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Biomedical subjects

C A Fisher

Publications and source records attributed to C A Fisher.

At least 19 recordsLinked to original sources
PubMed2001

Genetic determinants of jaundice and gallstones in haemoglobin E beta thalassaemia.

Chronic hyperbilirubinaemia, gallstone formation, and gall bladder disease are unusually common in people with haemoglobin E beta thalassaemia in Sri Lanka. To determine whether this has a genetic basis we compared the bilirubin levels and frequency of gallstones in patients with different alleles of the UGT*1 gene. There was a significantly higher bilirubin level in those with the 7/7 genotypes compared with 6/6 and 6/7 genotype (p=0.032 and 0.0015 respectively), who also appeared more prone to gallstone formation. These results suggest that the UGT*1 genotpe is of importance in the genesis of gallstones in this population of patients.

Alleles↗
PubMed2001

Age-specific and age-adjusted penetrance as poison center outcome measures.

BACKGROUND: The American Association of Poison Control Centers defines penetrance as the number of exposures per 1000 residents of a population during one year. This metric fails to account for confounding by age group variability. We demonstrate the error caused by using raw penetrance and present two alternate methods of calculation, age-specific penetrance and age-adjusted penetrance. METHODS: Data from the toxic exposure surveillance system were collected from calls to our 41 county regional poison centers in 1998. Age-specific penetrance (ASP) was calculated by dividing the number of exposures (E) in age interval "i" in county "A" by 1000 population in age interval "Pi" in county "A" or ASP = Ei/Pi. AAP is the summation of the weighted age-specific penetrance. Weights (w(si)) represent the relative age distribution of a standard population, in this case the 1998 US population. AAP = sigma(i) w(si) * Ei/Pi. RESULTS: [table: see text] County C has low raw penetrance and is known to have relatively fewer toddlers and presumably a lower incidence of poisoning. This demonstrates that raw penetrance misrepresents populations with small proportions of children and should not be used to compare promotion or prevention activities between populations. CONCLUSION: We recommend poison centers and the American Association of Poison Control Centers replace raw penetrance with age-adjusted penetrance as one measure of the effectiveness of a poison center's awareness efforts.

Adolescent↗
PubMed2000

The lipid-associated conformation of the low density lipoprotein receptor binding domain of human apolipoprotein E.

Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130-150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1-183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation.

Apolipoproteins E↗
PubMed2000

Thalassaemia in Sri Lanka: implications for the future health burden of Asian populations. Sri Lanka Thalassaemia Study Group.

BACKGROUND: Thalassaemias pose an increasing problem for the Indian subcontinent and many Asian countries. We analysed the different types of thalassaemia in the Sri Lankan population, surveyed gene frequencies in schoolchildren, and estimated the burden of disease and requirements for its control. METHODS: We analysed blood samples from patients attending clinics in nine hospitals and defined the different types of beta thalassaemia by high-performance liquid chromatography (HPLC) and DNA analysis. The range of mutations was obtained by analysis of beta-globin genes. Capillary blood was obtained from schoolchildren from different parts of the island and analysed by HPLC to provide an approximate assessment of the carrier frequency of beta thalassaemia and haemoglobin E (HbE). To estimate the frequency of alpha thalassaemia the alpha-globin genotypes were also analysed when it was possible. FINDINGS: Blood samples were obtained from 703 patients with beta thalassaemia and from 1600 schoolchildren. The thalassaemia mutations were unevenly spread. Although 23 different beta-thalassaemia mutations were found, three accounted for the thalassaemia phenotype in about 70% of the patients, most whom are homozygotes or compound heterozygotes for IVS1-5 (G-->C) or IVS1-1 (G-->A). The third common mutation, codon 26 (G-->A), which produces HbE, interacts with one or other of these mutations to produce HbE/beta thalassaemia; this comprises 13.0-30.9% of cases in the main centres. Samples from 472 patients were analysed to determine the alpha-globin genotype. Overall, 15.5% patients were carriers for deletion forms of alpha+ thalassaemia. Average gene frequencies showed that there will be more than 2000 patients requiring treatment at any one time, in the future, of whom those with HbE/beta thalassaemia will account for about 40%. INTERPRETATION: In Sri Lanka, interactions of the two common beta-thalassaemia alleles will nearly always result in a transfusion-dependent disorder. However, about 40% of patients will have HbE/beta thalassaemia, which has a variable course. The management of these disorders could require about 5% of the total health budget. We need to learn more about the natural history and appropriate management of HbE/beta thalassaemia if resources are to be used effectively.

