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Biomedical subjects

M Read

Publications and source records attributed to M Read.

At least 37 records · Page 2Linked to original sources
PubMed1997

Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization.

Sulfadoxine/pyrimethamine (Fansidar) is widely used in Africa for treating chloroquine-resistant falciparum malaria. To clarify how parasite resistance to this combination arises, various lines of Plasmodium falciparum were used to investigate the role of naturally occurring mutations in the target enzyme, dihydropteroate synthetase (DHPS), in the parasite response to sulfadoxine inhibition. An improved drug assay was employed to identify a clear correlation between sulfadoxine-resistance levels and the number of DHPS mutations. Moreover, tight linkage was observed between DHPS mutations and high-level resistance in the 16 progeny of a genetic cross between sulfadoxine-sensitive (HB3) and sulfadoxine-resistant (Dd2) parents. However, we also demonstrate a profound influence of exogenous folate on IC50 values, which, under physiological conditions, may have a major role in determining resistance levels. Importantly, this phenotype does not segregate with dhps genotypes in the cross, but shows complete linkage to the two alleles of the dihydrofolate reductase (dhfr) gene inherited from the parental lines. However, in unrelated lines, this folate effect correlates less well with DHFR sequence, indicating that the gene responsible may be closely linked to dhfr, rather than dhfr itself. These results have major implications for the acquisition of Fansidar resistance by malaria parasites.

Alleles↗
PubMed1997

The managed care of early pregnancy problems.

There is an increasing trend towards non-invasive diagnosis and conservative treatment for early pregnancy problems. Although there are enormous potential benefits for women being treated thus, the ideal treatment methods and diagnostic algorithms have yet to be elucidated. Further large-scale studies are required.

Abortion, Spontaneous↗
PubMed1996

The amplicons in HL60 cells contain novel cellular sequences linked to MYC locus DNA.

We have previously determined that the amplified DNA present in the HL60 promyelocytic leukaemia cell line contained 70 kb of continuous DNA sequences around the c-myc gene. In the work presented here we have further defined the HL60 amplicon and find it to be of the order of 160 kb and to contain a large region of DNA from chromosome 8q24 that is located at least 260 kb telomeric to the c-myc gene, joined to the 70 kb of DNA from the c-myc gene region. The novel chromosome 8 DNA coamplified sequences are not lost during multiple passage of HL60 cells since the composition of the chimeric amplicon is the same in both early passage HL60 cells containing only double minutes (DMs) and late passage isolates containing homogeneous staining regions (HSRs) at different chromosomal locations. This shows that the HL60 HSRs found in late passage cells are not generated anew but are directly derived from the precursor DMs. The HL60 cells contain two copies of chromosome 8, each with different polymorphic markers. Both these chromosome 8 homologues retain a c-myc gene as well as the region containing the coamplified DNA sequences indicating that the HL60 amplicons were not generated by simple DNA deletion. The constraint to maintain the novel DNA sequences coamplified with c-myc gene DNA suggests that these sequences may play some role in maintaining the growth potential and/or differentiation capacity of the HL60 cells.

Chromosomes, Human, Pair 8↗
PubMed1996

The human L35a ribosomal protein (RPL35A) gene is located at chromosome band 3q29-qter.

Using a polymerase chain reaction (PCR)-based strategy that distinguishes functional intron-containing genes from pseudogenes, the chromosomal location of the human intron-containing L35a ribosomal protein gene (rpL35a, HGMW-approved symbol RPL35A) was previously deduced to be onchromosome 18 using DNAs from a panel of human-rodent somatic cell hybrids. The inability to detect rpL35a in a human-hamster somatic cell hybrid containing only human chromosome 18 led to a reinvestigation of the chromosomal location of the human rpL35a gene. A clone containing intron 2 of rpL35a was isolated, sequenced, and used to isolate a P1 clone containing the human intron-containing rpL35a gene. By fluorescence in situ hybridization (FISH) analysis with the P1 clone as a probe, the rpL35a gene was located on chromosome band 3q29-qter. This location was confirmed by positive PCR analysis of a human-hamster somatic cell hybrid containing only human chromosome 3. The problem of using only human-rodent somatic cell hybrids for chromosome location is discussed.

