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Biomedical subjects

A J Clark

Publications and source records attributed to A J Clark.

At least 127 records · Page 7Linked to original sources
PubMed1996

Genetic modification of milk proteins.

It is now possible to change the protein composition of milk by direct manipulation of the germline. At present it is feasible only to add new milk-protein genes to dairy animals. If embryonic stem cells become available then it will also be possible to remove or replace endogenous genes. Various modifications made to milk proteins by using these germline-manipulation techniques are described. These are expected to enhance the processing, nutritional, and biomedical properties of milk.

Animals↗
PubMed1996

Differential suppression of priA2::kan phenotypes in Escherichia coli K-12 by mutations in priA, lexA, and dnaC.

First identified as an essential component of the phi X174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec-, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

Bacterial Proteins↗
PubMed1996

Linkage of the gene for the triple A syndrome to chromosome 12q13 near the type II keratin gene cluster.

The triple A or Allgrove's syndrome (MIM*231550) is an autosomal recessive disease characterized by the triad of adrenocorticotropic hormone (ACTH) resistant adrenal insufficiency, achalasia and alacrima. Since its first description by Allgrove et al. (1978) more than 70 cases from all over the world have been reported. The syndrome manifests itself during the first decade of life with severe hypoglycaemic episodes which can cause sudden death. The frequent association with neurological disorders presenting as a mixed pattern of upper and lower motor neuropathy, sensory impairment, autonomic neuropathy and mental retardation may result in a severely disabling disease. As an additional feature some patients have hyperkeratosis of their palms and soles. We have performed a systematic genome linkage scan in eight triple A families of which three were consanguineous [including the large highly inbred kindred described by Moore et al. (1991)]. We obtained conclusive evidence for linkage of the triple A syndrome locus to markers on chromosome 12q13 (D12S368, theta max = 0, Zmax = 10.81) with no indication of genetic heterogeneity. Haplotype and multipoint analyses suggest that the gene is located on a chromosomal segment flanked by the markers D12S1629 and D12S312 which are 6 cM apart. This region harbors the type II keratin gene cluster, and potential candidate genes include SCN8A and HOXC genes.

Adrenal Insufficiency↗
PubMed1996

Angiotensinogen in human essential hypertension.

The candidacy of angiotensinogen for a role in the genetic basis of hypertension is supported by the observation that plasma angiotensinogen levels track with raised blood pressure through families. In addition, transgenic mice with overexpression of a rat angiotensinogen gene develop hypertension, and knockout mice with a disrupted gene and absent angiotensinogen production develop low blood pressure. There are now two studies in populations of white European origin and one in African Caribbeans providing support for a role of the angiotensinogen gene locus in human essential hypertension.

Angiotensinogen↗
PubMed1996

Agonist and receptor binding properties of adrenocorticotropin peptides using the cloned mouse adrenocorticotropin receptor expressed in a stably transfected HeLa cell line.

The cloned mouse ACTH receptor was expressed in stably transfected human HeLa cells that lack an endogenous melanocortin receptor. ACTH[1-39] and several N- and C-terminally truncated analogues of ACTH were studied for their ability to stimulate cAMP generation and to displace bound 125I-ACTH. Only three of the peptides tested, ACTH[1-24], ACTH[1-39], and ACTH[1-17] were found to have agonist activity with EC50 values of 7.5, 57, and 49 x 10(-12) M respectively. Two peptides, ACTH[11-24] and ACTH[7-39], were devoid of agonist activity but had substantial competitive antagonist activity with IC50 values of approximately 10(-9) M. In binding studies, ACTH[1-39] and ACTH[1-24] were able to fully displace bound ligand, and Scatchard analysis indicated a dissociation constant (KD) of 0.84 and 0.94 x 10(-9) M for the two peptides, respectively. ACTH[1-17], ACTH[11-24], and ACTH[7-39] were only capable of displacing 60-70% of bound ligand. A three-site model for the interaction of ACTH and its receptor is proposed on the basis of these findings.

Adrenocorticotropic Hormone↗
PubMed1996

A homozygous splice site mutation affecting the intracellular domain of the growth hormone (GH) receptor resulting in Laron syndrome with elevated GH-binding protein.

