Biomedical subjects

A J Clark

Publications and source records attributed to A J Clark.

At least 145 records · Page 8Linked to original sources

PubMed1995

Cloning, characterization and expression of a functional mouse ACTH receptor.

A polymerase chain reaction-generated mouse ACTH-R probe was used to screen a mouse genomic library, and a clone of 13kb containing the entire coding sequence was isolated. The coding sequence shows 84% homology with the human gene at the DNA level and encodes a peptide with 89% homology to the human ACTH-R. This gene is expressed as a major transcript of 1.8kb in the mouse adrenal gland. The gene was expressed in HeLa cells and cAMP production in response to either ACTH or alpha-MSH was measured. cAMP increased in an ACTH dose dependent manner suggesting an EC50 of 7 x 10(-10)M ACTH. alpha-MSH was without effect on this receptor. In conclusion we have cloned a mouse ACTH receptor gene and demonstrated for the first time its expression and functional effect in HeLa cells.

Amino Acid Sequence↗

PubMed1995

recO and recR mutations delay induction of the SOS response in Escherichia coli.

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage: therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of beta-galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.

Bacterial Proteins↗

PubMed1995

A DNA variant at the angiotensin-converting enzyme gene locus associates with coronary artery disease in the Caerphilly Heart Study.

BACKGROUND: We analyzed an insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene in 1226 subjects from the Caerphilly Prospective Heart Disease Study. Amplification of genomic DNA using the polymerase chain reaction yielded the genotypes II, ID, and DD. Distribution of the polymorphism was analyzed among the whole group and within subgroups (specified following multiple risk factor analysis) for coronary artery disease (CAD) and against multiple risk factors. METHODS AND RESULTS: Allele frequencies were I = 0.413 and D = 0.587. No association was observed between the polymorphism and CAD in the whole group. Among subjects defined at lower risk of CAD by total cholesterol/HDL cholesterol (TC/HDL) ratios, we found significant associations of the DD genotype with CAD (P < .0053, n = 586 for TC/HDL < 5.654 [median] and P < .009, n = 385 for TC/HDL < 5.0 [clinical threshold]). On further exclusion of subjects with blood pressures > or = 140/90 or on hypotensive medications, the DD genotype still associated with CAD (P < .07, n = 210, TC/HDL < 5.654 and P < .016, n = 135, TC/HDL < 5.0). Further stratification of risk incorporating other risk factors, except body mass index, did not alter or enhance this association. Although similar association was observed when risk was specified by using HDL and apo B levels instead of TC/HDL, this association was lost when body mass index was included in the low-risk stratification. CONCLUSIONS: The DD genotype is a linkage marker for an etiologic mutation at or near the ACE gene that may confer risk of CAD detectable in subjects previously unidentifiable with "classic" risk factors. However, this risk may be quantitatively small among the general male population.

Alleles↗

PubMed1995

Epithelial proliferation and differentiation in the mammary gland do not correlate with cFABP gene expression during early pregnancy.

Cardiac fatty acid binding protein (cFABP) is abundantly expressed in the nondividing, functionally differentiated mammary epithelium. It is very closely related, if not identical to, a previously described protein termed mammary derived growth inhibitor (MDGI). In vitro studies suggest that low concentrations of diffusible cFABP/MDGI may play a hormone-like role in limiting proliferative activity and promoting functional differentiation of this tissue, but no in vivo data to support this idea have been published. To test this hypothesis, we compared the levels of cFABP mRNA with both the epithelial DNA labelling index and levels of beta-casein mRNA in wild-type mice. We also investigated the effect of a precocious experimental increase of cFABP levels in the mammary gland of transgenic mice on the labelling index and beta-casein mRNA levels. This was accomplished by expressing a bovine cFABP cDNA under the control of the ovine beta-lactoglobulin (BLG) gene promoter. We found that although both the DNA labelling index, beta-casein mRNA levels, and cFABP mRNA levels in wild-type mice are developmentally regulated, they do not correlate with each other during early pregnancy in individual mice. Moreover, a three- to fourfold increase of total cFABP mRNA in two transgenic lines did not affect the DNA labelling index or the levels of beta-casein mRNA, an established marker of differentiation of the mammary epithelium, at this developmental stage. These data suggest that epithelial DNA synthesis, beta-casein gene expression, and expression of the cFABP gene are regulated independently in the proliferatively active mammary gland and that the rapidly dividing mammary epithelial cells are not susceptible to the action of cFABP during early pregnancy.

