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M A Phillips

Publications and source records attributed to M A Phillips.

At least 73 records · Page 4Linked to original sources
PubMed1988

Trypanosoma brucei ornithine decarboxylase: enzyme purification, characterization, and expression in Escherichia coli.

Ornithine decarboxylase from the African trypanosome is an important target for antitrypanosomal chemotherapy. Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein. In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite. T. brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000. The isoelectric point of the protein was 5.0. The Km for ornithine was 280 microM and the Ki for the irreversible inhibitor alpha-difluoromethylornithine (DFMO) was 220 microM with a half-time of inactivation at saturating DFMO concentration of 2.7 min. T. brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins. Although a small quantity of T. brucei ornithine decarboxylase was purified from T. brucei, extensive structural and kinetic studies will require a more ample source of the enzyme. We therefore expressed our previously cloned T. brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible lambda promoter. T. brucei ornithine decarboxylase activity was induced in E. coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T. brucei. Ornithine decarboxylase activity in the crude E. coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein. The recombinant T. brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E. coli. The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme.

Animals↗
PubMed1988

Speech recognition by a deaf-blind multichannel cochlear implant patient.

A case study of the first deaf-blind patient implanted with a Nucleus 22 channel cochlear prosthesis is presented. The patient's preimplant evaluation data, using a powerful hearing aid and vibro-tactile device, are compared with results obtained 8 months postimplant. Preimplant testing indicated no consistent response to sound. Postimplant testing showed ability to comprehend some open set speech with hearing alone through the cochlear implant including ability to utilize the telephone without a code system. The postimplant data suggest potential benefit for other postlingually deaf-blind individuals from multichannel cochlear stimulation.

Blindness↗
PubMed1987

Cloning and sequencing of the ornithine decarboxylase gene from Trypanosoma brucei. Implications for enzyme turnover and selective difluoromethylornithine inhibition.

Ornithine decarboxylase of the African trypanosome Trypanosoma brucei brucei had an estimated native molecular weight of 100,000 by gel filtration and a subunit molecular weight of 45,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding this enzyme, present in a single copy in T. brucei, was identified by mouse ornithine decarboxylase cDNA under relatively stringent conditions of hybridization and subcloned in a 5.9-kilobase (kb) SstI fragment from a cosmid clone into the plasmid pUC 19. This clone encompassed a 2.8-kb SstII fragment that contained the entire T. brucei ornithine decarboxylase gene. The 2.8-kb SstII fragment hybridized to a 2.4-kb mRNA that presumably encodes the parasite enzyme. The 2.8-kb SstII fragment was partially sequenced and found to contain an open reading frame of 445 amino acids that has 61.5% homology with the corresponding sequence of the mouse enzyme. The only major discrepancies between the two enzymes are the addition of a 20-amino acid N-terminal peptide and the deletion of a 36-amino acid C-terminal peptide and the T. brucei ornithine decarboxylase. The C terminus has been postulated to be one of the structural factors associated with rapid in vivo turnover of mammalian ornithine decarboxylase. The absence of this C-terminal peptide in T. brucei ornithine decarboxylase predicts a slow turnover for the parasite enzyme in vivo, and this is supported by our experimental data. The lack of turnover of ornithine decarboxylase in trypanosomes may constitute the basis of selective antitrypanosomal action of the irreversible enzyme inhibitor DL-alpha-difluoromethylornithine.

Amino Acid Sequence↗
PubMed1987

A Trypanosoma brucei mutant resistant to alpha-difluoromethylornithine.

Procyclic Trypanosoma brucei brucei strain 366D is susceptible to DL-alpha-difluoromethylornithine (DFMO) with an in vitro ED50 value of 225 microM. A mutant of the procyclic strain resistant to 20 mM of DFMO was isolated by serial in vitro passages of the organisms in increasing concentrations of the drug. Drug resistance remains unchanged after at least ten serial passages in the absence of DFMO. The mutant contains the same level of ornithine decarboxylase activity as the wild-type procyclic, and the mutant enzyme exhibits a similar susceptibility toward DFMO as the wild type. Neither the rate of decarboxylation of ornithine, nor the membrane potential in the mutant cell is changed. The only observed change in the mutant is its significantly decreased uptake of DFMO which reaches a saturating level of 18 microM inside the cells; a concentration seven times below the Ki value of DFMO on T. brucei ornithine decarboxylase (130 microM). Apparently, the failure of DFMO uptake in the mutant strain has provided the basis of drug resistance. The results also raise the question on whether the uptake of DFMO by T. brucei is by passive diffusion or by transporter(s) mediation. DFMO does not compete with the uptake of ornithine, arginine or putrescine, and the reverse holds also true. However, the mutant strain cultivated under DFMO for several generations has a greatly enhanced uptake of ornithine and a moderately heightened uptake of putrescine. Both are reduced to the normal level upon further propagations of the mutant strain in the absence of DFMO.

