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Biomedical subjects

D B Ramsden

Publications and source records attributed to D B Ramsden.

At least 37 records · Page 2Linked to original sources
PubMed2000

Reduced hepatic content of dehydroepiandrosterone sulphotransferase in chronic liver diseases.

AIMS/BACKGROUND: Dehydroepiandrosterone sulphotransferase (DHEA ST) is the enzyme responsible for sulphation of lithocholic acid and other potentially hepatotoxic steroids. We have previously shown that DHEA ST activity is reduced in cytosol of liver from miscellaneous patients with chronic liver disease. The aim of this study was to investigate the cause of diminished sulphotransferase activity in order to further our understanding of whether a reduction in the ability to sulphate potentially hepatotoxic bile acids might play a role in the aetiology of primary cholestatic liver disease. METHODS: We quantified DHEA ST in human liver cytosol from groups of patients with chronic liver diseases and normal subjects using a semiquantitative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE)/ immunoblotting method, and an enzyme-linked immunosorbent assay (ELISA). We determined DHEA ST enzyme activity and correlated it with its immunoreactive concentration in 87 samples of human liver tissue. RESULTS: DHEA ST activity and concentration were significantly reduced in primary biliary cirrhosis, primary sclerosing cholangitis, chronic active hepatitis and alcoholic cirrhosis but not in cryptogenic cirrhosis when compared to normal liver. There were no significant differences among disease groups. In all groups enzyme activity and cellular concentration correlated, suggesting that no aberrant non-functional enzyme was produced. CONCLUSION: These results confirm that DHEA ST activity is diminished in liver disease and that the reduction is due to diminished enzyme presence. Further studies are required to show whether the reduction has any pathogenetic significance or is merely a consequence of disease.

Adult↗
PubMed1999

A study of five candidate genes in Parkinson's disease and related neurodegenerative disorders. European Study Group on Atypical Parkinsonism.

OBJECTIVE: To determine whether reported genetic association of polymorphisms in the CYP2D6, CYP1A1, N-acetyltransferase 2 (NAT2), DAT1, and glutathione s-transferase M1 (GSTM1) genes with PD were evident in a population of 176 unrelated patients with sporadic PD and to extend these findings to other disease groups (familial PD [n = 30], ALS [n = 50], multiple system atrophy [n = 38], progressive supranuclear palsy [n = 35], and AD [n = 23]). METHODS: A combination of allele-specific PCR and analysis of restriction fragment length polymorphisms were performed. RESULTS: We genotyped 1,131 individuals. After matching each patient with a control subject by age, sex, ethnicity, and geographic origin, there was no association of any allele/genotype with any of the six disease groups. There was an increased frequency of NAT2 slow acetylators in the ALS group compared with controls (70% versus 50%; OR 2.33 [95% CI, 1.03 to 5.30]), but this was not significant after adjusting for multiple testing. CONCLUSIONS: This is one of the most extensive candidate gene studies performed in PD and the first time that some of these loci have been studied in multiple system atrophy and progressive supranuclear palsy. In contrast with previous studies, we found no role for these polymorphisms in the etiology of PD, ALS, multiple system atrophy, progressive supranuclear palsy, or AD.

Aged↗
PubMed1999

Cytochrome P4502D6 (debrisoquine 4-hydroxylase) and Parkinson's disease in Chinese and Caucasians.

Four polymorphic sites (C/T188, C/T2938, G/C4268, G/A1934) in the cytochrome P4502D6 (debrisoquine 4-hydroxylase) gene were investigated for their association with sporadic Parkinson's disease (PD). Three mutant alleles (C/T188, C/T2938 and G/C4268) result in amino acid changes which could alter the substrate specificity or alter its ability to metabolize their substrates; the fourth (G/A1934) causes a loss of enzyme activity. The study was carried out in two ethnically homogenous populations: Chinese (123 PD patients, 124 controls); and Caucasian (95 PD patients, 62 controls). Haplotype status, which took into account amino acid changes at three polymorphic sites, was deduced from genotyping results in order to investigate whether substrate specificity was important rather than loss of enzyme activity. There was no gender difference in the distribution of the alleles in either race. There was, however, significant association among the three polymorphic sites (C/T188, C/T2938, G/C4268) in both ethnic groups. T/T188:C/C2938:C/C4268 is the most common genotype in the Chinese population, in contrast to C/C188:C/T2938:C/G4268 (followed by C/C188:C/C2938:G/G4268) in Caucasians. All 69 of the sub-group of Chinese patients tested were homozygous for the wild-type allele at the G/A1934 polymorphic site. Neither the CYP2D6 allele nor haplotype was associated with PD in either ethnic group.

