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C R Newton

Publications and source records attributed to C R Newton.

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

The production of PCR products with 5' single-stranded tails using primers that incorporate novel phosphoramidite intermediates.

We have prepared several novel phosphoramidites and have synthesised oligonucleotides incorporating them internally. The presence of these residues in an oligonucleotide template presents an impossible barrier to primed synthesis by Taq DNA polymerase. When extended as polymerase chain reaction products, these oligonucleotides no longer serve as templates for the polymerase beyond the insertion sites of the modified intermediates, thereby producing single-stranded tails on amplification products. These tails can then be used for solid phase capture and colorimetric detection of PCR products.

Alkaline Phosphatase↗
PubMed1992

Childhood deaths in Africa: uses and limitations of verbal autopsies.

The verbal autopsy (VA) is an epidemiological tool that is widely used to ascribe causes of death by interviewing bereaved relatives of children who were not under medical supervision at the time of death. This technique was assessed by comparison with a prospective survey of 303 childhood deaths at a district hospital in Kenya where medically confirmed diagnoses were available. Common causes of death were detected by VA with specificities greater than 80%. Sensitivity of the VA technique was greater than 75% for measles, neonatal tetanus, malnutrition, and trauma-related deaths; however, malaria, anaemia, acute respiratory-tract infection, gastroenteritis, and meningitis were detected with sensitivities of less than 50%. There may have been unwarranted optimism in the ability of VAs to detect some of the major causes of death, such as malaria, in African children. VA used in malaria-specific intervention trials should be interpreted with caution and only in the light of known sensitivities and specificities.

Cause of Death↗
PubMed1992

Motives for parenthood and response to failed in vitro fertilization: implications for counseling.

The purpose of this investigation was to identify in vitro fertilization (IVF) candidates' motives for parenthood and hence for seeking treatment. The relationship among identified motives, pretreatment emotional adjustment, and reaction to treatment failure was then examined. Women as a group placed greatest emphasis on fulfilling gender-role requirements, and those strongly endorsing such motives showed the poorest adjustment before IVF and the most negative reaction to first-cycle failure. In contrast, men in general were more likely to stress a desire for marital completion, although this motive was not predictive of emotional status before or after IVF. However, men experiencing social pressures to have children were at greater risk when treatment failed. The results indicate that greater consideration of cognitive factors may enhance understanding of emotional reactions to IVF failure and provide important insights for therapeutic intervention.

Adult↗
PubMed1992

The disposition of oral and intramuscular pyrimethamine/sulphadoxine in Kenyan children with high parasitaemia but clinically non-severe falciparum malaria.

1. H.p.l.c. methods are described for the measurement of pyrimethamine and sulphadoxine in small volumes of plasma dried on filter paper strips. 2. Pyrimethamine/sulphadoxine (Fansidar, Hoffman LaRoche) was given by mouth and by intramuscular injection to children with uncomplicated falciparum malaria but with high parasitaemia (n = 8 for both routes; pyrimethamine 1.25 mg kg-1, sulphadoxine 25 mg kg-1). 3. Plasma concentrations of pyrimethamine and sulphadoxine associated with synergistic effects against pyrimethamine-resistant strains of Plasmodium falciparum in vitro were achieved within 1 h of administration and were maintained beyond the end of sampling. 4. After both oral and parenteral administration the plasma concentrations of both compounds were lower than those predicted by data from healthy subjects. 5. Areas under the plasma concentration-time curves of sulphadoxine after oral and i.m. administration did not differ significantly, although maximum plasma drug concentrations were higher after the i.m. route (P = 0.03). 6. The AUC values of pyrimethamine did not differ significantly between the two routes of administration. However, after i.m. administration AUC(0,24 h) values were smaller (P = 0.03), and the time to maximum plasma drug concentration (tmax) was longer (P = 0.004) than when the drug was given orally.

Administration, Oral↗
PubMed1992

Prophylactic phenobarbitone in young children with severe falciparum malaria: pharmacokinetics and clinical effects.

1. A method is described for the measurement of phenobarbitone (PB) by reversed phase high performance liquid chromatography (h.p.l.c.) from small samples of whole blood dried onto filter paper strips. 2. The disposition of PB given prophylactically to young children with severe malaria on parenteral quinine is contrasted with that in aparasitaemic Kenyan children on no antimalarial drugs. There were no differences in the disposition of PB between the two groups. 3. Peak blood PB concentrations were equal to or greater than 15 mg l-1 in 27% of the patients on quinine and 23% of those not on quinine; a concentration of 10 mg l-1 was achieved or exceeded by 100% and 92% of each group, respectively, and was maintained for 39 +/- 24 h (mean +/- s.d.), and 33 +/- 21 h, respectively. 4. In an open, dose-finding study, the progress of young children with cerebral malaria given prophylactic PB (10 mg kg-1), was contrasted with that of controls given no seizure prophylaxis. 5. The drug had no apparent effect on depth or duration of coma, but neither was the incidence of seizures reduced. 6. A controlled trial of prophylactic PB in young children with cerebral malaria is needed, but a larger dose than 10 mg kg-1 should be studied.