Animals↗
PubMed1987

Why do costings?

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Costs and Cost Analysis↗
PubMed1987

Carrier detection of haemophilia A using DNA markers in families with an isolated affected male.

The carrier status of women in five families with an isolated haemophilia male was assessed by pedigree analysis, coagulation factor assays and DNA markers. In three families, ten women could be given very low risks of being carriers based on DNA analysis. In two of the families the DNA markers identified the mutation as originating in either the maternal or maternal grandfather's germ cell. Combined DNA and coagulation data suggested that the affected male in a third family was a de novo mutation. DNA analysis of the affected male in another family identified a large deletion of the F8 gene which was present in his mother and three sisters, suggesting that the grandmother was a carrier. A combination of coagulation factor data and DNA marker assessment can determine the carrier status of the majority of females in families with isolated affected haemophilia A males.

DNA↗
PubMed1986

Regional localization of 18 human X-linked DNA sequences.

A series of human probes with unique sequences has been isolated from a recombinant phage library constructed with DNA obtained from a human-hamster hybrid cell line. This cell line contained the X chromosome as the only human component. For 18 of these probes, a human X-chromosome origin has been confirmed and they have been regionally assigned by a combination of techniques: dosage studies utilizing DNA from human fibroblasts carrying X-chromosome duplications and deletions; the presence or absence of hybridization to digested DNA from hybrid lines carrying fragments of the X chromosome; and in situ hybridization to metaphase chromosomes. The use of dosage as a means to regionally assign probes significantly improves resolution of the X chromosome.

Animals↗
PubMed1985

Genetic mapping of DNA segments relative to the locus for the fragile-X syndrome at Xq27.3.

We have tested linkage between the locus for the fragile-X [fra(X)] syndrome at Xq27.3 and five polymorphic restriction sites identified by four DNA probes mapping distal to Xq26.1. A maximum distance of approximately 15 centimorgans (cM) between Xq27.3 and the marker loci mapping to this region was predicted based on the physical chromosome length. Close linkage between the disease and marker loci was excluded for probes DXS19 and DXS37 (theta = .05, Z = -2.94 and Z = -4.17, respectively). These marker loci were estimated to be less than five cM apart but approximately 40 cM proximal to the fragile site, indicating that there is a significantly greater frequency of recombination in this region of the X chromosome than expected from the physical length. Linkage results for the other marker loci and the fra(X) syndrome were inconclusive. However, the pX45d probe locus appears very closely linked to the factor IX locus (Z = 1.94 at theta = 0) and is approximately 20 cM proximal to Xq27.3. A relative map of the polymorphic restriction sites, fra(X) syndrome locus, and factor IX locus was constructed by maximizing lod scores over the Xq26.1----q27.3 region.

Animals↗
PubMed1984

Food intake in multiple sclerosis.

The usual dietary intake of 142 people with multiple sclerosis (MS), from different areas of Great Britain, has been assessed using the 7-day weighed intake method. This sample represents those subjects who said they had not altered their diets since diagnosis, ie, one-third of those who originally joined the management programme. Despite the low energy intakes of the subjects, intakes of other nutrients were similar to values of the general British population. The relationship of disability to energy and nutrient intake was studied. The relevance of dietary fatty acid intakes in MS is discussed with reference to epidemiological data and regional variations. The possibility that people with MS have specific requirements is considered and the need for nutritional guidance in MS is stressed.

Adult↗
PubMed1983

Dihydropyridine Ca2+ entry blockers selectively inhibit peak I cAMP phosphodiesterase.

The vasodilatory action of Ca2+ entry blockers is due primarily to slow Ca2+ channel inhibition; however, these drugs may have additional sites of action that contribute to vasodilation. Eleven Ca2+ entry blockers were evaluated for their inhibition of the two major forms of bovine heart cAMP phosphodiesterase which were separated by DEAE cellulose chromatography. Nifedipine and four other dihydropyridine Ca2+ entry blockers selectively inhibited peak I phosphodiesterase activity with IC50 values between 2 and 3 microM but were weak inhibitors of peak II phosphodiesterase with IC50 values of 100 microM or greater. The selective inhibition of peak I phosphodiesterase activity by these dihydropyridine Ca2+ entry blockers may be an intracellular mechanism for producing vasodilation in addition to slow Ca2+ channel inhibition.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Convergent differentiation in cultured rat cells from nonkeratinized epithelia: keratinocyte character and intrinsic differences.

