Biomedical subjects
R Harris
Publications and source records attributed to R Harris.
Hepatitis C in human immunodeficiency virus-coinfected patients: increased variability in the hypervariable envelope coding domain.
Patients coinfected with the hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) were studied with regard to nucleotide sequence variability in the E2/NS1 first hypervariable region of the HCV genome. The nucleotide variability within individual patients was compared to patients infected only with HCV. The proportion of predicted synonymous and nonsynonymous amino acid changes, and the relationship to putative high-antigenicity sites, were evaluated in the hypervariable envelope domain. Ninety-one clones from 10 patients with HCV/HIV coinfection were sequenced, following polymerase chain reaction (PCR) amplification of the hypervariable region. The control HCV group included 53 clones from 7 patients. Sequence analysis encompassed the region coding for amino acids 384 to 414. Consensus sequences from each patient were used as the internal standard for nonsynonymous amino acid codon variability. Cumulative proportional comparison at each amino acid site revealed increased variability in HCV RNA from patients with HCV/HIV coinfection versus HCV alone (P < .05). The greatest variability was observed at amino acids 386, 397, 400, 402, 405, 407, and 414, with >l0 percent clonal variation at these sites. Jameson-Wolf plots were used to predict putative high-antigenicity domains. Nonsynonymous clonal variation resulted in alteration of putative antigenic sites within the hypervariable region. All clones had at least one high-probability site. Clones with unique predicted antigenic domains were observed more frequently in HIV/HCV coinfected patients, and, independent of viral titer, were consistent with increased sequence variability. These data suggest an accumulation of envelope variants in the HCV/HIV coinfected patients, which could be related to ineffective viral clearance, and may help explain prior reports of interferon (IFN) resistance in this patient group.
A compendium and hydropathy/flexibility analysis of common reactive sites in proteins: reactivity at Asn, Asp, Gln, and Met motifs in neutral pH solution.
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Research in the community: recruiting and retaining practices.
Medical investigators increasingly attempt to demonstrate the effectiveness of treatments given in routine practice settings as well as in research settings. We identified and describe several key issues in practice-based research: identifying the appropriate population; drawing the sample; recruitment of physicians and patients; maintenance of the physician-researcher relationship; and data management. Three on-going practice-based projects are described from the perspective of these key issues. Two are practice-based randomized trials, one is a larger cohort study. Common characteristics are identified which contributed to the success of the three projects: direct recruitment of clinicians by clinicians; close liaison with local medical organizations; on-going personal contact with the practices; and recognition of the value of the practicing physician's time.
commissureless controls growth cone guidance across the CNS midline in Drosophila and encodes a novel membrane protein.
The commissureless (comm) gene was identified previously in a large-scale screen for mutations that disrupt CNS axon pathways in Drosophila. The comm gene has a unique mutant phenotype: the complete absence of most axon commissures, while midline cells and other aspects of CNS fate and patterning are left unchanged. Here, we report on the molecular cloning, characterization, and expression of the comm gene. comm encodes a novel protein of 370 amino acids that lacks a signal sequence, has a transmembrane domain, and biochemically copurifies with membranes. COMM mRNA and COMM protein are dynamically expressed during embryogenesis, including by CNS midline glia during the formation of the axon commissures. Anti-COMM antibodies reveal strong staining of organelles likely to include the Golgi complex and endosomes and weaker staining of the cell surface. As commissural growth cones contact and traverse the CNS midline, COMM protein is apparently transferred from midline glia to commissural axons.
Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila Netrin/UNC-6 homologs.
Netrins are chemotropic guidance signals that play important roles in circumferential axon guidance in C. elegans and in the developing vertebrate spinal cord. We have identified two Drosophila homologs of this protein family (Netrin-A and Netrin-B). Both Netrins are dynamically expressed throughout embryogenesis, including CNS midline expression at the time of commissure formation. Both Netrin genes map close to each other on the X chromosome, and embryos deficient for this region exhibit defects in commissure formation. This CNS phenotype can be rescued by expression of either Netrin at the CNS midline, confirming an important role for Drosophila Netrins in commissural growth cone guidance. A localized source of Netrin protein at the midline is apparently important for function, since ectopic expression of either Netrin throughout the CNS results in phenotypic defects similar to the loss-of-function phenotype.
Observation of albumin resonances in proton nuclear magnetic resonance spectra of human blood plasma: N-terminal assignments aided by use of modified recombinant albumin.
Two-dimensional total shift correlation spectroscopy (TOCSY) and double-quantum-filtered phase-sensitive homonuclear shift-correlated spectroscopy (DQF-COSY) 1H NMR spectra are used to assign peaks for about one sixth of the amino acids residues of isolated human serum albumin (67 kDa) to amino acid types. Sequential assignments are presented for 1H NMR resonances of the N-terminal residues Asp1, Ala2 and His3 of human serum albumin (HSA). These are based on pH-dependent chemical shifts reflecting the titrating N-terminal NH2 and the His3 imidazole ring, in addition to DQF-COSY and TOCSY experiments. Studies of variant recombinant human albumin with Asp1 deleted, rHA(2-585), aided the assignments. The structural nature of the N-and C-termini of HSA are discussed and pKa values of 7.9 and 6.3 were determined for the N-terminal amino group and His3 imidazole ring, respectively. About 20 spin systems for albumin, including those for the N-terminal amino acids, were assigned in 1H NMR spectra of blood plasma buy comparison with isolated albumin. Resonances for lipids within lipoproteins and also several low molecular mass components can also be assigned in 2D TOCSY 1H NMR spectra of plasma.