Animals↗

PubMed1995

Restricted tissue-specific but correct developmental expression mediated by a short human alpha 1AT promoter fragment in transgenic mice.

The tissue-specific and developmental pattern of expression controlled by the proximal promoter (position-348 to +15) derived from the human alpha-1-antitrypsin (h alpha 1AT) gene was studied in transgenic mice. The short promoter segment was linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The transgene showed highly specific expression in the liver and the correct developmental pattern of regulation. Interestingly, this short promoter targets expression to the liver with a greater specificity than that reported for larger alpha 1AT promoter fragments.

Animals↗

PubMed1995

Are activating mutations of the adrenocorticotropin receptor involved in adrenal cortical neoplasia?

The objective of this study was to investigate the presence of activating mutations of the ACTH receptor gene in benign and malignant adrenocortical tumours. Genomic DNA was extracted from a variety of adrenocortical neoplasms from 16 patients and the entire ACTH receptor gene was then amplified by the polymerase chain reaction and sequenced. No mutations were detected within the coding region of the ACTH receptor gene in the 16 adrenocortical tumours studied. It is likely that mutations of the coding region of the ACTH receptor gene do not play a major role in adrenocortical tumourigenesis.

Adenoma↗

PubMed1995

The proximal milk protein binding factor binding site is required for the prolactin responsiveness of the sheep beta-lactoglobulin promoter in Chinese hamster ovary cells.

To identify cis-acting prolactin (PRL) response elements within the sheep beta-lactoglobulin (BLG) promoter, CHO cells were co-transfected with a rabbit PRL-receptor (PRL-R) expression plasmid and a number of BLG-CAT constructs. Resection through the 4200 bp BLG promoter diminished the PRL response. Mutation of the proximal binding site for milk protein binding factor (MPBF), a previously described mammary gland transcription factor, abolished the PRL inducibility of full length and shorter forms of the promoter. MPBF was shown to be similar to the Stat protein mammary gland factor (MGF) which has been shown to mediate PRL responsiveness of the rat beta-casein gene in mammary cells. MPBF binding activity was detected in the nucleus of CHO cells and was increased 2-6-fold in cells stably transfected with the PRL-R. The lactating mammary gland has high levels of MPBF binding activity and it is likely that this has an important role in the PRL induction of a variety of milk protein genes.

Animals↗

PubMed1995

The molecular pathology of pituitary hormone deficiency and resistance.

In this chapter, we have reviewed the fast-moving area of the molecular pathology of pituitary hormone deficiencies and resistance. Examples have been described affecting all levels of pituitary function, i.e. the releasing hormone, its receptor, the pituitary hormone and its receptor, and the development of the pituitary gland. Other examples in these genes, and in those in which no mutation has yet been found, will undoubtedly be discovered in the next few years, throwing light on the structural basis of the gene product's function and allowing a greater understanding of endocrine physiology and pathophysiology. The main reason for this rapid progress in knowledge is the recent technological advances in mutation detection, which bring this activity within the grasp of the majority of reasonably equipped laboratories. Technological advancement, however is not all that it takes to carry out this work. The conditions caused by genetic damage such as we have described are rare, and there is clearly a requirement for great awareness on the part of the clinical endocrinologist. Patients in whom it is suspected that mutations such as these may occur require careful clinical and biochemical work-up. Indeed, in many instances, careful thought has to go into deciding what the phenotype of a particular mutation might be. Thus, the requirement for close collaboration between clinical and molecular endocrinologists has to be the important message for the future in this area of research.

Adrenocorticotropic Hormone↗

PubMed1995

Hormone-dependent expression of the ovine beta-lactoglobulin gene.

