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C A Brown

Publications and source records attributed to C A Brown.

At least 145 records · Page 8Linked to original sources
PubMed1993

beta-Hexosaminidase isozymes from cells cotransfected with alpha and beta cDNA constructs: analysis of the alpha-subunit missense mutation associated with the adult form of Tay-Sachs disease.

In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the alpha-subunit of beta-hexosaminidase A (alpha beta) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of alpha-chain mutations is not straightforward. We examine three approaches utilizing previously identified mutations affecting alpha-chain folding. These involve transfection of (1) the alpha cDNA alone; (2) a beta cDNA construct encoding a beta-subunit substituted at a position homologous to that of the alpha-subunit, and (3) both alpha and beta cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an "active" alpha Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. We conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some alpha-chain mutations. Using this technique, we demonstrate that the adult-onset Tay-Sachs mutation, alpha Gly --> Ser269, does not directly affect alpha beta dimerization but exerts an indirect effect on the dimer through destabilizing the folded alpha-subunit at physiological temperatures. Two other alpha mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit.

Adult↗
PubMed1992

Characterization of two HEXB gene mutations in Argentinean patients with Sandhoff disease.

Beta-hexosaminidase A (beta-N-acetyl-D-hexosaminidase, EC 3.2.1.5.2) is a lysosomal hydrolase composed of an alpha- and a beta-subunit. It is responsible for the degradation of GM2 ganglioside. Mutations in the HEXB gene encoded beta-subunit cause a form of GM2 gangliosidosis known as Sandhoff disease. Although this is a rare disease in the general population, several geographically isolated groups have a high carrier frequency. Most notably, a 1 in 16-29 carrier frequency has been reported for an Argentinean population living in an area contained within a 375-km radius from Córdoba. Analysis of the genomic DNA of two patients from this region revealed that one was homozygous for a G to A substitution at the 5' donor splice site of intron 2. This mutation completely abolishes normal mRNA splicing. The other patient was a compared of the intron 2 G-->A substitution and a second allele due to a 4-bp deletion in exon 7. The beta-subunit mRNA of this allele is unstable, presumably as a result of an early stop codon introduced by the deletion. Two novel PCR-based assays were developed to detect these mutations. We suggest that one of these assays could be modified and used as a rapid screening procedure for 5' donor splice site defects in other genes. These results provide a further example of the genetic heterogeneity that can exist even in a small geographically isolated population.

Argentina↗
PubMed1992

Two small deletion mutations of the HEXB gene are present in DNA from a patient with infantile Sandhoff disease.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes hexosaminidase A (alpha beta) and B (beta beta). The alpha subunit is encoded by the HEXA gene and the beta subunit by HEXB gene. Defects in the alpha or beta subunits lead to Tay-Sachs or Sandhoff disease, respectively. While many HEXA gene mutations have been reported only three HEXB gene mutations are known. We report the characterization of two rare HEXB mutations present in genomic DNA from a single fibroblast cell line, GM203, taken from a patient with the infantile form of Sandhoff disease. The first is a single base pair deletion in exon 7 changing the codon for Gly-258, GGA, to GA and the second, a two base pair deletion in exon 11 changes the codons for Arg-435/Val-436, AGA/GTC, to AGTC. Each mutation produces a frame shift in the affected allele that results in a premature stop codon 17 or 20 codons downstream, respectively. These mutations also result in the inability to detect beta-mRNA by Northern blot analysis of total mRNA. These data are consistent with the idea that the severe infantile form of Tay-Sachs or Sandhoff disease is associated with a total lack of residual hexosaminidase A activity.

Amino Acid Sequence↗
PubMed1992

Fatal toxoplasmosis in domestic rabbits in the USA.

Three rabbits from two sources died after an acute illness characterized by fever, lethargy and diarrhea in one rabbit and no clinical signs in two rabbits. The most striking lesion in all three rabbits was foci of necrosis of the spleen and liver associated with massive presence of multiplying Toxoplasma gondii tachyzoites. The diagnosis was confirmed by specific staining with anti-T. gondii serum in an avidin-biotin complex immunohistochemical stain.

Animals↗
PubMed1992

Metamorphic changes in the vagal innervation to the alimentary tract in the green frog, Rana clamitans (Ranidae).

The alimentary tract of typical anurans shortens extensively at metamorphosis, when the microphagous tadpole transforms into a macrophagous frog. We used methylene blue vital stain and Sihler stain, in conjunction with microdissection, to map the distribution of the gastric branch of the vagus nerve in green frogs (Rana clamitans) before and after metamorphosis. After metamorphosis, terminal divisions of the gastric branch can be observed on the tunica musculosa extending as far as the pylorus. In the tadpole, the gastric branch ends abruptly on the esophagus. The premetamorphic pattern of innervation is consistent with the absence of digestive activity in the foregut before metamorphosis. Preliminary experiments, involving stimulation of the gastric branch of the vagus in the tadpoles, confirm the anatomical observation of the absence of vagal innervation before metamorphosis.

Animals↗
PubMed1992

Changes in linoleic acid during follicular development and inhibition of spontaneous breakdown of germinal vesicles in cumulus-free bovine oocytes.