Prenatal genetic services for Down's syndrome: access and provision in 1990-1991. Steering Committee of the National Confidential Enquiry into Counselling for Genetic Disorders.
OBJECTIVE: To examine access to and provision of prenatal genetic services relating to Down's syndrome. DESIGN: Retrospective review of obstetric casenotes. SAMPLE: Pregnancies involving Down's syndrome in England and Wales in 1990-1991 in women aged 38 or over. Information was obtained in 430 cases from a questionnaire completed by the obstetric team who were asked to provide details based only on documentation in the antenatal casenotes. The outcome of pregnancy was a termination in 268 (62%) cases, a liveborn child with Down's syndrome in 144 (34%), a stillbirth in 9 (2%), a miscarriage in 8(2%) and in one case was not known. RESULTS: Overall, prenatal diagnosis was not offered in 7% pregnancies (95% CI: 4.4-9.2%) with late booking given as the main reason. Of women offered prenatal diagnosis, 76% accepted (95% CI: 72.3-80.6%). Counselling was documented before prenatal diagnosis in 89% of cases (95% CI: 86.0-92.3%) and after the procedure, to discuss the results, in 73% (95% CI: 67.5-77.7%). In 10% of pregnancies terminated for Down's syndrome, fetal products were not sent to the laboratory. There was no report of a normal fetus having been terminated as a consequences of incorrect prenatal diagnosis. However, in 10% (95% CI: 5.9 to 14.0%) of cases examined in the laboratory the diagnosis of Down's syndrome could not be confirmed. Details of prenatal diagnosis were not provided in five cases where a child with Down's syndrome was born. Of the remaining 139 livebirths, prenatal diagnosis was not offered in 27 (19%) cases, offered and declined in 92 (66%) and accepted in 20 (14%). In two cases a normal fetal karyotype was reported following prenatal diagnosis. CONCLUSIONS: The study has demonstrated that in 1990-1991: 1. There were certain shortcomings in the documentation of antenatal care; 2. Late booking was the main factor precluding the offer of prenatal diagnosis to women aged 38 or over, and 3. The rate of confirmation of Down's syndrome in terminated fetuses was incomplete.
Florid oral manifestations in an atypical familial adenomatous polyposis family with late presentation of colorectal polyps.
Early identification and prophylactic surgery are essential in preventing development of malignancy in colorectal polyps in familial adenomatous polyposis (FAP). Previous studies indicate a 100% cumulative risk of developing colonic polyps in individuals with FAP by the age of 34 yr. However, we have previously reported late-onset and non-penetrance of polyposis in four families. We describe here in detail one of these families with florid oral manifestations detectable on panoramic radiography of the jaws, which were instrumental in the diagnosis of FAP in the index patient and for ascertainment of her family for screening. The pathological adenomatous polyposis coli (APC) gene mutation in this family was shown to segregate with dento-osseous changes. Panoramic jaw radiography is a common examination carried out prior to dental or oral surgical procedures. This case illustrates the importance of appropriate investigations, including family history and colonoscopy, even in relatively older patients with radiological findings such as those described here and in members of their families at risk of FAP.
Implications for medical genetics of the House of Commons Science and Technology Committee's report on human genetics.
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Genetics in primary care. Report on workshop of EC Concerted Action on Genetics Services in Europe (CAGSE) in association with the Royal College of GP Spring Meeting, Blackpool, UK, 28 April 1995.
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Safety and activity of zalcitabine and zidovudine combination in HIV-positive people with CD4 cell counts < or = 300 cells/mm3. The Roche M50002 Study Group.
We carried out an open-label, single-arm, multicentre 1-year study of the safety and activity of therapy with a zalcitabine-zidovudine combination in 561 patients with CD4 cell counts < or = 300 cells/mm3 who were either established on zidovudine or had not previously received antiretroviral therapy. Additionally, we assessed the impact of baseline characteristics on clinical outcome during therapy. The study used specialist HIV-care centres in Argentina, Belgium, Brazil, Hungary, Italy, Luxembourg, Mexico, Peru, Spain and Venezuela. Participants were regularly assessed for adverse experiences, changes in laboratory values and clinical progression. A total of 561 patients entered the study, with 353 completing 12 months of follow-up. The 492 zidovudine-experienced patients had a mean duration of previous therapy of 18.5 months. No unexpected adverse events were reported. Peripheral neuropathy was observed in 12.7% of patients and was more common in participants with CD4 cell counts < or = 100 cells/mm3 at baseline. Clinical disease progression was significantly associated with lower baseline CD4 cell counts, a history of previous AIDS-defining events and a baseline haemoglobin < 11 g/dl. The median CD4 cell count at baseline was 141.5 cells/mm3, rising to 174 cells/mm3 after 12 weeks and to 148 cells/mm3 at 12 months. In conclusion, zalcitabine-zidovudine combination therapy was found to be well tolerated in this patient population, although patients with CD4 cell counts < or = 100 cells/mm3 were found to require more intensive monitoring for toxicities and disease events. The changes in the CD4 cell count seen in this study and in others provide evidence of the therapeutic activity of this combination.