Amino Acid Sequence↗
PubMed1995

Sea-level physical activity and acute mountain sickness at moderate altitude.

The effect of previous physical conditioning on young well-conditioned mountaineers in relationship to acquiring acute mountain sickness is controversial. Data show both increased and decreased effects on the incidence of altitude illness. How general tourists at moderate altitudes are affected is unknown. To determine the influence of sea-level habitual physical activity on the incidence of mountain sickness, we surveyed 205 participants in a scientific conference at 3,000 m (9,840 ft). A 36-item questionnaire was distributed to the subjects 48 hours after arrival at altitude. Their sea-level physical activity (SLPA) was measured by a published and validated instrument that included questions about patterns of work, sporting, and leisure-time activities. Acute mountain sickness was defined as the presence of 3 or more of the following symptoms: headache, dyspnea, anorexia, fatigue, insomnia, dizziness, or vomiting. Most of the respondents were male (62%) from sea level (89%) with a mean age of 36 +/- 8.7 (standard deviation) years (range, 22 to 65). Nearly all (94%) were nonsmokers, and 28% had acute mountain sickness. The mean SLPA score was 8.0 +/- 1.3 (range, 5.1 to 12.0). No statistically significant difference in mean SLPA scores was found between those with and without acute mountain sickness (8.1 versus 7.8), nor in the individual indices (work, 2.5 versus 2.4; sport, 2.9 versus 2.7; leisure, 2.8 versus 2.7). We conclude that habitual physical activity performed at sea level does not play a role in the development of altitude illness at moderate altitude in a general tourist group.

Acute Disease↗
PubMed1995

von Willebrand factor in plasma, platelets, and selected tissues of ferrets.

By standard laboratory methods the presence and activity of von Willebrand factor (vWF) was detected and characterized in the ferret (Mustela putorius furo); vWF in plasma, platelets, and selected tissues (thoracic aorta, cranial vena cava, thoracic portion of caudal vena cava, and lung) was documented. Activity, antigenic concentration, plasma multimeric distribution, and localization within tissues were similar to those features in other species. Two differences were apparent: multimeric distribution of platelet vWF was skewed toward the smaller molecular weight multimers, and mucous goblet, but not ciliated, cells of the bronchial epithelium stained positive for vWF. Larger molecular weight multimers were not released subsequent to administration of 1-deamino-8-D-arginine-vasopressin. The ferret may be a useful animal model in studying the role of vWF in hemostasis, thrombosis, and atherosclerosis. In particular, the role of small molecular weight multimers found in ferret platelets may provide further insight into the roles of platelet vWF multimeric distribution, platelet adhesion, and thrombosis.

Animals↗
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

Molecular characterisation of the enolase gene from the human malaria parasite Plasmodium falciparum. Evidence for ancestry within a photosynthetic lineage.

We have isolated and characterised the gene encoding the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase) from the human malaria parasite Plasmodium falciparum. This was achieved using a combination of cDNA sequencing and inverse-PCR techniques. The gene maps to chromosome 10 of the parasite. We have also mapped two further glycolytic enzyme genes, glyceraldehyde-3-phosphate dehydrogenase and triose-phosphate isomerase, to chromosome 14. The enolase gene encodes a protein of 446 amino acids (48.7 kDa), and all amino acid residues implicated in substrate/cofactor binding and catalysis are conserved in the malarial enolase molecule. The predicted protein sequence displays approximately 60-70% identity to enolase molecules of other eukaryotes, the closest relationship with its homologues seen amongst the seven fully described glycolytic pathway enzymes of P. falciparum. Of particular significance in this well conserved molecule is a characteristic 5-amino-acid insertion sequence that is identical in position and virtually identical in primary structure to that which is otherwise found uniquely in plant enolase proteins. This pentapeptide, together with other features of the plasmodial sequence, points to a common ancestry with photosynthetic organisms at the level of a protein-encoding nuclear gene, thus extending earlier analyses of nuclear small-subunit ribosomal RNA genes, and of an extrachromosomal circular 35-kb DNA element found in P. falciparum, which have also indicated such a relationship.

