Biomedical subjects
Publications and source records attributed to D B Ramsden.
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.
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.
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.
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).
We have shown previously that certain analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are potent inhibitors of human and bovine plasma benzylamine oxidase (BzAO: EC 1.4.3.6). Inhibition was competitive, reversible and allosteric. Under certain conditions competitive inhibitors of allosteric enzymes can act as allosteric activators. In the present work, 1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH3MPTP) was found to activate bovine plasma BzAO at low substrate and 2'-CH3MPTP concentrations. At higher 2'-CH3MPTP concentrations, the activation was negated.
Alcoholic liver disease is associated with abnormalities in circulating levels of thyroid, adrenal and gonadal steroid hormones. The relative importance of ethanol consumption and severity of liver disease in the aetiology of these changes and their relationship to clinical abnormalities are unclear. We studied 31 subjects with alcohol-induced liver disease divided into three groups according to the severity of histological features: fatty change, hepatitis and cirrhosis. Circulating concentrations of thyroid, adrenal and gonadal steroid hormones, together with their major binding proteins, were measured in all subjects, and changes related to histology and tests of liver function, as well as clinical endocrine status. A reduction in circulating free tri-iodothyronine (fT3) was seen in subjects with alcoholic hepatitis and cirrhosis, in association with normal or reduced levels of thyrotrophin (TSH). The absence of abnormalities in subjects with fatty change despite similar ethanol intake to the other groups, and correlations between fT3 and liver function tests, suggest that changes in fT3 reflect the severity of underlying liver disease. Similarly, marked increases in circulating cortisol in the hepatitis and cirrhosis groups, and correlations between cortisol and liver function, suggest that changes largely reflect hepatic disease. The absence of clinical features of hypothyroidism or Cushing's syndrome in these groups, despite abnormalities of fT3 and cortisol, suggest an altered tissue sensitivity to hormone effects. In contrast, increases in circulating oestradiol and reductions in testosterone were found in all three groups in males. These findings suggest that both direct effects of ethanol and hepatic dysfunction determine changes in gonadal steroids in males.
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Amplification and sequencing of the four transthyretin (TTR) exons of a subject with a variant TTR with four-fold increased affinity for thyroxine revealed a heterozygous G to A point mutation at base 7 of exon 2. This results in a serine for glycine change at residue 6 of the mature TTR monomer. No other mutations were found in any exon. Amplification and MspI digestion of TTR exon 2 from the leucocyte DNA of eight members of the subject's family revealed that all but one member were also heterozygous for [Ser6]-TTR.
Classification of familial amyloidosis by the chemical nature of the fibrillar protein has become possible. Most such amyloidogenic proteins so far recognized are variant transthyretins, but two kindreds with the same apolipoprotein AI modification have been reported. We describe the clinical features of another such family in whom petechial skin rash appeared to be a marker for the disease, which was non-neuropathic and of the Ostertag-type. Immunohistochemistry showed the protein to be apolipoprotein AI, but allele-specific DNA amplification indicated that it was not the Arg26 variant previously identified.
A sensitive assay for human plasma BzAO, involving the conversion of 14C-benzylamine to 14C-benzaldehyde, was developed. MPTP and several of its analogues were found to be competitive inhibitors of the enzyme. Ki values for the MPTP analogues in the presence of human plasma BzAO were determined. The analogues had a different rank order of inhibition of human plasma BzAO compared with the rank order of inhibition of bovine plasma BzAO found previously. MPTP and 1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH3-MPTP), which are potent nigrostriatal toxins, were weak inhibitors of human plasma BzAO.
The human thyroxine-binding prealbumin (TBPA) gene was examined for restriction fragment length polymorphism (RFLP) in normal subjects and a subject with euthyroid hyperthyroxinaemia, due to increased thyroxine binding by TBPA, using 16 restriction enzymes. Only Taq I and Msp I were shown to detect RFLPs. In a male of the normal population and one of his daughters, an additional Taq I site was found in the 3'-flanking region of the TBPA gene. The RFLP in a subject with euthyroid hyperthyroxinaemia was due to the deletion of a MspI site. All three subjects with RFLPs were heterozygous.
Thyroid and steroid hormones act by similar mechanisms to influence gene expression in the anterior pituitary gland. The genes encoding the common alpha and TSH-beta glycoprotein subunits are known to be regulated by thyroid hormones; we report here the effects of androgen administration on levels of alpha and TSH-beta mRNA in pituitary cytoplasm in the euthyroid and hypothyroid female rat. Dihydrotestosterone (DHT) suppressed both alpha and TSH-beta mRNAs to levels lower than those found in untreated animals; a similar reduction was seen in hypothyroid animals treated with DHT. A biphasic response of TSH-beta mRNA was seen following administration of tri-iodothyronine (T3) to hypothyroid rats, with early stimulation followed by later inhibition; these changes were also evident after administration of T3 to androgen-treated animals, although mRNA levels were again suppressed. The effects of testosterone were similar to those of DHT. In contrast to the changes in mRNA levels, androgen administration did not lead to significant alterations in serum TSH concentrations or pituitary TSH content. These results indicate that, like thyroid hormones, androgens suppress both alpha and TSH-beta subunit mRNA levels in the female rat. Androgens, however, exert differential effects on TSH synthesis and release which contrast with those of thyroid hormones.