5-fluorouracil and allopurinol combined with recombinant interferon-alpha 2b in the treatment of patients with advanced prostate cancer: a phase I/II study.
PURPOSE: We determined the toxicity and preliminary response rate of escalating doses of 5-fluorouracil (670 to 1,500 mg./m.2 per day) combined with a fixed dose of interferon-alpha 2b (5 million units) and allopurinol (300 mg. every 8 hours) in cohorts of patients with metastatic prostate cancer. MATERIALS AND METHODS: The trial included 11 men with metastatic prostate cancer. Cohorts of patients received a 5-day constant infusion of 5-fluorouracil combined with subcutaneous interferon-alpha 2b 3 times weekly and allopurinol for 1 week during 5-fluorouracil infusion. Treatment was repeat every 3 weeks. RESULTS: Of 10 patients evaluable for treatment response and toxicity 3 had a partial response as judged by significant decreases in prostate specific antigen measurements (mean followup 13.5 months). Significant dose limiting toxicities encountered included mucositis, diarrhea and leukoneutropenia. CONCLUSIONS: Further evaluation of this treatment to determine overall response rates and benefit should take into consideration the significant toxicity experienced.
Pilot study of the acceptability of cystic fibrosis carrier testing during routine antenatal consultations in general practice.
BACKGROUND: In 1989, the gene for cystic fibrosis was cloned and it became possible to detect carriers of the gene among the general population, including pregnant women. AIM: The aim of the pilot study was to assess the acceptability of integrating cystic fibrosis carrier testing into antenatal care by general practitioners at the first booking appointment. METHOD: Between 1 September 1991 and 31 August 1992, inclusive, all patients receiving routine antenatal care in a two-partner training practice in south Manchester were offered carrier testing for cystic fibrosis using a computer protocol for antenatal care developed by the practice. A questionnaire including a Spielberger state-trait anxiety inventory was sent to patients 2 weeks after they received the results of their carrier test, and interviews with the patients in their home were carried out 4 weeks and one year after they received the result. RESULTS: All but one patient (75 out of 76) booking before 14 weeks of pregnancy accepted the offer of cystic fibrosis carrier testing, and 96% (72 out of 75) felt that they had made the right decision and that they had enough time for discussion with their general practitioner before testing. CONCLUSIONS: Cystic fibrosis carrier testing can be successfully integrated into the antenatal booking appointment in general practice and is acceptable to patients. This is a model for other genetic screening opportunities resulting from advances in medical genetics.
A clinical, genetic and audiological study of patients and families with unilateral vestibular schwannomas. I. Clinical features of neurofibromatosis in patients with unilateral vestibular schwannomas.
Ninety-three patients with unilateral vestibular schwannomas were examined in a clinical, genetic and audiological study, to determine whether they had features associated with neurofibromatosis Type 1 or neurofibromatosis Type 2. In 91 families, one patient only was found to be affected with a unilateral vestibular schwannoma. Patients did have a few café-au-lait macules, but fewer than six in number. None of the patients satisfied the cutaneous diagnostic criteria for neurofibromatosis Type 1. Neither Lisch nodules nor presenile posterior subcapsular lenticular opacities or cortical opacities were a feature. Five patients with unilateral vestibular schwannomas are described where the clinical findings raised the possibility of neurofibromatosis Type 2. It is suggested that certain individuals with unilateral vestibular schwannomas are at risk of developing neurofibromatosis Type 2. Furthermore, the possibility of neurofibromatosis Type 2 should be considered if more than one individual in a family is found to be affected with a unilateral vestibular schwannoma.
Comparative studies of recombinant human albumin and human serum albumin derived by blood fractionation.
Recombinant human albumin produced in the yeast Saccharomyces cerevisial and human serum albumin derived from blood fractionation were compared by a series of analytical techniques. These demonstrated that the two proteins were equivalent structurally. However, differences observed in some of the assays indicated that the recombinant product had lower levels of structural heterogeneity than the blood-derived protein.
Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.
Na+/H+ exchanger (NHE) activity is regulated by several types of receptors directly coupled to distinct classes (i.e. Gs, Gi, Gq, and G12) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G proteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four isoforms of the rat Na+/H+ exchanger were identified by molecular cloning. To examine their intrinsic responsiveness to G protein and second messenger stimulation, three of these isoforms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubation of cells with either AIF4-, a general agonist of G proteins, or cholera toxin, a selective activator of G alpha s that stimulates adenylate cyclase, accelerated the rates of amiloride-inhibitable 22Na+ influx mediated by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PKC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMPi on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMPi, elevation of cGMPi by treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.
Primary care for patients at genetic risk.
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