectable by currently available HPLC methods. This decomposition is temperature dependent and occurs after relatively brief artemether (ARM)-catalyst contact. Using radiolabelled [16-14C]ARM, we have demonstrated that the extractability of total radioactivity into the organic phase diminishes with increasing FP-IX concentration with an appreciable reduction in the organic extractability as ARM in the presence of FP-IX and associated increase in water solubility of radioactivity when the concentration of FP-IX increases from 0 to 7.7 mM. This effect appears to be temperature dependent for incubations with haematin. Characterisation of the organically extractable radioactivity from incubations of [16-14C]ARM with FP-IX has shown a loss of ARM and the formation of two radioactive products which occurs in proportion to the concentration of FP-IX. We believe these findings are of particular relevance to the determination of plasma concentrations of ARM and dihydroartemisinin (DHA) in malaria patients where extensive haemolysis and the presence of breakdown products of haemoglobin may contribute to a reduction in the circulating concentration of these substances. In these circumstances, measurement of ARM and DHA by conventional methods may be meaningless.

Antimalarials↗
PubMed1993

Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder arising in a multipotent hemopoietic stem cell. PNH manifests clinically with intravascular hemolysis resulting from an increased sensitivity of the red cells belonging to the PNH clone to complement-mediated lysis. Numerous studies have shown that surface proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor (including proteins protecting the cell from complement) are deficient on the cells of the PNH clone, leading to the notion that GPI-anchor biosynthesis may be abnormal in these cells. To investigate the biochemical defect underlying PNH we have used lymphoblastoid cell lines (LCLs) with the PNH phenotype obtained by Epstein-Barr virus immortalization of lymphocytes from nine patients with PNH. By labeling cells with myo-[3H]inositol we have found that PNH LCLs produce phosphatidylinositol normally. By contrast, PNH LCLs fail to incorporate [3H]mannose into GPI anchor precursors. When cell-free extracts of PNH LCLs and normal LCLs obtained from the same patients (and expected therefore to be isogeneic except for the PNH mutation) were incubated with uridine diphospho-N-acetyl[3H]glucosamine (UDP-[3H]GlcNAc), we observed complete failure or marked reduction in the production of N-acetylglucosaminyl(alpha-1,6)phosphatidylinositol and glucosaminyl(alpha-1,6)phosphatidylinositol by the PNH LCLs in all cases. These findings pinpoint the block in PNH at an early stage in the biosynthesis of the GPI anchor, suggesting that the defective enzyme is UDP-GlcNAc:phosphatidylinositol-alpha-1,6-N- acetylglucosaminyltransferase. The existence of PNH type III cells and type II cells is probably explained by the transferase deficiency being total or partial, respectively.

Acetylglucosamine↗
PubMed1993

Purification and properties of the alpha-3/4-L-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line.

A soluble alpha-3/4-fucosyltransferase secreted into the growth medium of the human A431 epidermoid carcinoma cell line has been purified 700,000 fold by a series of steps involving chromatography on Phenyl Sepharose 4B, CM-Sephadex C-50 and GDP-hexanolamine Sepharose 4B. The untreated spent culture medium transferred almost ten times more fucose to the subterminal N-acetylglucosamine residue in the Type 1 (Gal beta 1-3GlcNAc) disaccharide than to the subterminal sugar in the Type 2 (Gal beta 1-4GlcNAc) disaccharide; the relative activity with these two substrates remained virtually unchanged throughout the purification procedure. At no stage was any alpha-3-fucosyltransferase species acting solely on N-acetylglucosamine residues in Type 2 chains separated from the bulk of the alpha-3/4-fucosyltransferase activity. The purified enzyme preparation showed insignificant activity with glycoprotein substrates having N-linked oligosaccharide chains with terminal Type 2 sequences but transferred fucose to a mucin-type glycoprotein with O-linked oligosaccharide chains with terminal Type 1 structures. Lactose was a poor substrate but the activity of the enzyme was influenced by the presence of substituents on the terminal beta-galactosyl residue and 2'-fucosyllactose was almost as good an acceptor as the Type 1 disaccharide. The properties of the purified enzyme with regard to specificity, divalent cation requirements, pH optimum, and M(r), closely resembled those of the Lewis-blood-group gene associated alpha-3/4-fucosyltransferase isolated from human milk.

Carbohydrate Metabolism↗
PubMed1993

Possible genetical pathways for the biosynthesis of blood group mucopolysaccharides. Vox Sang 1959:4:97-119.

This paper put forward possible biosynthetic pathways for the formation of the blood group A, B, H and Lewis antigens based on the limited knowledge of their chemistry and genetics that was available in 1959. The schemes proposed that genes at four independent loci ABO, HH, Lele and Sese interacted to give the five specificities A, B, H, Lea and Leb found in secretions and that the primary products of the blood group genes were not the antigens but enzymes that catalysed the sequential addition of single sugars to complete the determinants.

ABO Blood-Group System↗
PubMed1993

Falciparum malaria: differential effects of antimalarial drugs on ex vivo parasite viability during the critical early phase of therapy.

A method of monitoring Plasmodium falciparum viability ex vivo was used to compare the ability of different antimalarial drugs to arrest the progression of young parasites to mature, potentially damaging stages. Neither pyrimethamine-sulfadoxine nor quinine, the treatment of choice for severe, life-threatening malaria, had a demonstrable effect on circulating parasites during the first 24 hr of therapy. In contrast, in vivo exposure to halofantrine for as little as six hours was sufficient to arrest parasite development. The method of assessing ex vivo parasite viability permits a comparison of antimalarial drug action at a time that may be critical for the therapy of life-threatening disease. If parenteral formulations of halofantrine prove to be safe and effective, they may have a role in the therapy of severe malaria.

Animals↗
PubMed1992

Measurement of halofantrine and its major metabolite desbutylhalofantrine in plasma and blood by high-performance liquid chromatography: a new methodology.

A new high-performance liquid chromatographic assay for the measurement of halofantrine and desbutylhalofantrine in plasma and whole blood is described. The method involves a smaller sample volume, simplified sample pre-treatment and a shorter run-time, and is adaptable to the measurement of samples dried onto filter paper strips. Using this method, which is both selective and sensitive, plasma concentration versus time profiles for both substances have been investigated following a single oral dose (500 mg) of halofantrine hydrochloride to a healthy adult volunteer. In addition, a clinical study designed to evaluate the disposition and elimination of the two compounds in children with non-severe falciparum malaria is in progress.

Antimalarials↗
PubMed1992

Purification of the Lewis blood-group gene associated alpha-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to type 1 and lactose-based oligosaccharide chains.

A soluble Lewis blood-group gene associated alpha-3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated alpha-3-L-fucosyltransferase activity directed towards N-acetylglucosamine in Type 2 (Gal beta 1-4GlcNAc-R) acceptors from an alpha-3/4-fucosyltransferase fraction acting on both Type 1 (Gal beta 1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose to N-acetylglucosamine in both Type 1 and Type 2 acceptors and to the O-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described alpha-3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of alpha-3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual alpha-3/4-fucosyltransferase that retained strong alpha-4 activity with the Type 1 acceptor, lacto-N-biose 1, and alpha-3 activity with 2'-fucosyllactose, but had relatively little alpha-3 activity with N-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins with N-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.

Chromatography, Affinity↗