Aged↗
PubMed1998

A direct correlation between nicotinamide N-methyltransferase activity and protein levels in human liver cytosol.

Phenotypic differences in nicotinamide N-methyltransferase (NNMT, E. C. 2.1.1.1) activity may be due to a genetic polymorphism. We report the characterisation of the hepatic NNMT activity in cytosol from normal human livers, enzyme protein levels determined by Western blotting and ELISA and mRNA levels determined by SDS-PAGE/Northern blotting. Subjects with high NNMT activity had high levels of NNMT protein and NNMT mRNA levels in hepatic cytosol and the converse was true for individuals with low NNMT activity. No differences in sequences were seen when cDNAs of individuals with high and low NNMT activity were compared. Thus phenotypic differences in the general population are due to differences in steady-state mRNA levels and not because of a polymorphism in the coding region of the NNMT gene.

Adolescent↗
PubMed1998

Cysteine dioxygenase: regional expression of activity in rat brain.

The levels of expression of cysteine dioxygenase (CDO) protein and activity were investigated in nine functionally distinct regions of the rat brain before and after induction with methionine by Western analysis and an activity assay. Activity expression ranged from no activity in the brain-stem to high activity expression in the olfactory bulb and basal ganglia. Upon exposure to 400 mg/l methionine for 5 days, significant induction of expression was observed in the basal ganglia, brain-stem, cerebellum, hippocampus, midbrain and olfactory bulb. Protein expression appeared to correlate with activity expression when levels before and after induction were compared. This non-uniformity of expression may reflect different physiological functions of CDO in these areas.

Animals↗
PubMed1998

Hepatic localisation of rat cysteine dioxygenase.

BACKGROUND/AIM: Cysteine dioxygenase (CDO, E.C. 1.13.11.20) is the main catabolic enzyme of cysteine, metabolising cysteine to cysteinesulphinic acid. CDO abnormality has been implicated in a number of neurological and non-neurological diseases, with CDO deficiency possibly leading to excitotoxic damage to the brain and impaired Phase II metabolism in the liver. METHODS: Two novel anti-CDO antibodies raised against linear synthetic peptides corresponding to two distinct epitopes on the 22 kDa gene product of the CDO-I gene were used for immunohistochemistry and Western blotting. These antibodies were characterised by their ability to both block and precipitate CDO enzyme activity as well as the ability of the respective antigenic peptides to absorb the antibodies and prevent the immunodetection of CDO. RESULTS: The antibodies were found to detect the presence of a 68 kDa protein, which was subsequently shown to be CDO. Distribution was found to be centrilobular and did not alter when CDO was induced with cysteine or methionine; however, the intensity of staining increased, indicating an increase in the levels of CDO in that region. CONCLUSIONS: These results suggest that the 68 kDa Type II is the predominant isoform in vitro and in vivo and that its centrilobular localisation may allow CDO to initiate the production of sulphate and taurine for Phase II conjugation in the liver.

Animals↗
PubMed1998

Cytochrome P450 1B1 mRNA in the human central nervous system.