The minimal hormonal requirements for inducing the ovine beta-lactoglobulin gene have been investigated using mammary gland explants from ewes in the first half of pregnancy. Quantification of beta-lactoglobulin mRNA showed that a combination of insulin, cortisol and prolactin was required to stimulate the expression of the gene and that this response could not be enhanced by the addition of oestrogen and thyroid hormone to the culture medium. Explants cultured in the presence of insulin, cortisol and prolactin also demonstrated the capacity to synthesize the protein. Progesterone did not inhibit the induction of the gene, which is consistent with the increase in beta-lactoglobulin mRNA observed in vivo in the mammary gland during the final 2 months of pregnancy when the circulating level of progesterone is elevated.

Animals↗

PubMed1995

Sleep disturbance in mid-life women.

A correlational field survey was used to describe sleep disturbance and explore the relationship of that sleep disturbance to the psychological and menopausal status of 23 mid-life women. Subjects were 40-55 years of age and had self-reported sleep problems. Instruments included the sleep disturbance questionnaire (SDQ), Centre for Epidemiologic Studies depression scale (CES-D), Speilberger state-trait anxiety scale (STAI), a demographic form, 4 cc blood sample for follicle stimulating hormone analysis, and a 7-night sleep diary ('self report of sleep', or SRS). Ten of the 23 subjects reported periodic limb movement on the SDQ. Sleep diaries (SRS) revealed a characteristic sleep disturbance among 13 of the 23 subjects, which paralleled in some respects the changes in sleep efficiency observed with advancing age, but was more pronounced and was present at an earlier age. There was no statistically significant relationship between sleep disturbance and menopausal status or symptoms, or anxiety and depression among subjects, as shown by correlational coefficient and chi-square statistical tests. Results suggest that a characteristic sleep disorder accompanied by periodic limb movement may be responsible for sleep disturbance in mid-life women. What remains unclear is whether this sleep disturbance is related to aspects of the perimenopause not addressed in this study or to other aspects of ageing.

Adult↗

PubMed1995

Absence of mutations in the MEN2A region of the ret proto-oncogene in non-MEN 2A phaeochromocytomas.

OBJECTIVE: To determine the presence of abnormalities of the MEN2A region of the ret proto-oncogene in phaeochromocytomas/paragangliomas (PHAEO) of different aetiologies. DESIGN: Total RNA was extracted from tumours and used as templates for reverse transcriptase polymerase chain reactions. A ret primer pair, which encompasses the region which is mutated in the germ-line of patients with MEN 2A, was used. The resulting 262-bp product was sequenced. PATIENTS: Ten PHAEOs were examined. Four tumours were from von Hippel-Lindau disease patients; five were sporadic, isolated tumours; one from a patient with multiple endocrine neoplasia type 2A (MEN 2A). The medullary thyroid cancer from the single MEN 2A patient was also examined. RESULTS: A heterozygous TGC to CGC mutation of codon 634 (cysteine to arginine) was found in the PHAEO and medullary thyroid cancer from the MEN 2A patient. The 262-bp ret fragment was not found in two tumours (one malignant PHAEO and one secretory paraganglioma), although the intra-cellular ret tyrosine kinase domain was detected in these tumours. The cysteine codons were normal in all other non-MEN 2A PHAEOs. CONCLUSION: Mutations of key cysteine codons of the ret proto-oncogene may be specific to MEN 2A.

Adrenal Gland Neoplasms↗

PubMed1995

Linkage of the angiotensinogen gene locus to human essential hypertension in African Caribbeans.

The renin-angiotensin system regulates blood pressure and sodium balance. The angiotensinogen gene which encodes the key substrate within this system has been linked to essential hypertension in White Europeans. It has been suggested that people of West African ancestry may have a different genetic basis for hypertension. In this study we have tested whether there is linkage of the angiotensinogen gene to essential hypertension in African Caribbeans from St. Vincent and the Grenadines. DNA from 63 affected sibling pairs with hypertension was tested for linkage by analyzing whether there was excess allele sharing among siblings genotyped using an angiotensinogen dinucleotide repeat sequence. There was significant support for linkage (T = 3.07, P = 0.001) and association of this locus to hypertension (chi 2 = 50.2, 12 degrees of freedom, P << 0.001). A DNA polymorphism which alters methionine to threonine at position 235 (M235T) within the angiotensinogen peptide has been associated previously with hypertension. However, we found no association of this variant with hypertension in this study. These findings provide support for linkage and association of the angiotensinogen locus to hypertension in African Caribbeans and suggest some similarities in the genetic basis of essential hypertension in populations of different ethnicity.