Adult↗
PubMed2000

Rapid detection of alpha-thalassaemia deletions and alpha-globin gene triplication by multiplex polymerase chain reactions.

We describe a sensitive, reliable and reproducible method, based on three multiplex PCR assays, for the rapid detection of seven common alpha-thalassaemia deletions and one alpha-globin gene triplication. The new assay detects the alpha0 deletions - -SEA, - (alpha)20.5, - -MED, - -FIL and - -THAI in the first multiplex PCR, the second multiplex detects the -alpha3.7 deletion and alphaalphaalphaanti3.7 variant, the third multiplex detects the -alpha4.2 deletion. This simple multiplex method should greatly facilitate the genetic screening and molecular diagnosis of these determinants in populations where alpha-thalassaemias are prevalent.

Gene Deletion↗
PubMed2000

Reducing time in bed after percutaneous transluminal coronary angioplasty (TIBS III).

BACKGROUND: This study is the third in a series of investigations on the requisite length of time that patients should be restricted to bed after coronary arteriography or percutaneous transluminal coronary angioplasty using a femoral artery approach. METHODS: A prospective, experimental-control group design with randomization was used initially to compare the incidence of bleeding between patients who remained in bed for 4 hours and patients who remained in bed for 6 hours after sheath removal following percutaneous transluminal coronary angioplasty. RESULTS: Rapid changes in the healthcare environment led to nurses collecting complete data sets for the experimental group only. The experimental group (n = 51) was 73% male and 27% female; mean age was 57 years (SD = 11.4 years). Mean time in bed was 4.1 hours (SD = 0.27 hours). Most patients (98%) did not bleed from the femoral artery access site after remaining in bed for 4 hours following sheath removal. Ninety-two percent of patients required analgesics while in bed. Mean length of stay after the angioplasty was 1.4 days (SD = 0.79 days). Bleeding occurred in one subject and was related to multiple invasive procedures and an activated clotting time of greater than 200 seconds. CONCLUSIONS: Requisite time in bed after percutaneous transluminal coronary angioplasty has been reduced to 4 hours at the University of Virginia Medical Center, the same time required for patients undergoing cardiac catheterization. Discomfort after the procedure remains to be addressed.

Angioplasty, Balloon, Coronary↗
PubMed2000

The relationship between heavy alcohol use and work productivity loss in active duty military personnel: a secondary analysis of the 1995 Department of Defense Worldwide Survey.

This cross-sectional study examines the association between heavy alcohol use among active duty military personnel and five work productivity loss events that may have an adverse effect on military performance and readiness. Data for light (N = 3,147) and heavy (N = 2,242) drinkers, categorized by gender and pay grade, were obtained from the 1995 Department of Defense Worldwide Survey. Drinking classification was predefined using a standard algorithm that factored quantity and frequency of wine, beer, and liquor consumed. The relative risks of experiencing a productivity loss event at a particular level and 95% confidence intervals were calculated by applying the Mantel-Haenszel method after adjusting for age. The relative risks for increased self-reported lateness, leaving early, low performance, and on-the-job injury were all higher for heavy drinkers than for light drinkers. This association between the heavy-drinking population and four of five work productivity loss events indicates that prevention programs should target all personnel.

Absenteeism↗
PubMed1999

Early fungal endocarditis in homograft recipients.

Retrospective analysis of 200 homograft valve recipients at our institution revealed two cases of fungal endocarditis. Pathogenesis appears to be related to either recipient seeding in one elderly immunocompromised patient or a previously contaminated donor valve implanted in an otherwise healthy recipient. Therefore, our experience underscores the need for both meticulous prevention of fungal infection preoperatively in the recipient and elimination of previously contaminated homograft valves from the donor pool.

