Biomedical subjects
Publications and source records attributed to N D Carter.
Stroke is a very common cause of death and disability. Several lines of evidence point to stroke as a polygenic multifactoral phenotype and there are also a number of well studied single gene Mendelian stroke conditions. Rat models have identified several chromosomal sites linked to stroke, and this together with other background information provides a strategy for both cross-sectional and gene scanning approach to defining the genetics of human stroke.
BACKGROUND: Impaired joint position sense (JPS) has been shown in anterior cruciate ligament (ACL) deficient and osteoarthritic knees. The relation between JPS and function is uncertain. The aim of this study was to determine further if ACL deficient knees show abnormal JPS and the effect of exercise therapy on JPS, and also to assess the relation between JPS, functional stability, and strength. METHODS: Fifty patients (46 men and four women, mean age 26.3 years) with unilateral ACL deficient knees were assessed on admission and after rehabilitation (5 hours a day for four weeks). JPS was assessed by reproduction of passive positioning using a visual analogue incorporating a goniometer. Knee stability was analysed by self report questionnaire (score 0-280) and functional activity test (single leg hop and figure of eight run). Isokinetic dynamometry was performed to evaluate quadriceps and hamstring peak torque strength. Controls were either age and sex matched individuals or the contralateral knee. Statistical analysis was by Wilcoxon signed rank test and Spearman rank order correlation coefficient. RESULTS: JPS was impaired in ACL deficient knees. The mean (SD) errors in reproducing angles were 9.4 (3.1) degrees and 7.1 (2.3) degrees for the ACL deficient knee and control knee respectively (P < 0.0005). There was no improvement in JPS after rehabilitation (9.4 (3.1) degrees and 8.5 (3.2) degrees before and after rehabilitation respectively, P = 0.14). There was improvement as ascertained from the questionnaire (on admission 202 (32.1), after rehabilitation 243 (25.4), P < 0.0001) and functional activity testing (hop: on admission 148.7 (37.3) cm, after rehabilitation 169.8 (31.1) cm, P < 0.0005; figure of eight: on admission 48.4 (16.6) seconds, after rehabilitation 41.6 (3.4) seconds, P < 0.0001). Quadriceps strength improved (peak torque on admission 198.5 (58.9) Nm, after rehabilitation 210.5 (54.2) Nm, P < 0.05), but not hamstring strength (peak torque on admission 130.6 (28.1) Nm, after rehabilitation 135.5 (27.7) Nm, P = 0.24). JPS did not correlate with the functional activity tests (hop and figure of eight run), the responses to the questionnaire, or strength. There was no correlation between the responses to the questionnaire and functional activity tests or muscle strength. CONCLUSIONS: JPS was impaired in ACL deficient knees. Although knee stability improved with exercise therapy, there was no improvement in JPS. The role of JPS in the stability of ACL deficient knees remains unclear.
Atherosclerotic renal artery disease is an important secondary cause of hypertension. Currently, there is great interest in possible genetic determinants of cardiovascular disease. The ACE-D allele has been reported to be associated with increased risk of myocardial infarction as well as coronary re-stenosis after angioplasty. We therefore assessed whether this allele is also linked to renovascular disease by studying 56 Caucasian subjects with atherosclerotic renal artery stenosis and 74 age, sex and race matched control subjects. Genetic analysis for the ACE I/D polymorphism was performed on peripheral leukocytes using PCR techniques, including insertion-specific primers. The distribution of I and D alleles was: renal artery stenosis 8 II, 25 ID, 23 DD; and controls, 16 II, 41 ID, 17 DD. The frequency of the D allele in the renal artery stenosis group was significantly higher (D/total 71/112 = 0.66) than that of the control population [75/148 = 0.51; chi 2 = 4.17, P = 0.04; odds ratio 1.69 (95% CI 1.02 to 2.78)]. Our results suggest that the ACE-D allele may be associated with increased risk of vascular disease at sites other than the coronary circulation.
