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Biomedical subjects

C A Brown

Publications and source records attributed to C A Brown.

At least 19 recordsLinked to original sources
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

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↗
PubMed1990

Translation initiation in the HEXB gene encoding the beta-subunit of human beta-hexosaminidase.

The human lysosomal enzyme beta-hexosaminidase (EC 3.2.1.52) is a glycoprotein composed of dimers of alpha- and/or beta-subunits. The subunits of the enzymes are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the lysosome. As such, each subunit contains an amino-terminal signal peptide that directs the nascent polypeptide into the lumen of the endoplasmic reticulum. The signal peptide cleavage site of the beta-polypeptide is known, but its NH2 terminus has not been determined due to the presence of three candidate initiation codons upstream of the cleavage site. In this study, we identified the mRNA cap site, confirming the presence of all three AUGs in the majority of HEXB mRNA. To identify the site of translation initiation, we mutated the three ATGs by deletion and site-directed mutagenesis and showed that all three AUG codons can be used for translation initiation after expression in COS cells. Furthermore, in each case, a fully processed, i.e. mature lysosomal, and enzymatically active beta-hexosaminidase was produced indicating that a functional signal peptide was synthesized. However, expression of a frameshift mutation in the normal construct, created by insertion of a single nucleotide between the first and second ATG, resulted in no significant enzyme activity or beta-subunit protein. We conclude, therefore, that the first in-frame ATG is used exclusively in vivo, in keeping with the scanning model of eukaryotic translation initiation. Interestingly, substitution of all three ATGs with CTG resulted in a significant amount of mature beta-hexosaminidase, showing that under these conditions, initiation could occur from non-AUG codons. Translation initiation from the first AUG gives the prepro-beta-polypeptide a signal peptide of 42 amino acids that has an unusually long hydrophobic core more typical of membrane spanning domains. Such a large hydrophobic core has not been found in other cleavable signal peptides.

Amino Acid Sequence↗
PubMed1990

Suspected familial renal disease in chow chows.

Renal disease was diagnosed in 6 young Chow Chows. Clinical abnormalities included vomiting, polyuria, polydipsia, and weight loss. Common abnormal laboratory findings were azotemia, hyperphosphatemia, hypocalcemia, nonregenerative anemia, and low urine specific gravity. All 6 dogs had similar microscopic renal lesions. characterized by interstitial fibrosis, a population of small glomeruli, and lack of inflammatory cells. A familial basis for the renal disease is suggested because of its development in 4 closely related dogs.

Animals↗
PubMed1990

Mitogenic effects of transforming growth factor type e on epithelial and fibroblastic cells--comparison with other growth factors.

Transforming growth factor type-e (TGFe) is a novel TGF which was first described as a growth factor possibly involved in autocrine stimulation of anchorage-independent growth of carcinoma cells. Its later identification in normal tissues, plasma, and platelets suggested a role for TGFe in normal cell growth. This study shows that TGFe stimulates both anchorage-dependent and -independent growth of epithelial and fibroblastic cells of nonneoplastic origin. The mitogenic activity of TGFe in monolayer is slightly less than that of basic fibroblast growth factor, equipotent to that of epidermal growth factor, and greater than that of IGF-1. TGFe acts as a progression factor for both AKR-2B and Balb-3T3 cells. TGFe is also a potent mitogen for normal human epidermal keratinocytes and may therefore play a role in epidermal growth and regeneration.

Animals↗
PubMed1990

Partitioning of amino acids in lactating cows: oxidation to carbon dioxide.

The rate and extent of oxidation of 20 different amino acids has been evaluated as part of a study on partitioning of amino acids in normal lactating cows. Only four amino acids--glutamate, aspartate, alanine and glutamine--were extensively oxidized, and when expressed as a percentage of the injected dose oxidized during 3 h after intravenous injection, the value for these four amino acids was not significantly different from those obtained with the volatile fatty acids. The oxidation of all other amino acids was significantly slower, effectively prolonging their availability for protein synthesis. The pattern of oxidation among the amino acids supports the hypothesis that catabolized protein can provide a protracted source of anaplerotic precursors that augment the metabolic role of the tricarboxylic acid cycle and would have survival advantage in both acute and chronic exigencies. Glutamate and aspartate provide the earliest and largest influx to the tricarboxylic acid cycle, with alanine and, especially, glutamine appearing to serve as reservoirs of amino-nitrogen. Several dispensable amino acids are metabolized as slowly as the indispensable ones, indicating that metabolic conservation is not restricted to the latter group.

Amino Acids↗
PubMed1989

Introduction of the alpha subunit mutation associated with the B1 variant of Tay-Sachs disease into the beta subunit produces a beta-hexosaminidase B without catalytic activity.

Lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) occurs in two major isozyme forms, hexosaminidase A (alpha beta) and hexosaminidase B (beta beta). Although dimer formation is required for enzymatic activity, both subunits contain active sites which share many common substrates. However, the alpha subunit alone confers on hexosaminidase A the specificity for negatively charged substrates, e.g. GM2 ganglioside. Recently, a point mutation, producing a single amino acid substitution in the alpha subunit (Arg178-His), has been found to be associated with the B1 variant phenotype of Tay-Sachs disease (Ohno, K., and Suzuki, K. (1988) J. Neurochem. 50, 316-318). This variant is characterized by normal levels of hexosaminidase A as measured by a common artificial substrate, but an absence of activity toward alpha subunit-specific substrates. However, because of the presence of an active beta subunit in the mutant hexosaminidase A, it has not been possible to determine whether the affected alpha subunit has undergone a change in substrate specificity or become totally inactive. In order to define the full effect of the B1 mutation we have taken advantage of the common evolutionary origin of the genes coding for the alpha and beta subunits. Since the B1 mutation occurs in a region of extended identity between the two subunits, we have duplicated the Arg178-His mutation in a cDNA coding for the human beta subunit (Arg211-His). By expression of the mutant construct in monkey COS cells we have been able to examine the effect of this mutation on beta subunits which are capable of forming stable, active homodimers, an experiment that could not readily be accomplished with heterodimeric hexosaminidase A. Our data show that beta homodimers containing the Arg211-His substitution are formed and are transported into the lysosome in a manner identical to that of normal pro-hexosaminidase B. However, the mutant homodimers are processed at a slower rate and are less stable in the lysozyme. Their most striking feature was a total lack of normal hexosaminidase B activity. We conclude that while the effect of the Arg178-His substitution is not strictly limited to the active site, the severe B1 phenotype results from a totally inactive alpha-subunit in hexosaminidase A.

Amino Acid Sequence↗
PubMed1989

Membrane topology of mammalian cytochromes P-450 from liver endoplasmic reticulum. Determination by trypsinolysis of phenobarbital-treated microsomes.

We have studied the membrane topology of liver microsomal cytochromes P-450 derived from phenobarbital-treated rabbits via trypsinolysis of intact microsomes, recovery of solubilized peptide fragments by ultracentrifugation and liquid chromatography, primary structure determination by Edman microsequence analysis, and database searching to match isolated fragments with parent sequences. Relative to the primary structure of isozyme 2, the major phenobarbital-inducible form, fragments were isolated beginning at residues Glu86, Ile101, Arg126, Cys152, Leu198, Ser211, Asn237, Glu327, Asn385, Thr407, Phe408, Phe413, and Thr444. Such results show that this family of structurally related cytochromes is bound to the endoplasmic reticulum membrane by only one or two transmembrane segments, located at the NH2-terminal end of the polypeptide chain. The remainder of the protein, from residue approximately 50 to the COOH terminus must exist as a catalytic, heme-containing domain exposed on the cytosolic side of the membrane. Furthermore, our results indicate that the catalytic domain must be peripherally associated with the membrane surface. This would imply that substrates might have access to the active site of the cytochrome P-450 either by diffusion from the cytosol or from within the lipid bilayer.

Amino Acid Sequence↗
PubMed1989

A Canadian multicenter study with Zaditen (ketotifen) in the treatment of bronchial asthma in children aged 5 to 17 years.

One hundred thirty-eight children with chronic asthma, requiring daily treatment with bronchodilators, took part in a 7-month, double-blind, multicenter clinical study. Patients were randomized into two groups, and after a 1-month baseline, were administered Zaditen (ketotifen), 1.0 mg twice daily, or an identical placebo for a period of 6 months. After 10 weeks of receiving the study medication, bronchodilator use was reduced or stopped. In the Zaditen-treated group, 60% of the children taking theophylline were able to stop its use completely, compared to 34% of the patients taking placebo (p less than 0.05). Of the patients who were unable to stop taking theophylline, the Zaditen-and placebo-treated groups recorded average dosage reductions of 62% and 26%, respectively. These differences were statistically significant (p less than 0.05). Thus, a high percentage of patients in the placebo-treated group maintained asthma symptom control with theophylline, whereas most of the Zaditen-treated patients could stop using this medication. Although pulmonary function readings improved in both groups, those patients taking Zaditen demonstrated earlier improvement and greater changes from baseline. Significant differences (p less than 0.05) in favor of Zaditen were found for reduction of concomitant medications, patient's global evaluation, physician's clinical evaluations, incidence of emergency room visits for asthma, and upper respiratory tract infections. No unexpected side effects were observed. It is concluded that Zaditen is an effective medication for long-term control of asthma in children.

Adolescent↗