AIMS: To study the expression of CYP1B1 in a variety of human and rat cell lines as a means of identifying a new tool for the investigation of gene regulation. In addition, to identify the expression of cytochrome P450 1B1 (CYP1B1) in different regions of the central nervous system (CNS). METHODS: Reverse transcription-polymerase chain reaction followed by cloning and sequencing were used to detect the expression of CYP1B1 in human cell lines. Poly A+ mRNA from the human spinal cord and from different brain regions was analysed using a CYP1B1 probe labelled with 32PdCTP. RESULTS: Expression of CYP1B1 was shown in a human astrocytoma cell line (MOG-G-CCM). CYP1B1 mRNA was expressed in a variety of regions of the CNS but with a distinct regional specificity. Expression was highest in the putamen. CONCLUSIONS: The expression of CYP1B1 in a human astrocytoma enables this cell line to be used in further studies of regulation and function of this gene. The demonstration that CYP1B1 mRNA is expressed in a variety of regions of the CNS suggests a role for this gene in brain and spinal cord metabolism. The regional specificity of expression might explain the focal damage of certain human neurodegenerative diseases.

Animals↗
PubMed1997

Human cysteine dioxygenase type I (CDO-I; EC 1.13.11.20): 5' flanking region and intron-exon structure of the gene.

AIM: The elucidation of the structure of the 5' flanking region and the exonic organisation of the human cysteine dioxygenase type I gene. METHODS: Material for sequence studies was generated by polymerase chain reaction (PCR) methods using human genomic DNA, a cosmid clone containing the entire gene, and a commercial gene walking kit. RESULTS AND CONCLUSIONS: The gene was found to be--12 kb in length and made up of five exons (418, 78, 155, 170, and 731 base pairs, respectively). The immediate 5' flanking region did not contain the canonical TATAA box. One DNA source (Clontech promoter finder kit) contained a liver specific nuclear factor response element approximately 550 base pairs from the transcription start site, whereas a second source did not. This and other cis acting factors in the 5' flanking region were identified by computer analysis. The physiological significance of such elements requires detailed experimental evaluation.

Base Sequence↗
PubMed1997

Deoxyribonucleic acid binding and transcriptional silencing by a truncated c-erbA beta 1 thyroid hormone receptor identified in a severely retarded patient with resistance to thyroid hormone.

We describe the analysis of a thyroid hormone receptor (TR) beta causing resistance to thyroid hormone, the patient exhibiting hypothyroid symptoms (severe mental retardation, hypoactivity, obesity) and hyperthyroid symptoms (tachycardia, low serum cholesterol) and, additionally, relative early puberty, advanced bone age, and short stature. The patient was heterozygous, with a point mutation producing a premature stop-codon in TR beta-gene exon 10, resulting in a 28-amino acid carboxy-terminal deletion in the cognate TR beta (TR beta-EZ). T3 binding was abolished. Homodimer binding of TR beta-EZ to DR4- and F2-T3 response elements (TREs) was weaker, and to a palindromic TRE (PAL) was stronger than that of wild-type TR beta (TR beta-WT) in the absence of T3. T3 dissociated TR beta-WT, but not TR beta-EZ homodimer, from DR4, F2, and Pal. Heterodimerization of TR beta-EZ with retinoid x receptor beta was seen. TR beta-EZ repressed basal thymidine kinase-promotor activity, coupled to DR4, F2, or PAL. Silencing of basal gene transcription via PAL was weaker, and via DR4 and F2 was more pronounced, compared with TR beta-WT. TR beta-EZ had a strong dominant negative effect on TR beta-WT, attenuated in a TRE- and cell-specific manner by high T3 concentrations. Finally, the degree of TR beta-EZ homodimer-binding affinity to DNA did not correlate with the degree of transcriptional dominant negative activity.

Adolescent↗
PubMed1996

Chromosomal localisation of genes coding for human and mouse liver cytosolic cysteine dioxygenase.

A panel of 22 hybrids was tested for the presence of the gene coding for human cysteine dioxygenase (CDO) by using human specific oligonucleotide primers in the polymerase chain reaction. Detection of human CDO completely correlated with the presence of human chromosome 5. A human total genome cosmid library was screened with a PCR product from the coding region of human CDO cDNA and the two positive clones identified were used in fluorescent in situ hybridisation (FISH) analysis on metaphase chromosome spreads. Fluorescent signals were seen on chromosome 5q22-23. Interspecific backcross mapping in the mouse indicated that Cdo, the mouse homologue of CDO, is situated in the central region of mouse chromosome 18 which shares a region of homology with human chromosome 5.

Animals↗
PubMed1996

Complete structural characterisation of the human aryl hydrocarbon receptor gene.