Membrane-bound carbonic anhydrase (CA) has recently been identified in mammalian cardiac tissue. In this study, we have investigated the histochemical location and functional role of CA in the ferret heart. Heart sections stained by a modified Hansson's technique showed CA to be located on capillary endothelial membranes as well as on sarcolemmal membranes. In the Langendorff-perfused heart, washout of CO2 brought about by switching perfusion between 25 mM HCO3(-)-5% CO2-buffered solution and nominally HCO3(-)-CO2-free solution caused a transient rise in intracellular pH (pHi) measured by the chemical shift of 2-deoxy-D-glucose 6-phosphate with 31P nuclear magnetic resonance spectroscopy. The initial rate of change of pHi, measured over the first 60-75 s of CO2 efflux, was significantly reduced from 0.41 +/- 0.03 pH units/min (n = 9) in control hearts to 0.28 +/- 0.02 pH units/min (n = 5) in the presence of the membrane-permeable CA inhibitor 6-ethoxzolamide (P < 0.05 compared with control) and to 0.22 +/- 0.04 pH units/min (n = 5) in the presence of the membrane-impermeable CA inhibitor CL-11,366 (P < 0.01 compared with control). After reperfusion of the ischemic myocardium, both CA inhibitors caused a significant slowing of initial rate of change in pH (and initial rate of recovery of contractile function) compared with control hearts. These results suggest that CA, by facilitating the hydration-dehydration of CO2-H2CO3, alters the relative concentrations of CO2 inside and outside the cells, thus enhancing the rate of CO2 transfer from the intracellular to extracellular compartments, which contributes significantly to pHi recovery after reperfusion of the ischemic myocardium.
The objectives of this study were to assess relations between ACE gene I/D polymorphism, essential hypertension, plasma renin activity and aldosterone in white (European descent) and black (Afro-Caribbean descent) peoples. Measurements were carried out on a total of 320 subjects (210 white: 116 men, 94 women; 110 black: 65 men, 45 women); all were on their usual sodium intake; none was on anti-hypertensive therapy and none had secondary hypertension. Genomic DNA was isolated from blood cells and ACE I/D genotype was established using polymerase chain reaction. Plasma hormones were measured by radioimmunoassay and blood pressure (BP) with an ultrasound sphygmomanometer. All subjects were grouped into normotensive, borderline and hypertensive according to WHO guidelines. The distribution of the I/D genotype in the white people was approximately 1:2:1; by contrast, in the Afro-Caribbean people there was a significantly higher frequency of the D allele (chi 2P = 0.04). Within the white people there was no significant association between ACE genotype and high BP; however, within the black people there was a positive association between the frequency of the D allele and increasing BP ( chi 2 for trend P = 0.03). In either group, there were no associations between ACE I/D genotype and plasma renin activity and aldosterone suggesting that ACE genotype does not contribute to the expression of the circulating renin-angiotensin system. This study highlights differences in ACE I/D polymorphism between white and black peoples and suggests the possibility of racial differences in the association between ACE genotype and BP.
BACKGROUND: LysB28ProB29-human insulin (Humalog), a fully potent insulin analog in which the prolyl, lysyl sequence at the C-terminal end of the B-chain is inverted, exhibits a decreased association of monomers to dimers leading to rapid in vivo absorption. This provides important benefits for the insulin-requiring diabetic. In spite of its monomeric nature, LysB28ProB29-human insulin can exist as a discrete hexameric structure in the presence of both zinc and phenol. Studies of the crystal structure of LysB28ProB29-human insulin in a hexameric complex were initiated to gain a molecular understanding of the effect of the sequence inversion on the analog's self-association properties and, consequently, its in vivo efficacy. RESULTS: Under the conditions reported, LysB28ProB29-human insulin crystallized as a T3Rf3 hexamer that is isomorphous with the uncomplexed T3Rf3 native human insulin hexamer previously known as '4Zn insulin'. The three-dimensional structure of the T3Rf3 hexamer was determined by X-ray crystallographic methods to a resolution of 2.3 A. The prolyl, lysyl sequence inversion leads to local conformational changes at the C termini of the B-chains which eliminate two critical hydrophobic interactions and weaken two terminal beta-sheet hydrogen bonds that stabilize the dimer. CONCLUSIONS: The loss of these native dimer interactions weakens the hexameric LysB28ProB29-human insulin complex formed in the presence of phenolic ligands. Thus, it is hypothesized that the diffusion of the phenolic ligands from the site of injection results in the dissociation of hexamers directly to monomers, thereby maintaining the rapid time-action of the monomeric analog in spite of the hexameric conformation in therapeutic formulations.