The fatty-acid composition of follicular fluid from small and large developing follicles was analysed and the effects of saturated and unsaturated fatty acids on spontaneous breakdown of germinal vesicles were investigated. Fatty acids were bound to bovine serum albumin and cultured with oocytes at 100 mumol/l. Linoleic acid (18:2) was the only fatty acid tested that significantly inhibited breakdown of germinal vesicles (P less than 0.01). The effect was dose-dependent and was greatest at 50 mumol fatty acid/l (% breakdown of control, 81.1 +/- 6.8 vs. 50 mumol linoleic acid/l, 35.4 +/- 7.3; P less than 0.02). Linoleic acid was the major fatty acid, constituting about a third of the total fatty acid in the follicular fluid; followed by 18.9 +/- 1.0% and 16.9 +/- 1.3% oleic acid (18:1) in small and large follicles, respectively. Saturated fatty acids accounted for less than 30% of the total fatty acid composition. There was a marked absence of tetraenoic acids in small and large follicles. Proportions of linoleic acid were significantly lower in follicular fluid from large follicles (31.1 +/- 1.2% of total fatty acid) than from small follicles (34.8 +/- 0.7% of total fatty acid) (P less than 0.05) and there was a significant inverse correlation between follicle diameter and percentage of linoleic acid in the follicular fluid (r = -0.6966; P less than 0.05). There was no significant alteration in any other fatty acid during follicular development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Active arginine residues in beta-hexosaminidase. Identification through studies of the B1 variant of Tay-Sachs disease.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isoenzymes, hexosaminidases A (alpha beta) and B (beta beta). The alpha- and beta-subunits are encoded by the HEXA and HEXB genes, respectively. Extensive homology in both the gene structures and deduced primary sequences demonstrate their common evolutionary origin. Defects in the alpha- or beta-subunits lead to Tay-Sachs of Sandhoff disease, respectively. The B1 variant of Tay-Sachs disease is characterized by an unusual phenotype. Patient samples contain both isoenzymes; however, hexosaminidase A lacks activity toward alpha-specific substrates. In a previous report, we analyzed the biochemical consequences of an Arg178----His substitution in the alpha-subunit, causing the B1 phenotype, by in vitro mutagenesis of the homologous codon for Arg211 in the beta-subunit to produce His. We found that the substitution did not affect dimer formation or cellular targeting but caused a near total loss of activity toward a common alpha- and/or beta-substrate. Additional effects were also noted that suggested a perturbation had occurred to the protein's secondary structure. In this report, we investigate further the role of Arg in the catalysis of hexosaminidase substrates. The introduction of more or less conservative amino acid substitutions at the beta-Arg211 site were evaluated in terms of their effects on the protein's catalytic activity and susceptibility to the arginine-specific reagents and on its stability and rate of maturation in the cell's lysosome. These data demonstrate that the changes in the in vivo stability and rate of maturation, previously noted with the Arg211----His substitution, are independent of the loss in enzymatic activity. Whereas treatment of purified normal human placental hexosaminidases A and B with arginine-specific modifying reagents produced a time-dependent loss of enzymatic activity toward both alpha-specific and common substrates, these reagents failed to significantly decrease the residual activities of mutant proteins lacking Arg at position 211. Kinetic analysis of the residual enzyme activity from our most conservative construct, Arg211----Lys, determined an apparent Vmax approximately 400-fold reduced from that of the wild type enzyme but detected no change in the apparent Km. Additionally, the pH optimum of this mutant enzyme was narrower and slightly more basic than that of the normal enzyme. Thus, Arg211 in the beta-subunit and, by extrapolation, the Arg178 in the alpha-subunit of beta-hexosaminidase are "active" residues, i.e. part of the catalytic sites, but do not participate in substrate binding.

Arginine↗
PubMed1991

Duchenne's cardiomyopathy in a canine model: electrocardiographic and echocardiographic studies.

Thirteen dogs affected with X-linked Duchenne's muscular dystrophy and 11 female carrier dogs were studied by electrocardiography (ECG) and echocardiography. Twelve of the affected dogs were studied as immature animals and followed at 1 to 6 month intervals until they were 7 to 46 months of age. Compared with control dogs, affected dogs had significantly increased (p less than 0.02) Q/R ratios in ECG leads II, III, aVF, CV6LL (V2) and CV6LU (V4). Carrier dogs had significantly increased (p less than 0.02) Q/R ratios in leads V2 and V4. The Q/R ratio increased in three of six dogs followed up from age 6 months to greater than 2 years. The PR intervals were significantly shorter (p less than 0.02) in affected dogs. Ventricular arrhythmias were identified in four of six mature affected dogs. Two-dimensional echocardiography revealed distinctive hyperechoic lesions in 12 of the 13 affected dogs and in 6 of the 11 carrier dogs. Hyperechoic lesions corresponded to calcified myocardium and surrounding dense connective tissue. This study establishes the dog affected with Duchenne's muscular dystrophy as an animal model of Duchenne's cardiomyopathy and demonstrates that the heart in carrier dogs is affected by the dystrophic process.