Adult↗

PubMed1995

An alternatively spliced human insulin-like growth factor-I transcript with hepatic tissue expression that diverts away from the mitogenic IBE1 peptide.

An alternatively spliced transcript of the human insulin-like growth factor-I (IGF-I) gene is described. The transcript was identified in human liver RNA by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. It contained IGF-I exons 3 and 4, 49 basepairs of exon 5, then exon 6 (exon 4-5-6). The 5'-donor site at the exon 5-6 junction was a cryptic 5'-donor splice site (IGF633). The 3'-acceptor site of the splice was the usual intron-exon 6 junction. A second pair of primers across the exon 5-exon 6 junction was used to confirm the presence of the transcript by reverse transcriptase-polymerase chain reaction. Cloning and sequencing this second fragment confirmed the presence of this splice in human liver. The exon 4-5-6 transcript was quantified at about 10% relative to the exon 4-6 transcript in human livers (n = 7 subjects), but was not detected in other tissues. The exon 4-5-6 transcript was found in cultured human hepatoma HepG2 cells and increased, relative to exon 4-6 transcripts, in response to GH, but not in cultured human lymphoblast IM-9 cells. The exon 4-5-6 splice predicts a prepro-IGF-I of 158 amino acid residues, with an E-peptide sequence of 24 residues (Ec). The deduced Ec peptide sequence is 73% homologous to the rat Eb-peptide sequence. The predicted final residues of the Ec peptide are frameshifted exon 6 codons ending in an in-frame stop codon. The predicted peptide sequences of Ec and Eb differ at the cleavage site of the Eb-peptide fragment (IBE1), which has been shown to have mitogenic activity. These data suggest that 1) the exon 4-5-6 splice has hepatic tissue expression and occurs by the use of a cryptic 5'-donor consensus splice site (IGF633) in exon 5; 2) exon 4-5-6 can be hormonally regulated in cultured human HepG2 cells; 3) exon 4-5-6 is the human counterpart of the rat IGF-IEb, because the complementary DNA and predicted sequences are homologous; and 4) the production of IBE1 is potentially regulated by alternative splicing.

Alternative Splicing↗

PubMed1995

Adrenocorticotropin receptor gene mutations in familial glucocorticoid deficiency: relationships with clinical features in four families.

Familial glucocorticoid deficiency is an autosomal recessive syndrome of adrenal unresponsiveness to ACTH characterized by glucocorticoid deficiency, high plasma ACTH levels, and a normal renin-aldosterone axis. Defects of the ACTH receptor have been suggested as a possible cause, and we have previously reported a number of novel mutations of the ACTH receptor gene in some, but not all, cases, suggesting that familial glucocorticoid deficiency may have a heterogeneous molecular etiology. Here we report the clinical features and ACTH receptor gene analysis in four patients from different families. We found that two patients were compound heterozygotes for the S74I and R128C mutations (patient A) and I44M and L192fs frame shift mutations (patient B). The other two patients (C and D) were of different ethnic ancestry, but were both homozygous for a R146H mutation. Segregation studies within families revealed heterozygosity in the parents and several other family members. Human CRH tests in the parents of patients A and B showed normal cortisol and ACTH responses in the S74I, R128C, and I44M heterozygotes and exaggerated cortisol and ACTH responses in the L192fs heterozygote, suggesting that the physiological ACTH increment induced in this test did not reveal evidence of subclinical ACTH resistance, and that this test may not be of value in ascertaining heterozygosity.

Adrenocorticotropic Hormone↗

PubMed1995

Tissue-specific, temporally regulated expression mediated by the proximal ovine beta-lactoglobulin promoter in transgenic mice.