BACKGROUND AND PURPOSE: A deletion (D)/insertion (I) polymorphism in the angiotensin-converting enzyme gene has been associated with myocardial infarction. Its relations to both stroke and atheroma remain uncertain. We examined its role as a risk factor in patients with cerebrovascular disease and its relation to carotid atheroma. METHODS: One hundred one patients with symptomatic carotid artery territory cerebral ischemia were compared with 137 age-matched control subjects. In the patient group, carotid atheroma was assessed by measurement of degree of carotid stenosis and intima-media thickness with high-resolution duplex ultrasound. The D/I polymorphism was examined using the polymerase chain reaction. RESULTS: D:I allele frequency was 0.59:0.41 in case subjects and 0.48:0.52 in control subjects (P = .01). The DD genotype was more common in patients with cerebrovascular disease compared with control subjects (36/101 versus 30/137, P = .02). The DD genotype conferred a relative risk of any type of cerebrovascular disease of 1.98 (95% confidence interval [CI], 1.11 to 3.51; P = .02). However, this was largely due to a strong association in the 18 patients with lacunar stroke, in whom the D:I ratio was 0.75:0.25 (P = .0097 versus control subjects). The odds ratio for lacunar stroke associated with the DD genotype was 5.6 (95% CI, 2.0 to 15.7) and was still significant at 4.40 (95% CI, 1.45 to 12.6; P < .009) after controlling for other risk factors. There was no significant association between angiotensin-converting enzyme genotype and cerebrovascular disease due to large-vessel stenosis. There was no association between genotype and age, sex, smoking history, diabetes, or cholesterol level. CONCLUSIONS: The deletion polymorphism in the angiotensin-converting enzyme gene is a new independent risk factor for lacunar stroke but is not a risk factor for stroke associated with carotid stenosis.
Insulin-like growth factor I (IGF-I) is thought to play a role in the development of proliferative diabetic retinopathy. Proliferative diabetic retinopathy is characterised by the formation of fibrovascular epiretinal membranes, and IGF-I may initiate and/or potentiate this epiretinal proliferation. To evaluate further the part played by IGF-I in the development of epiretinal tissue, we investigated the presence of IGF-I receptor/binding protein in proliferating diabetic fibrovascular epiretinal membranes. Five fibrovascular epiretinal membranes were obtained by vitrectomy from five patients with proliferating diabetic retinopathy. The presence of IGF-I receptors was investigated by autoradiography using 125I-labeled IGF-I on frozen sections. To characterise binding specificity, some sections were preincubated with either insulin or unlabeled IGF-I. Sections of post-mortem liver were used as controls. Strong labeling of cells with 125I-labeled IGF-I was observed in all epiretinal membranes and in liver cells. Almost no autoradiographic labeling was observed in sections that had been blocked with non-radioactive IGF-I, and very little labeling was found following blockage with insulin. Our preliminary study suggests the presence of IGF-I receptor/binding protein in human diabetic epiretinal membranes. These results support the hypothesis that IGF-I may be involved in the formation of proliferative diabetic membranes.