Amino Acid Sequence↗
PubMed1994

Efficient transfection of primary cells in a canine hemophilia B model using adenovirus-polylysine-DNA complexes.

We have used molecular conjugates containing combinations of DNA, adenovirus, polylysine, and transferrin to transfect primary cells derived from canines with hemophilia B (factor IX deficiency), as well as a canine epithelial cell line. Transfection of canine hemophilia B fibroblasts with molecular conjugates resulted in efficient transfection and expression of luciferase DNA-adenovirus-polylysine (AdpL) conjugates or luciferase DNA-adenovirus-polylysine-transferrin (hTfpL/AdpL) conjugates. No expression in canine hemophilia B fibroblasts was evident after exposure to DNA alone, or DNA conjugated with polylysine and transferrin. Transfection efficiencies of 50% or more could be demonstrated in cells transfected with a beta-galactosidase reporter gene as part of an hTfpL/AdpL molecular conjugate. Transfection with canine factor IX AdpL conjugates or canine factor IX hTfpL/AdpL conjugates resulted in factor IX expression for more than 2 weeks in vitro in hemophilia B canine fibroblasts. Maximum levels of expression of over 700 ng of canine factor IX/10(6) cells/24 hr were demonstrated in fibroblasts after transfection with canine factor IX hTfpL/AdpL conjugates. Similar conjugates were used to transfect hemophilia B canine bone marrow stromal cells and Madin-Darby canine kidney cells that also expressed canine factor IX. The use of molecular conjugates to transfect primary cells may be feasible as a means of in vitro or in vivo gene therapy for hemophilia B, and can be tested in the canine hemophilia B model.

Adenoviridae↗
PubMed1994

Patient satisfaction studies: methodology, management and consumer evaluation.

Discusses how consumer evaluation studies of health-care services might be undertaken to provide valid assessments of consumer opinions and priorities, amenable to subsequent, effective management action. To do so, provides an account of the history of patient satisfaction surveys, presents a detailed examination of key examples, and discusses the well-documented strengths and weaknesses of this approach. Draws attention to recent critiques of survey methods and growing interest in qualitative research focusing on the rationale that the latter provides more useful data for managers. Argues that the search for a "best" consumer evaluation method is misplaced since appropriate methods should be determined by research objectives which themselves may vary. Points to existing analyses of relevant research which offer sound methodological guidelines and concludes by offering explicit suggestions for the future conduct of consumer evaluation research.

Health Services Research↗
PubMed1994

The DNA region around the c-myc gene and its amplification in human tumour cell lines.

In order to study amplicons containing the c-myc locus in human tumour cells we have analysed the region around the human c-myc gene. Using pulsed field gel electrophoresis we have constructed a 1.8 Mb map of the c-myc locus using six rare-cutting endonucleases. In addition we have cloned 130 kb of continuous DNA around the c-myc gene which includes 65 kb of 5' sequence and 65 kb of 3' sequence. The first exon of the pvt-1 gene was located 59 kb 3' of the c-myc gene. Using nine neighbouring probes, covering a region of about 395 kb around the c-myc gene, we were able to determine the extent and composition of c-myc amplicons in the HL60, NCI-N417, Colo320HSR, SkBr3 and the Sk-N-MC human tumour cell lines. Furthermore we analysed the chromosomal location of amplified c-myc DNA in NCI-N417 and SkBr3 cells. Similarities and differences between the amplified DNA in the different cell lines are discussed.