Adult↗
PubMed1999

Human apolipoprotein E N-terminal domain displacement of apolipophorin III from insect low density lipophorin creates a receptor-competent hybrid lipoprotein.

The surface of Manduca sexta low density lipophorin (LDLp) particles was employed as a template to examine the relative lipid binding affinity of the 22 kDa receptor binding domain (residues 1-183) of human apolipoprotein E3 (apo E3). Isolated LDLp was incubated with exogenous apolipoprotein and, following re-isolation by density gradient ultracentrifugation, particle apolipoprotein content was determined. Incubation of recombinant human apo E3(1-183) with LDLp resulted in a saturable displacement of apolipophorin III (apo Lp-III) from the particle surface, creating a hybrid apo E3(1-183)-LDLp. Although subsequent incubation with excess exogenous apo Lp-III failed to reverse the process, human apolipoprotein A-I (apo A-I) effectively displaced apo E3(1-183) from the particle surface. We conclude that human apo E N-terminal domain possesses a higher intrinsic lipid binding affinity than apo Lp-III but has a lower affinity than human apo A-I. The apo E3(1-183)-LDLp hybrid was competent to bind to the low density lipoprotein receptor on cultured fibroblasts. The system described is useful for characterizing the relative lipid binding affinities of wild type and mutant exchangeable apolipoproteins and evaluation of their biological properties when associated with the surface of a spherical lipoprotein.

Animals↗
PubMed1999

Multicentric origin of hemochromatosis gene (HFE) mutations.

Genetic hemochromatosis (GH) is believed to be a disease restricted to those of European ancestry. In northwestern Europe, >80% of GH patients are homozygous for one mutation, the substitution of tyrosine for cysteine at position 282 (C282Y) in the unprocessed protein. In a proportion of GH patients, two mutations are present, C282Y and H63D. The clinical significance of this second mutation is such that it appears to predispose 1%-2% of compound heterozygotes to expression of the disease. The distribution of the two mutations differ, C282Y being limited to those of northwestern European ancestry and H63D being found at allele frequencies>5%, in Europe, in countries bordering the Mediterranean, in the Middle East, and in the Indian subcontinent. The C282Y mutation occurs on a haplotype that extends </=6 Mb, suggesting that this mutation has arisen during the past 2,000 years. The H63D mutation is older and does not occur on such a large extended haplotype, the haplotype in this case extending </=700 kb. Here we report the finding of the H63D and C282Y mutations on new haplotypes. In Sri Lanka we have found H63D on three new haplotypes and have found C282Y on one new haplotype, demonstrating that these mutations have arisen independently on this island. These results suggest that the HFE gene has been the subject of selection pressure. These selection pressures could be due to infectious diseases, environmental conditions, or other genetic disorders such as anemia.

Alleles↗
PubMed1999

The association of Hb Khartoum [beta124(H2)Pro-->Arg] with gamma+-thalassemia is responsible for hemolytic disease in the newborn of a Sudanese family.

The unstable Hb Khartoum with a Pro-->Arg replacement at position beta124 was identified by isoelectrofocusing, high performance liquid chromatography, and peptide mapping in a mother and two male children of a Sudanese family. All three were heterozygous for the abnormal hemoglobin; the father and a third male child did not carry the mutation. The mother was also homozygous for two putative gamma+-thalassemia point mutations, one affecting both Agamma and Ggamma genes at IVS-II-115 (A-->G), and one affecting the Ggamma gene at the 3' untranslated region (-A) at position -6 from the polyadenylation site. The father had normal gamma genes. All three children were heterozygous for both the gamma+-thalassemia mutations. The two older children, who were compound heterozygotes for Hb Khartoum/gamma+-thalassemia, presented at birth with severe neonatal jaundice which necessitated exchange blood transfusions. Other causes of neonatal jaundice were excluded. The third male child, who did not carry the Hb Khartoum anomaly but was heterozygous for gamma+-thalassemia, did not develop neonatal jaundice. It is concluded that the instability of Hb Khartoum in combination with gamma+-thalassemia is responsible for neonatal hemolytic anemia in this family.