To determine the relative importance of factors influencing tumour uptake of antibodies, we used a mathematical model to simulate intravenous injection of substances of varying molecular sizes and tumour-binding affinities at several dose levels. The FACSIMILE program was used to simulate the time course of tumour uptake of the tumour-binding substance by calculating the instantaneous tumour content (TC) and tumour:background uptake ratios (UR). Relative total doses to tumour and normal tissue were calculated by integration of TC/time curves. The model was used to make theoretical predictions on the effects of altering different parameters. The size of the injected dose in relation to the number of tumour receptors was crucial:if too low, uptake could not be improved by manipulating other variables, and if too high, the UR for large binding molecules was reduced. Using the standard scanning dose of labelled antibody, absolute numbers of labelled molecules binding to tumour could be increased by injection of a large excess of unlabelled molecules. Given an adequate dose, peak tumour content increased with increasing affinity up to receptor saturation. The peak uptake ratio rose progressively with affinity for a small ligand, but reached a relatively low plateau for antibody due to constant high background levels. At low doses such as those currently administered for diagnostic scanning with antibody, no effect of increasing affinity was predicted.
Thyroid hormones regulate the transcription of a number of genes in the anterior pituitary gland. A thyroid hormone response element in the regulatory region of the rat growth hormone gene has previously been shown to mediate the effects of thyroid hormones on growth hormone gene transcription. The 5' flanking regions of the thyrotrophin alpha and beta subunit and prolactin genes have now been examined for the presence of sequences similar to this response element. Southern blotting reveals that no such sequences are present in the regions of the thyrotrophin subunit and prolactin genes examined, suggesting that the thyroid response elements of these genes differ from that of the rat growth hormone gene.
A mathematical model has been developed to determine the best approach to improving tumor targeting with antibody. The amount of antibody in the tumor (tumor content) and the tumor:normal tissue antibody concentration ratio (uptake ratio) were calculated over 12 days from injection, using the computer program FACSIMILE to solve the stiff nonlinear differential equations describing the system. Results indicate that success requires an optimal combination of dose, size, and binding affinity of antibody. Increasing the dose to 100 times that presently used for scanning increased both the percentage of injected antibody in the tumor and the uptake ratio by up to 2 orders of magnitude to maximal values determined by affinity. This result could be achieved by coinjecting unlabeled antibody. Increasing affinity from Keq = 10(9) to 10(13)M-1 increased the uptake ratio from 5 to 100 for whole antibody and to 550 for a small ligand, at the calculated optimal dose, but had no effect at the current scanning dose. With decreasing molecular size at average affinity, the same maximum tumor content and uptake ratio were achieved but progressively earlier. At high affinity there was a substantial advantage for a small ligand compared with whole antibody in terms of uptake ratio (550 versus 100) and tumor:normal tissue integral dose ratio (330 versus 60). The uptake of a small ligand was not increased by binding to plasma protein but with increasing time the tumor content was higher than without protein binding.
Glucocorticoid hormones suppress circulating concentrations of thyrotrophin (TSH), but their effect on synthesis of TSH in the pituitary gland is unclear. We have examined the influence of the glucocorticoid dexamethasone on serum TSH, pituitary TSH content and TSH beta- and alpha-subunit mRNA concentrations in pituitary cytoplasm in both the euthyroid and hypothyroid rat, and following triiodothyronine (T3) treatment in the hypothyroid rat. The rise in serum TSH in hypothyroidism was attenuated in animals treated with dexamethasone; in addition the suppression of serum TSH 6 h after T3 administration to hypothyroid rats was enhanced by dexamethasone. In contrast to the changes in serum TSH, pituitary TSH content was unaffected by dexamethasone. Furthermore dexamethasone had no significant effect on changes in pituitary cytoplasmic TSH beta- and alpha-subunit mRNA levels with thyroid status. These findings demonstrate that dexamethasone exerts differential effects on serum TSH levels and TSH biosynthesis which contrast with those of thyroid hormones.
The possibility that oleic acid is the thyroxine binding inhibitor in the serum of seriously ill patients was investigated. 3H-Oleic acid was shown to bind directly to human thyroxine-binding globulin (TBG) by the techniques of one and two-dimensional immunoelectrophoresis in combination with autoradiography. However, no correlation was seen between serum thyroxine concentration and oleic acid concentration in two groups of patients, one of which underwent routine cholecystectomy, whilst the other group was admitted to an intensive therapy unit (mortality 75%). No correlation was seen between serum total thyroxine concentration and either stearic, palmitic, linoleic or arachidonic acid concentrations in these groups. Therefore, it was concluded that oleic acid was unlikely to be the circulating inhibitor of thyroxine binding.
Interactions between DNA sequences and nuclear proteins are important in the regulation of gene expression. We have applied a gel retardation method to examine the binding of nuclear proteins from the rat pituitary gland to regulatory sequences of the rat TSH beta-subunit gene. Binding between nuclear protein and DNA is demonstrated by an alteration in electrophoretic mobility of the DNA. A 0.4 M NaCl fraction of pituitary nuclear proteins bound to a fragment of DNA containing the promoter region of the TSH-beta gene, but not to plasmid DNA or insulin cDNA of comparable size. This nuclear protein fraction also bound to fragments of DNA containing sequences from the 5'-flanking region of the gene; binding of nuclear proteins was evident as far as 1 kb 5' to the structural gene. Each of these regions of the TSH-beta gene formed a number of distinct complexes with the nuclear protein extract, distinguished by their differing binding affinities in the presence of poly(dI-dC), and by their electrophoretic mobility. These findings suggest that transcriptional regulation of the rat TSH-beta gene may be mediated by interaction of a number of nuclear proteins with regulatory sequences of DNA up to 1 kb upstream of the transcriptional start site of the gene.