Cloning, Molecular↗
PubMed1993

Characterisation of the gene encoding an unusually divergent TATA-binding protein (TBP) from the extremely A+T-rich human malaria parasite Plasmodium falciparum.

The intergenic regions of the human malaria parasite, Plasmodium falciparum, are extreme in their base composition, averaging approx. 90% A + T. As a first step to investigating whether transcription in this organism follows conventional models based largely on yeast, we have isolated and characterised the gene (TBP) encoding its TATA-binding protein (TBP). The gene is present as a single copy on chromosome 5 and is expressed as a 1.8-kb mRNA encoding a protein of 228 amino acids (aa) (26 164 Da). The inferred protein product has a bipartite structure consisting of a 45-aa species-specific N-terminal domain and a 183-aa C-terminal domain. In the latter, the malarial protein contains two directly repeated, but imperfectly homologous regions, each approx. 78 aa in length, together with a highly basic region located between them. These features are characteristic of all TBPs studied to date. Moreover, hydropathy plots suggest that the overall folding of this C-terminal domain is very similar to that of other TBPs. However, TBP from P. falciparum is much less closely related at the primary sequence level to the archetypal yeast homologue than are all other characterised TBPs (42% identity, compared to 76-93%, respectively). Despite this divergence of the primary sequence, most residues known to be involved in DNA binding are conserved. Those instances where sequence variation at generally conserved residues is observed may reflect functional differences that could ultimately be exploited by selective chemotherapy.

Amino Acid Sequence↗
PubMed1993

Developmental expression of pancreatic gastrin and somatostatin in the sheep.

The nature and developmental profile of pancreatic gastrin and somatostatin were determined in the ovine fetus, a model considered relevant to human development. Gastrin and somatostatin peptide and mRNA were examined in the pancreas of fetal sheep at 80, 105, 125 and 140 days gestation (term: 145 days), 15-day-old lambs and adult sheep. Highest concentrations of gastrin (both amidated and the glycine-extended precursor) were observed in the lamb pancreas with gastrin mRNA levels highest at 140 days gestation. Amidated gastrin was present almost entirely as sulphated gastrin-17 while glycine-extended gastrin was mostly present as a high molecular weight form. Only glycine-extended gastrin was detected in the adult pancreas, indicating attenuated processing in mature adult pancreas. Up to 140 days gestation, gastrin mRNA correlated better with glycine-extended gastrin than with the amidated form, suggesting that amidation was a rate-limiting factor in the production of bioactive gastrin. Somatostatin mRNA and peptide reached a higher concentration and peaked before that of gastrin. Gastrin is a normal product of the fetal and adult pancreas although, when compared to the antrum, the levels are low and the processing to amidated forms is substantially reduced. Unlike in the stomach, the developmental profile of pancreatic gastrin and somatostatin does not appear to be linked.

Amino Acid Sequence↗
PubMed1993

Microtubular organization visualized by immunofluorescence microscopy during erythrocytic schizogony in Plasmodium falciparum and investigation of post-translational modifications of parasite tubulin.

We describe a novel procedure for the immunofluorescent investigation of Plasmodium falciparum. This has allowed us to visualize clearly microtubular structures and their changing conformation through the erythrocytic cell-cycle, to the stage of cytodifferentiation leading to merozoite release. The images of spindle development we observed, together with an analysis of nuclear body numbers in large numbers of parasites, indicate that there is an apparent asynchrony in chromosomal multiplication within a single parasite. Using antibodies specific for post-translational modification of alpha-tubulin, we also demonstrate that the C-terminal tyrosine-containing epitope of P. falciparum alpha-tubulin I is similar to that of other organisms. Lysine-40 in the same molecule, a target for highly specific in vivo acetylation in some organisms, is unmodified in the blood stages we examined here. After in vitro acetylation of this residue, however, the epitope to which it contributes was recognized by antibody, showing that the conformation of this part of the molecule is also conserved, despite a lack of primary sequence homology immediately downstream of the target lysine residue.

Animals↗