Laron syndrome (LS) is a severe autosomal recessive form of GH resistance resulting from molecular defects in the GH receptor (GHR). Affected individuals have extreme short stature and a typical facial phenotype. The point mutations in the GHR gene identified in this condition have until now been confined to the region encoding the extracellular domain of the receptor. We report here the first homozygous point mutation within the intracellular domain of the GHR in two LS cousins distinguishable from classical LS patients only by the presence of elevated GH-binding protein (GHBP) in their serum. A G to C transversion at the vital - 1 position in the splice donor site of exon 8 disrupts normal splicing, resulting in the complete skipping of exon 8, producing a mutant GHR protein lacking transmembrane and intracellular domains. We predict that this mutant protein would not be anchored in the cell membrane and would be measurable in the circulation as GHBP, hence explaining the phenotype of severe GH resistance combined with elevated circulating GHBP.

Base Sequence↗
PubMed1996

Regulation of the CYP1A1 promoter in transgenic mice: an exquisitely sensitive on-off system for cell specific gene regulation.

Mammalian cytochrome P-450s in the CYP1A gene family catalyse the oxidation of a wide range of drugs and foreign compounds resulting in their excretion. These enzymes are highly inducible by a range of compounds, including polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (3-MC) and dioxins. Analysis of the CYP1A1 promoter has identified dioxin responsive enhancer elements which mediate the induction response. In order to evaluate this promoter as an in vivo regulatable expression system and to gain further insights into the tissue specific regulation of this gene, an 8.5 kb genomic fragment of the rat CYP1A1 promoter was cloned upstream of the lacZ reporter gene. This construct was used to generate transgenic mice and three independent lines were expanded for further study. The regulation of beta-galactosidase expression was determined in mock and 3-MC-treated mice in an extensive range of tissues. In untreated animals no transgene expression was detectable over non-transgenic controls. Treatment with 3-MC caused a profound increase in transgene expression (> 1,000-fold) in many tissues including liver, adrenal, kidney and intestine. Inducible transgene expression was also detectable in many of the other tissues including the spleen, lung, pancreas and the reproductive organs. Although the absolute levels of induction varied, no significant differences in the pattern of transgene expression were observed between the three different transgenic mouse lines. In addition, the pattern of transgene expression correlated closely with the reported regulation of CYP1A1 protein. These results indicate that the CYP1A1 promoter can drive expression of heterologous genes in a truly on/off manner in a variety of tissues and cell types which will allow the expression of other proteins to be controlled in vivo. This reporter system also provides a model for establishing the environmental and hormonal factors regulating the expression of the CYP1A1 gene.

Animals↗
PubMed1996

Characterization of the ovine LH beta-subunit gene: the promoter is regulated by GnRH and gonadal steroids in transgenic mice.

We have previously demonstrated that 1.9 kb of ovine LH beta promoter fused to bacterial chloramphenicol transferase (CAT) coding sequence is sufficient to target expression of the transgene specifically to the gonadotroph cells of the anterior pituitary in mice with no expression being observed in other tissues. However, it is not known if this region of the ovine LH beta promoter contains the necessary elements that confer transcriptional regulation by gonadal steroids and GnRH. Following gonadectomy, both endogenous pituitary LH and CAT activity significantly (P > 0.001) increased as did plasma LH. This post-gonadectomy increase in CAT, pituitary and plasma LH could be suppressed in females by treatment with oestradiol alone or oestradiol and progesterone, with an additional significant (P < 0.05) reduction in CAT activity being observed in one line following the combined steroid treatment. In castrated males, testosterone suppressed CAT activity in one line. Treatment of transgenic ovariectomized females with oestradiol alone significantly suppressed plasma LH (P < 0.01) with no change in pituitary LH content. There was no difference in pituitary LH between oestradiol-treated ovariectomized transgenic and non-transgenic females. Treatment of intact females from both lines with either GnRH antiserum or agonist demonstrated a decrease in pituitary CAT activity whereas similar treatment in intact males had no effect. While endogenous pituitary LH concentrations were variable, plasma LH was lower in all treated animals irrespective of line, sex or expression of the transgene. In conclusion, these results indicate that (1) the presence of CAT protein is not toxic and does not compromise either endogenous LH synthesis, storage and secretion and (2) the ovine LH beta-CAT gene is regulated in a similar but more variable manner to the endogenous LH beta gene. This may relate to the use of CAT as a reporter where its release is not necessarily related to that of the endogenous hormone whose synthesis, storage and release may differ.

Animals↗
PubMed1996

Optimizing the intervention in research studies.