Child↗
PubMed1991

Direct haplotype determination by double ARMS: specificity, sensitivity and genetic applications.

We have developed a novel double Amplification Refractory Mutation System (double ARMS) using a highly polymorphic region 5' to the human delta-globin gene as a model system. The double ARMS approach involves using two allele-specific ARMS primers simultaneously during DNA amplification by the polymerase chain reaction (PCR). The resulting system is highly sensitive and more specific than single ARMS. In addition, this approach enables the elucidation of the relationship of polymorphic sites on the same chromosome and thus allows the direct determination of haplotypes. We have also demonstrated that this system can be used in conjunction with inverse PCR, the resulting double ARMS inverse PCR (DARMSI-PCR) may allow haplotype determination on polymorphic sites which are separated further apart than the length limit imposed by PCR. The double ARMS approach has numerous other applications in molecular biology including HLA typing, virology, forensic pathology and the investigation of the phenomenon of chimerism following bone marrow transplantation.

Alleles↗
PubMed1991

Intracranial pressure in African children with cerebral malaria.

Opening lumbar cerebrospinal fluid (CSF) pressure was measured with a paediatric spinal fluid manometer in 26 of 61 Kenyan children (mean age 39 months) with cerebral malaria. In all cases pressure was above normal (mean [SD]22.6 [7.4] cm CSF, range 10.5-36). Clinical features of our patients suggest that intracranial hypertension is important in the pathogenesis of cerebral malaria in children, especially as a cause of death. We suggest that raised intracranial pressure is secondary to increased cerebral blood volume. Lowering intracranial pressure may significantly reduce the mortality and morbidity of cerebral malaria. The potential risks and benefits of lumbar puncture should be considered carefully in patients with suspected cerebral malaria.

Animals↗
PubMed1991

Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells.

Primary cultures of bovine brain microvessel endothelial cells (BMECs) were used to examine the cycling kinetics of ferrotransferrin (Tf) and to provide evidence for a transcytotic pathway in vitro. Binding of 125I-Tf to BMECs grown on matrix-coated plastic was measured in the presence of saponin to calculate the total number of transferrin receptors (TfRs). Nonlinear regression analysis of the binding isotherm showed that there were 100,000 high-affinity receptors per cell and that expression was maximum at cell confluence. Binding of Tf at 4 degrees C indicated that there was a large intracellular receptor pool comprising 85-90% of the total cellular receptors. Accumulation of Tf at 37 degrees C, inhibited at low temperature and in the presence of metabolic poisons, occurred with an initial rate coefficient of 0.030 min-1 and this decreased by 83% after 60 min. Concomitant accumulation of 59Fe from Tf-59Fe was linear. In the absence of externally added ligand, 80% of the accumulated 125I-Tf was released into the medium with a rate coefficient of 0.017 min-1 and this was inhibited at low temperature. In the presence of the weak base primaquine, the accumulation of Tf and 59Fe and the efflux of Tf were decreased. Moreover, phorbol myristate acetate (PMA) caused a 30% increase in surface TfRs and an 82% increase in Tf accumulation, although the size of the recycling pool remained unchanged. Despite the low numbers of TfR expressed by post-confluent cells, filter-grown BMEC monolayers were used to measure transcytosis of Tf. A small portion of the Tf that was accumulated from the apical side entered a transcytotic pathway. Most of the Tf and all of an accumulated fluid-phase tracer were recycled towards the apical side. These results showed that cultured BMECs cycle Tf-TfR complexes slowly and vectorially and suggested that the large intracellular receptor pool may facilitate steady state accumulation and regulate transcellular transport of iron.

Animals↗
PubMed1991

Genetic analysis in cystic fibrosis using the amplification refractory mutation system (ARMS): the J3.11 MspI polymorphism.

A new method of genetic analysis has been devised. The method, amplification refractory mutation system (ARMS), has been used to genotype the J3.11 MspI restriction fragment length polymorphism (RFLP) closely linked to cystic fibrosis (CF). The DNA sequence for both alleles of this dimorphism has been used to design ARMS primers. These allow genotyping of DNA isolated from blood, Guthrie cards, and buccal cells.

Alleles↗
PubMed1991

Quinine treatment of severe falciparum malaria in African children: a randomized comparison of three regimens.