Adult↗
PubMed1999

Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E.

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.

Apolipoprotein E3↗
PubMed1998

The low density lipoprotein receptor active conformation of apolipoprotein E. Helix organization in n-terminal domain-phospholipid disc particles.

Lipid association is a prerequisite for receptor interactions of apolipoprotein E (apoE). Disc complexes of the N-terminal 22-kDa apoE3 receptor binding domain and dimyristoylphosphatidylcholine display full receptor binding activity. Studies have been performed to characterize conformational adaptations of the globular, lipid-free four-helix bundle structure that culminate in stable association of its amphipathic alpha-helices with a lipid surface. Helix-lipid interactions in bilayer disc complexes can conceivably adopt two orientations: parallel or perpendicular to the phospholipid acyl chains. Evidence based on infrared dichroism, geometrical arguments, and x-ray crystallography support the view that defined helical segments in the four-helix bundle realign upon lipid association, orienting perpendicular to the phospholipid fatty acyl chains, circumscribing the bilayer disc. Thus, it is likely that paired helical segments align in tandem, presenting a convex receptor-active surface.

Apolipoprotein E3↗
PubMed1997

Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.

The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single quantum correlation NMR spectroscopy. The data revealed that rApoE3(1-183) is an excellent candidate for solution structure studies by NMR, including conformational adaptations associated with lipid association.

Apolipoprotein E3↗
PubMed1996

Increased activation of the coagulation and fibrinolytic systems leads to hemorrhagic complications during left ventricular assist implantation.

BACKGROUND: Left ventricular assist devices (LVADs) have provided a new therapeutic option for patients with end-stage heart failure. Despite advances in device design, there remains an apparent bleeding diathesis, which leads to increased transfusion requirements and reoperative rates. The purpose of our study was to examine the abnormalities that might contribute to these clinical sequelae. METHODS AND RESULTS: To separate the effects of cardiopulmonary bypass (CPB), eight patients undergoing coronary revascularization (CABG) were compared with seven LVAD (TCI HeartMate) recipients intraoperatively and 2 hours postoperatively. We evaluated several well-characterized indexes of platelet activation: platelet count, platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), and thromboxane B2 (TXB2). We also measured activation of thrombin: thrombin-antithrombin III (TAT), prothrombin fragment 1 + 2 (F1 + 2), and fibrinopeptide A (FPA) as well as markers of fibrinolysis: plasmin-alpha 2-antiplasmin (PAP) and D-dimer. Patterns of intraoperative platelet adhesion and activation were not statistically different in the CABG control and LVAD groups. In the immediate postoperative period, however, there was significant release of PF4 and beta-TG and generation of TXB2. Compared with the CABG controls (TAT, 26 +/- 8 micrograms/L; F1 + 2, 4 +/- 1 nmol/L; mean +/- SEM), there was a significant increase in TAT (380 +/- 112 micrograms/L) and F1 + 2 (23 +/- 4 nmol/L) in LVAD patients 2 hours after surgery. Furthermore, a sharp rise in FPA was noted 20 minutes after LVAD initiation (CABG, 8 +/- 4 ng/mL; LVAD, 235 +/- 63 ng/mL; P < .05). A concomitant increase in both PAP (CABG, 987 +/- 129 micrograms/L; LVAD 3456 +/- 721 micrograms/L; P < .05) and D-dimer (CABG, 1678 +/- 416 ng/mL; LVAD, 15243 +/- 4682 ng/mL; P < .05) was observed. CONCLUSIONS: The additive effects of CPB and LVAD lead to platelet activation as well as elevation of markers of in vivo thrombin generation, fibrinogen cleavage, and fibrinolytic activity. The etiology of these findings may be secondary to the LVAD surface, flow characteristics, and/or operative procedure. Nevertheless, platelet alterations and exaggerated activation of the coagulation and fibrinolytic systems may contribute to the clinically observed hemostatic defect.

Adolescent↗