Epithelial cells derived from a variety of glandular and other nonkeratinized rat tissues (pituitary, thyroid, bladder, endometrium, trachea, seminal vesicle, prostate, and mammary epithelium) were serially cultivated using a feeder layer of lethally irradiated 3T3 cells. The epithelial cells grew as progressively expanding colonies, in some cases stratified, and were shown to form cornified envelopes upon ionophore-induced activation of cross-linking. Cultures derived from each tissue were distinguishable from the others by characteristic cellular appearance and colony morphology. Those examined in greater detail could be distinguished biochemically in three ways. (a) A majority of cells in sparse cultures of bladder, tracheal, endometrial, and vaginal epithelial cells were capable of envelope formation, whereas those from pituitary, thyroid, seminal vesicle, and mammary epithelia did not attain maximal envelope forming ability until after confluence. (b) Bladder, thyroid, and pituitary cells exhibited different electrophoretic profiles of keratins, which accounted for 20-50% of the cellular protein. (c) Bladder cells were distinguished from thyroid and pituitary cells by a greater suppression of envelope-forming ability by vitamin A. These observations showed that cells from many epithelia have the potential to express properties of keratinocytes in culture while maintaining morphological and physiological differences. Serial passage of these cells generated continuous lines.

Acyltransferases↗
PubMed1983

Ultralente based insulin regimens--clinical applications, advantages and disadvantages.

The prolonged action of daily injections of beef ultralente insulin provides a source for the basal, steady state insulin supply which diabetics need in addition to their meal requirements. The complete distinction between basal and meal insulin requirements, provided by two or three injections of soluble insulin per day, allows simple rules to guide both the physician and patient. Thus, the required ultralente dose needs to be continued daily, irrespective of illness or missing meals, whereas the soluble insulin requirements are given according to meals. When starting ultralente insulin therapy a loading dose is required. The doses of ultralente and soluble insulin needed for different severities of diabetes and degrees of insulin resistance can be predicted. A simple regimen to cover the decreasing insulin requirements of newly presenting, ketotic juvenile-onset diabetics has been developed. During surgical operations the continued basal insulin supply, from ultralente insulin, greatly facilitates diabetes control. Whilst many patients have improved nocturnal blood glucose control after transfer to ultralente insulin, optimal control of diabetes sometimes remains difficult in view of the pre-breakfast plasma glucose rise and the longer action of subcutaneous soluble insulin than the physiological meal insulin response. Purified monocomponent beef ultralente insulin is antigenic, and human ultralente insulin might be advantageous.

Diabetes Mellitus↗
PubMed1983

Inhibition of asparagine-linked glycosylation of pro-opiomelanocortin in mouse pituitary cells by DL-threo-beta-fluoroasparagine.

The actions of DL-threo-beta-fluoroasparagine (DL-beta-F-Asn) on the glycosylation of proteins were examined in AtT-20/D16v cells which synthesize several forms of the glycoprotein prohormone, pro-opiomelanocortin (POMC). Treatment with threo-beta-F-Asn(5-10 mM) resulted in: 1) a reduction in the amount of the more highly glycosylated form of POMC (Mr = 32,000) relative to the less glycosylated form (Mr = 29,000) and 2) the appearance of a new species of POMC (Mr = 27,000). 35S]Methionine-labeled tryptic peptides prepared from 27,000 POMC were identical to those from 29,000 and 32,000 POMC; however, 27,000 POMC was found to contain 10% as much [3H]glucosamine relative to [35S]methionine as the 32,000 molecule. Furthermore, 27,000 POMC comigrated with a previously characterized unglycosylated form of this prohormone produced by treatment of cells with tunicamycin. These findings indicate that treatment of cells with threo-beta-F-Asn results in the production of a species of POMC which contains little or no carbohydrate. The effects of beta-F-Asn were specific for the threo diastereomer, were reversible by equimolar concentrations of Asn, but not Asp, and were dose-dependent. Evidence that threo-beta-F-Asn can replace Asn in proteins was obtained by showing that an identified Asn-containing tryptic peptide from threo-beta-F-Asn-treated cells displayed an altered mobility during electrophoresis consistent with threo-beta-F-Asn substitution within this peptide. We conclude that threo-beta-F-Asn can inhibit the glycosylation of proteins in intact cells and that this effect is due to its ability to replace Asn at glycosylation sites.

Animals↗
PubMed1982

Selective synthesis of octadeuterated (+/-)-5-HETE for use in GC-MS quantitation of 5-HETE.

5(S)-hydroxy-6 trans-8,11,14 cis-eicosatetraenoic acid (5-HETE) is the major product of arachidonic acid metabolism via the 5-lipoxygenase pathway. A limiting factor in the quantitation of 5-HETE by GC-MS analysis is the availability of a stable isotope analog for use as an internal standard. In this report, we detail procedures for selective chemical synthesis of multimilligram quantities of octadeuterated (+/-)-5-HETE from octadeuterated arachidonic acid. The octadeuterated (+/-)-5-HETE is suitable for use as an internal standard for GC-MS quantitation of 5-HETE. Preparation of the octadeuterated analog of 5-HETE can be readily performed in most laboratory settings.

Arachidonic Acid↗