Sepsis induces a net catabolic state in gastrocnemius by increasing protein degradation and decreasing protein synthesis. To determine whether or not sepsis induces a preferential effect on the expression of individual proteins, proteins from gastrocnemius muscle of control and septic rats were separated by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laser densitometry of proteins stained with silver provided evidence that the relative abundance of thirty-five proteins was significantly (p < .05) and reproducibly increased during sepsis compared to control. No individual protein underwent significant down-regulation in their relative abundance during sepsis. Twenty-three of the 35 proteins identified in two-dimensional gels of the gastrocnemius were also present in the plasma of septic rats. The remaining 12 proteins, therefore, were taken to represent skeletal muscle proteins. One of the 12 proteins was identified by immunoblot analysis to be carbonic anhydrase III. Another of the proteins was identified as triosephosphate isomerase based upon microsequencing of the N terminus.
We examined the molecular forms of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) in endomyocardial right ventricular extracts from cardiac transplant recipients, using gel chromatographic separation coupled with specific radioimmunoassay techniques. Two distinct molecular forms of ANP and BNP were observed in the ventricular extracts consistent with the biologically active alpha-human peptides and the gamma-precursor peptides. Alpha-BNP was predominant and constituted 75 +/- 3% (n = 5) of the total immunoreactive BNP, while 21 +/- 2% (n = 5) of the total immunoreactivity comprised gamma-BNP. In contrast, gamma-ANP constituted 89 +/- 4% (n = 5) of the total immunoreactive ANP and was dominant over alpha-ANP, which made up 10 +/- 2% (n = 5) of the total immunoreactivity. There was no evidence for altered processing of BNP and ANP precursors in the right ventricular tissues from the de-nervated transplanted human heart. These findings provide further insight by identifying the molecular forms of BNP and ANP present in the transplanted human heart.
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) play a role in transepithelial fluid movement in the kidney and at the blood-aqueous barrier. We sought a similar natriuretic peptide-mediated regulatory system at the level of the blood-retinal barrier by investigating human neural retina and retinal pigment epithelium for the presence of ANP, BNP and their receptors. ANP and BNP binding to receptors could be demonstrated autoradiographically in all layers of the retina inclusive of the retinal pigment epithelium. Competitive preincubation with unlabeled peptides blocked the binding of the respective radioactive peptide. ANP and BNP could also be demonstrated immunohistochemically in both the neural retina and the retinal pigment epithelium. Our results suggest a role of these peptides both in the regulation of intraretinal fluid movement and--by analogy with other peptides--as possible neutrotransmittors. The localisation of ANP and BNP in the retinal pigment epithelium suggests that these peptides may influence ocular fluid homeostasis at the outer blood-retinal barrier by modulating pigment epithelial function.
OBJECTIVE: To determine the distribution of the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene in several ethnic groups: Caucasian Europeans, Black Nigerians, Samoan Polynesians and Yanomami Indians. RESULTS: The ratio of the frequencies of the II, ID and DD genotypes were 1:2:1 in the Europeans, but there was a tendency towards a higher frequency of the D allele in the Nigerians. In contrast, the Samoans and the Yanomami Indians displayed a much higher frequency of the I allele than of the D allele. CONCLUSION: The relationship between ACE genotype and disease in these latter groups is still not known, but the present results clearly suggest that ethnic origin should be carefully considered in the increasing number of studies on the association between I/D ACE genotype and disease aetiology.
We have previously developed a method for maintaining human cardiac explants in culture under serum-free conditions, for the assessment of cardiac endocrine function and myocardial growth factors. In order to assess the local role of dynorphin in the human heart, we studied the effects of dynorphin on the secretion of atrial natriuretic peptide and brain natriuretic peptide by human cardiac atrial explants. Dynorphin did not affect the basal secretion of brain natriuretic peptide, but clearly enhanced the release of atrial natriuretic peptide from the human cardiac explants in culture. The atrial content of brain natriuretic peptide was not significantly reduced, whereas the atrial content of atrial natriuretic peptide in cultured explants was reduced two-fold in the presence of dynorphin. These findings indicate that dynorphin may have a direct stimulatory effect on the release of atrial natriuretic peptide, but not brain natriuretic peptide, from human cardiac atria.