The pharmacokinetics and effectiveness of three dosage regimens of quinine were studied in a group of 59 children with severe malaria. The children were randomized to receive high-dose intravenous or intramuscular quinine (20 mg salt/kg loading, then 10 mg salt/kg every 12 hr), or low-dose intravenous quinine (10 mg salt/kg loading, then 5 mg salt/kg every 12 hr). In the group receiving the high-dose intravenous regimen, mean high and low quinine concentrations were consistently greater than 10 and 6.5 mg/l, respectively. Peak concentrations as well as the time required to achieve them were similar in the intramuscular and high-dose intravenous groups. The low-dose intravenous quinine regimen resulted in mean peak concentrations greater than 6 mg/l and mean low concentrations greater than 3.5 mg/l. All blood concentrations exceeded the 99% in vitro inhibitory concentration (EC99) of 0.89 mg/l or less of quinine for 60 isolates of Plasmodium falciparum, which were taken from children with malaria during the same period. Judged by a number of clinical criteria, the response was better in patients receiving the high-dose than the low-dose intravenous regimen. The time taken to clear parasites with both the high-dose intravenous and intramuscular regimens were significantly shorter than those obtained in the low-dose group. We have also shown for the first time that the rate of parasite clearance can be directly related to the area under the quinine concentration versus time curve. This applied to all three quinine regimens (r = 0.4252, P less than 0.02; n less than or equal to 35). Five patients, two on the low-dose regimen, two on the intramuscular regimen, and one on the high-dose regimen, developed hypoglycemia after admission, but in these cases, insulin concentrations were correspondingly low. No significant quinine toxicity was observed in any of the cases. The high-dose intravenous quinine regimen described here may be optimal for treatment of severe falciparum malaria in areas of chloroquine resistance in Africa. Our data provide no justification for reducing the dose of quinine in the treatment of severe malaria in Africa. The intramuscular regimen could provide a satisfactory alternative in areas where intravenous administration might be delayed or is impossible.

Child↗
PubMed1991

A plasmid cassette system that allows optimization of primers for use in the amplification refractory mutation system.

A plasmid "cassette" system is presented that allows the development of amplification refractory mutation system (ARMS) primers. Primer development may be accomplished even in the absence of appropriate clinical material from patients demonstrating mutated alleles. The use of such cassettes is demonstrated here in the assessment of primers prepared for ARMS analysis of the gene coding for the normal and Z variant alleles of the human serine protease inhibitor alpha-1-antitrypsin.

Adult↗
PubMed1991

Rapid diagnosis of familial defective apolipoprotein B-100 by Amplification Refractory Mutation System.

We report a method for the diagnosis of familial defective apolipoprotein (apo) B-100, using the Amplification Refractory Mutation System (ARMS) and either whole blood or extracted DNA in the polymerase chain reaction. Normal and mutant alleles are identified by using two allele-specific oligonucleotide primers, each with the same common primer, to amplify a 187-bp fragment of the apo B-100 gene. Fragment amplification occurs only when the allele-specific primer matches the nucleotide sequence of the template DNA. The amplification product is detected by agarose gel electrophoresis, followed by staining with ethidium bromide. The technique is simple, reliable, and robust. It avoids the use of radiation or hybridization with allele-specific oligonucleotide probes, and is well suited for use in the routine clinical chemistry department.

Apolipoprotein B-100↗
PubMed1991

Analysis of apolipoprotein E genotypes by the Amplification Refractory Mutation System.

The Amplification Refractory Mutation System (ARMS) has been successfully applied to the detection of apolipoprotein (apo) E genotypes in human DNA extracted from peripheral blood. By using four allele-specific oligonucleotide primers and one common primer, one can identify the three common alleles of the apo E genetic polymorphism, epsilon 2, epsilon 3, and epsilon 4. The system amplifies two sequences of the apo E gene, one of 181 bp and the other 319 bp. These sequences are amplified when DNA containing a particular allele is incubated with its allele-specific oligonucleotide primer and a common primer. The method is simple, reliable, and nonisotopic and obviates the need for digestion with restriction endonucleases or for hybridization with allele-specific oligonucleotide probes. Genotyping DNA by this method overcomes the problem of post-translational modification of the apo E phenotype encountered with isoelectric focusing of the mature plasma apo E protein.

Alleles↗
PubMed1990

Molecular characterisation of two alpha-1-antitrypsin deficiency variants: proteinase inhibitor (Pi) Null(Newport) (Gly115----Ser) and (Pi) Z Wrexham (Ser-19----Leu).

Two single point mutations in the alpha-1-antitrypsin gene, resulting in AAT deficiency, have been characterised in heterozygotes by DNA amplification and direct sequencing. The mutations result in amino acid substitutions, Gly115----Ser and Ser-19----Leu, in the leader sequence, respectively, and have been designated Pi Null(Newport) and Pi Z Wrexham. In the two families studied the mutations occur on chromosomes which also carry the common mutation causing Z deficiency. Individuals with such a deficiency are, therefore, compound heterozygotes. It is not known if these particular mutations would only cause a mild form of AAT deficiency in the absence of the Z mutation as they do not appear to cause predictable folding abnormalities. They do, however, result in severe deficiency when the Z mutation occurs in the same gene.

Amino Acid Sequence↗