Aims-To clone and characterise the complete structural gene for the human aryl hydrocarbon receptor (AhR). This gene, located on chromosome 7, encodes a cytosolic receptor protein which, upon activation by various xenobiotic ligands, translocates to the nucleus, where it acts as a specific transcription factor.Methods-Primers, based on the AhR cDNA sequence, were used in conjunction with recently developed long range PCR techniques to amplify contiguous sections of the cognate gene. The amplicons produced were then cloned and characterised. A cDNA probe was also used to screen a human P1 library.Results-Using the cDNA primers, DNA fragments which mapped the entire coding region of the gene were amplified and cloned. All but one of these fragments were amplified directly from human genomic DNA. The remaining fragment was amplified using DNA prepared from a P1 clone as the PCR template. This P1 clone, obtained by screening a human P1 library, also contained the entire Ah locus. Characterisation of amplified and cloned DNA fragments provided sufficient information for the construction of a complete structural map of the gene. This also included 150 base pairs of nucleotide sequence data at all intronic termini.Conclusions-These data indicate that the human AhR gene is about 50 kilobases long and contains 11 exons. The overall intron/exon structure of the human gene is homologous to that of the previously characterised mouse gene; however, it is probably some 20 kilobases larger. These results demonstrate the need for further characterisation and provide the data to facilitate this.

Journal Article↗
PubMed1995

An allelic association study of monoamine oxidase B in Parkinson's disease.

A single-stranded conformational polymorphism in the monoamine oxidase B gene was shown to be A or G, 36 bases upstream from the intron 13-exon 14 boundary. An allelic association study revealed no statistically significant associations between this single-base polymorphism and Parkinson's disease, unlike the results of a previous study.

Adult↗
PubMed1995

Toxicity of sulphur-containing compounds to neuronal cell lines.

Cysteine and its metabolites cysteine sulphinic acid (CSA) and taurine (TA) were shown to be toxic to human and rat neuronal cell lines. However, the mechanisms of action of CSA and TA appeared to be different. As MND/ALS patients have high plasma and CSF cysteine levels, they may have increased levels of neurotoxins in vivo, since cysteine was more toxic than CSA in the assay system used. Cysteic acid, homocysteic acid, BMAA and BOAA were also toxic to the cell lines used.

Animals↗
PubMed1995

The effects of ethanol, 3,5,3'-triiodothyronine (T3) and oestradiol (E2) on thyroid hormone nuclear receptor expression in human hepatocyte primary cultures.

The possibility that ethanol regulated the expression of thyroid hormone receptor mRNA levels was explored in a model involving human hepatocyte primary culture. Ethanol (20, 50 and 100 mM for 24 h was found to have no effect on the steady-state levels of either triiodothyronine (T3) receptor alpha 1 or alpha 2 mRNAs. In contrast both T3 (1, 10 and 100 nM) and oestradiol (0.1, 1 and 10 ng/ml) treatments affected either one or both of these mRNA levels in a complex manner, showing that the model was capable of responding to other stimuli. Triiodothyronine receptor beta 1 mRNA was not assayed. The hypermetabolic effects of long-term ethanol consumption in humans appears not to be due to the direct effect of ethanol on the regulation of these receptors.

Culture Techniques↗
PubMed1994

Human cysteine dioxygenase type I: primary structure derived from base sequencing of cDNA.

The base sequence of cDNA encoding the complete human liver cysteine dioxygenase type I (CDO-I; EC 1.13.11.20) message, and the derived amino-acid sequence are reported. CDO-I is encoded on a single mRNA species (approx. 1.5 kb). Human CDO-I clones were identified by screening a liver cDNA library, and inserts were isolated and sequenced. In addition, human liver total RNA was reverse-transcribed and CDO-I cDNA amplified by PCR using a modified poly-T primer and specific CDO-I primers to give a second source of sequencing template. The CDO-I message encodes a 200 amino-acid residue protein, the sequence of which has greater than 90% homology with the equivalent rat enzyme. The message was not expressed in a human hepatoma cell line (Hep G2).

Amino Acid Sequence↗