One of the many challenges of nursing intervention studies is to assure that significant effects of the intervention are detected in the research. Optimizing the intervention in the design of the study can increase the potential for finding positive outcomes, if they are, in fact, present. Attending to the operationalization, strength, integrity, and timing of the intervention, as well as to the contrast between the treatment and control groups, can optimize the intervention in intervention research.

Humans↗
PubMed1995

Identification of an orphan receptor gene as a type 1 calcitonin gene-related peptide receptor.

Calcitonin gene-related peptide (CGRP) is a 37 residue neuropeptide that is distantly related to adrenomedullin. We have recently reported the cloning and expression of an adrenomedullin receptor which is approximately 30% homologous to the canine orphan receptor RDC-1. Therefore we tested the hypothesis that RDC-1 was a CGRP receptor. The RDC-1 gene was expressed in COS-7 cells and showed a dose dependant increase of cAMP in response to CGRP and adrenomedullin (EC50 values of 3 x 10(-9) M and 1 x 10(-7) M respectively) which was inhibited by the CGRP antagonist CGRP[8-37]. There was no cAMP response to amylin or [Cys(acm)2,7]a-hCGRP. Ligand binding studies confirmed high affinity of this receptor for CGRP and CGRP[8-37] with KD values of 9.2 x 10(-9) M and 13.4 x 10(-9) M respectively.

Adrenomedullin↗
PubMed1995

Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.

Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.

Animals↗
PubMed1995

Cloning and expression of cDNA encoding a rat adrenomedullin receptor.

Adrenomedullin is a potent vasodilator peptide that exerts major effects on cardiovascular function. Its actions are mediated through an abundant class of specific binding sites that activate adenylyl cyclase through a G protein-coupled mechanism. We report here the identification of a cDNA clone for the adrenomedullin receptor that was originally isolated as an orphan receptor from rat lung. The cDNA encodes a polypeptide of 395 residues that contains seven transmembrane domains and has a general structural resemblance to other members of the G protein-linked receptor superfamily. When expressed in COS-7 cells, this receptor mediates a cAMP response to adrenomedullin with an EC50 of 7 x 10(-9) M, and binds 125I-adrenomedullin with a KD of 8.2 x 10(-9) M, properties that are consistent with those observed in cardiovascular and other target tissues. The receptor gene is expressed as several mRNA species of which the most prominent is a 1.8-kilobase transcript found in the lung, adrenal, heart, spleen, cerebellum, and other sites. Identification of this receptor cDNA should facilitate further investigation of the cellular actions of adrenomedullin and its regulatory effects in normal and disordered states of cardiovascular function.

Adrenomedullin↗
PubMed1995

Secretory proteins compete for production in the mammary gland of transgenic mice.

To explore the possibility that genes might compete for expression, we have studied transgenic mice producing high levels of the sheep milk protein, beta-lactoglobulin (BLG), in the mammary gland. Mice carrying one or more transgene loci expressed BLG in milk at levels ranging from 7 to 33 mg/ml. The effects of BLG synthesis on the levels of endogenous milk gene expression were examined. No significant increase in total milk protein concentration was recorded even in mice expressing the largest amounts of BLG. Measurement of individual milk proteins showed that transgene protein was manufactured at the expense of host protein synthesized in the gland. Whey acidic protein production was more suppressed than casein production. Suppression of endogenous proteins was matched by a reduction in the corresponding steady-state mRNA levels; in double-transgenic mice, which expressed the largest amounts of BLG, beta-casein and whey acidic protein mRNA populations were reduced to 75 and 56% of control levels respectively. We demonstrate that an exogenous gene competes effectively for expression with endogenous genes. Possible mechanisms of competition are discussed.

Animals↗
PubMed1995

Effects of the selective protein kinase C inhibitor Ro 31-7549 on human angiotensin II receptor desensitisation and intracellular calcium release.

The mechanism underlying type I angiotensin II (Ang II) receptor (AT1 receptor) desensitisation is unknown. Structural features suggest it may be a substrate for protein kinase C (PKC). The effects of a selective PKC inhibitor, Ro 31-7549, on receptor desensitisation were investigated in CHO cells expressing the human AT1 receptor. Desensitisation was demonstrated with respect to the calcium response to Ang II in Fura-2-loaded cells. Ro 31-7549 had no effect on desensitisation. However, pretreatment with Ro 31-7549 caused a dose-dependent reduction in calcium release from intracellular stores. PKC may therefore act at a locus distal from the receptor itself.

Angiotensin II↗