The ovine beta-lactoglobulin gene is expressed abundantly in the mammary gland. This study determines whether the ovine beta-lactoglobulin promoter is sufficient for targeting tissue-specific expression in transgenic mice. To address this, the expression profile of an ovine beta-lactoglobulin promoter driven bacterial chloramphenicol acetyltransferase reporter construct was analysed. Comparison of the expression frequency of this hybrid transgene to that of a genomic beta-lactoglobulin transgene indicates that additional sequences, downstream of the promoter, are required for position-independent expression in transgenic mice. Nevertheless, the hybrid transgene was expressed specifically in the mammary gland. Furthermore, the hybrid transgene was expressed in the appropriate temporal pattern during pregnancy and lactation. Thus, the proximal promoter of the ovine beta-lactoglobulin gene contains sufficient sequence information to target expression to the mammary. This construct constitutes the basis for a compact mammary expression vector.

Animals↗

PubMed1994

The mammary factor MPBF is a prolactin-induced transcriptional regulator which binds to STAT factor recognition sites.

Site-directed mutagenesis of the three binding sites for the mammary factor MPBF in the beta-lactoglobulin (BLG) promoter demonstrates that MPBF is a transcriptional activator of the BLG gene in mammary cells. MPBF requires phosphorylation on tyrosine for maximum binding activity and binds to GAS (interferon gamma-activation site) elements which are similar to the MPBF binding sites. Prolactin induces MPBF binding activity in CHO cells and is not antigenically related to Stat1 (p91) and Stat2 (p113), suggesting that this transcription factor is likely to be another member of the STAT family of cytokine/growth factor-induced transcription factors.

Animals↗

PubMed1994

Linkage of the angiotensinogen gene to essential hypertension.

BACKGROUND: The renin-angiotensin system is a powerful pressor system with a major influence on salt and water homeostasis. Angiotensinogen (also called renin substrate) is a key component of this system; it is cleaved by renin to yield angiotensin I, which is then cleaved by angiotensin-converting enzyme to yield angiotensin II. The observation that plasma angiotensinogen levels correlate with blood pressure and track through families suggests that angiotensinogen may have a role in essential hypertension. We therefore investigated whether there is linkage between the angiotensinogen gene on chromosome 1q42-43 and essential hypertension. METHODS: Samples of DNA from 63 white European families in which two or more members had essential hypertension were tested for linkage of the angiotensinogen gene to this disorder. Affected cousins, nephews, nieces, and half-siblings were included when possible. To test for linkage, we used as a marker a dinucleotide-repeat sequence flanking this gene, and we employed the affected-pedigree-member method of linkage analysis. Two molecular variants of the angiotensinogen gene, one encoding threonine instead of methionine at position 235 (M235T) and the other encoding methionine rather than threonine at position 174 (T174M), were also tested for possible association with essential hypertension. RESULTS: We found significant linkage (t = 5.00, P < 0.001) and association (chi-square = 53.3, P < 0.001) of the angiotensinogen-gene locus to essential hypertension in the 63 multiplex families. This linkage was consistently maintained in the subgroup of subjects with diastolic pressure above 100 mm Hg and in the subgroups classified according to sex. It has been proposed previously that T174M and M235T are associated with essential hypertension. However, we found no association in our population between either polymorphism and this disorder. CONCLUSIONS: This study provides strong and consistent support for the linkage to essential hypertension of regions within or close to the angiotensinogen gene. Precisely how mutations in this region may result in hypertension remains to be determined.

Aged↗

PubMed1994

Molecular cloning of the canine angiotensin II receptor. An AT1-like receptor with reduced affinity for DuP753.

Canine aortic strip studies revealed insensitivity of angiotensin II (AII)-induced aortic contraction to inhibition by the non-peptide antagonist DuP753 (pKB = 6.7 +/- 0.1). In order to determine the origin of this phenomenon we cloned the canine homologue of the AT1 AII receptor. Expression of this cDNA in COS-7 cells indicated a low affinity of DuP753 for the cloned receptor (KD = 92 nM). The predicted amino acid sequence is highly homologous to other mammalian AT1 receptors; sequence comparisons suggest the pharmacological difference may be the result of a threonine residue in position 163 in the IVth transmembrane domain.

Amino Acid Sequence↗