Animals↗
PubMed1991

The drinking, passive smoking, smoking deception and serum cotinine in the Scottish Heart Health Study.

Following a recent claim that the use of cotinine in body fluids, to assess passive smoking and smoking "deception", was confounded by metabolic individuality, and by non-tobacco sources of dietary nicotine, particularly tea, data were examined from a large cross-sectional survey in a tea-drinking population. In 3383 men and women aged 40-59 years from the Scottish Heart Health Study, defined as non-smokers, both by self-report and by low thiocyanate and expired air carbon monoxide levels, serum cotinine showed minimal association with self-reported daily average tea consumption. However, there was a strong correlation between degree of self-reported passive tobacco smoke exposure and median serum cotinine level. In the same survey, serum cotinine in 4144 self-reported non-smokers and in 3326 smokers showed entirely different distributions, but the same range, suggesting heavy nicotine intake in some "non-smokers". These analyses confirm that cotinine levels in true non-smokers reflect far more the nicotine in inhaled ambient tobacco smoke than they do nicotine in tea. Some smoking "deceivers" have the same degree of exposure to nicotine as heavy smokers. Despite individual variability, the claim of confounding is poorly supported, and cotinine is confirmed as an indicator both of passive smoking and of smoking deception.

Adult↗
PubMed1991

Cigarette tar content and symptoms of chronic bronchitis: results of the Scottish Heart Health Study.

STUDY OBJECTIVE: The aim was to determine if there was a relationship between cigarette tar yield and rates of chronic cough and chronic phlegm. SETTING: 22 districts across Scotland were used for the Scottish Heart Health Study (SHHS) which was conducted between 1984 and 1986 and from which the data for this analysis were obtained. SUBJECTS: 10,359 men and women aged 40-59 years were studied. Of these, 2801 current cigarette smokers whose brand of cigarette smoked was known were selected. MEASUREMENTS AND MAIN RESULTS: Data on self reported smoking habits and prevalence of chronic cough and chronic phlegm were obtained from the SHHS. Tar yield was divided into three groups: low (less than or equal to 12 mg/cigarette); middle (13-14 mg/cigarette); high (greater than or equal to 15 mg/cigarette). The average tar yield consumed per person was 13.2 mg/cigarette. Women in the middle and high tar groups had smoked for longer and had significantly higher breath carbon monoxide levels, serum thiocyanate levels, serum cotinine levels, and daily cigarette consumption than the women in the low tar group. This pattern was not seen in men for any of these five smoking variables. Rates of chronic cough and chronic phlegm were higher with higher tar yield of cigarettes smoked for women (low tar v high tar: p less than 0.001) but not for men. Daily cigarette consumption and the number of years of smoking were the most significant risk factors for chronic cough and chronic phlegm for both men and women. Tar was still a significant risk factor (p less than 0.05) for women after controlling for these two risk factors and social class. CONCLUSIONS: Both sexes show strong effects of daily cigarette consumption and years of smoking on respiratory symptoms; women show an additional effect of cigarette tar content while men do not. The spread of tar yield in both sexes was small but there were more women on low tar cigarettes and this may have enabled a weak effect of tar to be seen better in them. On the other hand, tar level in women was confounded with other factors. Statistical methods of controlling for this may not have removed this confounding completely.

Adult↗
PubMed1991

The impact of quitting smoking on symptoms of chronic bronchitis: results of the Scottish Heart Health Study.

Scotland has high rates of death from diseases of the respiratory system and high rates of smoking, especially among women. Data on self reported smoking and prevalence of chronic cough and chronic phlegm among 10,359 men and women aged 40-59 years were obtained from the Scottish Heart Health Study. Overall, current cigarette smokers had rates of chronic cough and chronic phlegm four to five times those of never smokers after standardisation for age (32.3% v 6.5% for men and 24% v 5.5% for women for chronic cough; 31% v 8.3% for men and 21% v 5.5% for women for chronic phlegm). Ex-smokers' symptom rates were a little above those of never smokers and were significant for chronic cough among women and chronic phlegm among men. Men had higher symptom rates than women and this was true for smokers, ex-smokers, and never smokers. The higher rates among men could not be explained by higher cotinine concentrations. Tests to detect "deceivers" among ex-smokers and never smokers using biochemical validation suggested that 87 (1.5%) respondents were in fact smoking; they were excluded from analyses. There were substantially lower rates of chronic cough and chronic phlegm within a year of stopping smoking, and two to four years after stopping 89-99% of the difference between current smokers and never smokers was accounted for (99% and 93% for men and women with chronic cough, 96% and 89% for men and women with chronic phlegm). Even 10 years after stopping, rates of symptoms among ex-smokers remained a little above those of never smokers (except for women with chronic phlegm), though these differences were not statistically significant. Former heavy smokers continued to have rates of chronic cough and chronic phlegm that were higher than those of former light and moderate smokers (though not significantly so). These are cross sectional data, but they emphasise the importance for chronic bronchitis symptoms of giving up cigarette smoking, though the amount previously smoked continues to exert a small influence.

Adult↗