There is increasing evidence to suggest that the gene encoding angiotensin 1-converting enzyme (ACE) plays a significant part in cardiac disease risk. It is not clear if this risk is due to atherosclerotic factors in general or whether its influence is local to cardiac tissue. Previous studies have suggested that there is a genetic control of plasma ACE levels in normal subjects. We sought to see if such a relationship also existed amongst computer tomography (CT) proven cerebral infarction. Twenty-eight patients with stroke and 19 control subjects were grouped according to their ACE genotype and their plasma ACE levels were assessed. Although there is a trend for plasma levels to be associated with the variant ACE alleles there was a wide overlap. However, when compared with their respective alleles in control subjects there was no difference. We conclude that any future evaluation of stroke risk from the ACE gene will need to determine both plasma ACE level and genotype.
Numerous factors have been reported to influence the pathogenesis of stroke. The risks arising from variations in genes between individuals have not been explored. The angiotensin 1-converting enzyme gene is a candidate gene for atherosclerotic related disease. In this work we studied the frequency of the deletion allele D and the polymorphism DD within the gene encoding angiotensin 1-converting enzyme in stroke patients compared with controls to determine if this deletion polymorphism was a potential independent risk factor for stroke. We report a study of 100 patients with cerebral infarction along with 73 normal age and sex matched controls. Personal medical histories associated with high risk factors for stroke were documented. Cholesterol and triglyceride levels for each individual were also measured. Using polymerase chain reaction techniques patients were divided into the three angiotensin 1-converting enzyme genotypes (Deletion D and Insertion I) II, ID and DD. The D/I frequency in patients with stroke (0.57/0.43) was compared with controls (0.48/0.52). This was not statistically different (P > 0.05, chi-squared test). Although strokes did occur at a younger age in those with the DD genotype compared with those with the insertion allele, the results did not approach acceptable levels of significance (II/ID) (P > 0.05, Mann-Whitney U-test). There were no differences between the three genotypes and other well accepted risk factors for stroke.(ABSTRACT TRUNCATED AT 250 WORDS)
PURPOSE: Inhibition of carbonic anhydrase (CA) by acetazolamide causes a decrease in the standing potential of the retinal pigment epithelium (RPE) and an increase in the rate of subretinal fluid absorption, and it may improve cystoid macular edema. These effects are thought to be mediated by the RPE. Given the solubility coefficient of acetazolamide, the drug is most likely to act by direct inhibition of membrane-bound CA (CA IV). To identify a substrate for acetazolamide in the RPE, the distribution of CA activity and the isoform of CA in the RPE membrane were investigated. METHODS: Carbonic anhydrase activity was determined by Hansson's technique in fresh human eyes from donors of both sexes and different ages. The presence of the membrane-bound isoform CA IV was investigated immunohistochemically at the light and electron microscopic level, as well as by Western blotting in fresh RPE, and in adult and fetal RPE cultures. RESULTS: Hansson's histochemical method demonstrated CA activity on the apical and basolateral cell membrane of the RPE. Using the gamma-globulin fraction of a polyclonal antibody against pure CA IV, immunocytochemistry showed labeling for CA IV on the apical RPE membrane or morphologically polarized human adult and fetal RPE cultures. Gel electrophoresis and Western blotting demonstrated a major immunoreactive band at 55 kDa in homogenates, which was consistently reduced to approximately 35 kDa by incorporation of 0.1% Triton X-100 detergent. CONCLUSIONS: These results suggest that the clinical effects of carbonic anhydrase inhibitors on RPE function may be mediated via membrane-bound carbonic anhydrase activity in RPE and that CA IV is responsible for activity on the apical surface.
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The detection, isolation and properties of the restriction endonuclease TspEI are described. The canonical recognition sequence (AATT) is the same as the 4-bp core of the 6-bp sequence (GAATTC) of EcoRI. Hydrolysis occurs 5' to the palindromic tetramer so that TspEI produces the same cohesive termini as EcoRI. TspEI therefore has an obvious application in producing partial digests of DNA for ligation to EcoRI-digested cloning vectors.