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Biomedical subjects

A J Clark

Publications and source records attributed to A J Clark.

At least 91 records · Page 5Linked to original sources
PubMed1998

[Phenotype-genotype relations in growth hormone insensitivity].

Growth hormone (GH) insensitivity is associated with several different mutations of the GH receptor gene and a recently described new genetic disorder of the IFGI gene. The phenotype and biochemical characteristics were studied in 82 patients with growth hormone insensitivity, from 23 different countries, with a mean age of 8.25 years. Mean height SDS was -6.09. SDS of the IGF binding protein -3 (IGF BP3) was 7.99. Twenty three per cent of the patients were GH binding protein (GHBP) positive (> 10%). Mean height SDS score was -6.5 in the GHBP negative patients and -4.9 in the GHBP positive patients (p < 0.001). Fifteen different mutations of the GH receptor gene were identified in 27 patients. There were no relationships between the type of mutation or the involved GH receptor gene exon and height or IGFBP-3 SDS. The new phenotype due to a partial deletion of the IGFI gene was described in a 15-year-old boy who presented with a severe intrauterine growth retardation, a very poor postnatal statural growth, a neurosensorial deafness and a mild mental retardation. He had elevated GH levels, normal levels of IGFBP3, undetectable levels of IGFI, and showed no response to GH treatment. A partial deletion concerning the exons 4 and 5 of the IGFI gene was found. Thus, GH insensitivity is associated with large variations in the clinical and biochemical phenotypes.

Adolescent↗
PubMed1998

The mammary gland as a bioreactor: expression, processing, and production of recombinant proteins.

A variety of transgenic animal species are being used to produce recombinant proteins. The general approach is to target the expression of the desired protein to the mammary gland using regulatory elements derived from a milk protein gene and then collect and purify the product from milk. Promoter sequences from a number of different milk protein genes have been used to target expression to the mammary gland, although significant problems remain with regard to achieving transgene expression levels consistent with commercial exploitation. The mammary gland appears to be capable of carrying out the complex posttranslational modifications. such as glycosylation and gamma-carboxylation required for the biological activity and stability of specific proteins. Effective purification protocols have been established and products produced by this route have now entered clinical trials.

Animals↗
PubMed1998

The effect of therapeutic touch on pain and anxiety in burn patients.

The purpose of this single-blinded randomized clinical trial was to determine whether therapeutic touch (TT) versus sham TT could produce greater pain relief as an adjunct to narcotic analgesia, a greater reduction in anxiety, and alterations in plasma T-lymphocyte concentrations among burn patients. Therapeutic touch is an intervention in which human energies are therapeutically manipulated, a practice conceptually supported by Rogers' (1970) theory of unitary human beings. Data were collected at a university burn centre in the south-eastern United States. The subjects were 99 men and women between the ages of 15 and 68 hospitalized for severe burns, and they received either TT or sham TT once a day for 5 days. Baseline data were collected on day 1, data were collected before and after treatment on day 3, and post-intervention data were collected on day 6. Instruments included the McGill Pain Questionnaire, Visual Analogue Scales for Pain, Anxiety and Satisfaction with Therapy, and an Effectiveness of Therapy Form. Blood was drawn on days 1 and 6 for lymphocyte subset analysis. Medication usage for pain in mean morphine equivalents, and mean doses per day of sleep, anxiety and antidepressant medications were recorded. Subjects who received TT reported significantly greater reduction in pain on the McGill Pain Questionnaire Pain Rating Index and Number of Words Chosen and greater reduction in anxiety on the Visual Analogue Scale for Anxiety than did those who received sham TT. Lymphocyte subset analyses on blood from 11 subjects showed a decreasing total CD8 + lymphocyte concentration for the TT group. There was no statistically significant difference between groups on medication usage.

Adolescent↗
PubMed1998

Transgenic mice secreting coronavirus neutralizing antibodies into the milk.

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.

Amino Acid Sequence↗
PubMed1998

Exclusion of the adrenocorticotropin (ACTH) receptor (MC2R) locus in some families with ACTH resistance but no mutations of the MC2R coding sequence (familial glucocorticoid deficiency type 2).

Several mutations in the coding exon of the ACTH receptor (MC2R) gene have been reported in cases of familial glucocorticoid deficiency or FGD. However, many patients with a similar syndrome do not present any mutation in the coding region of this gene. This is the case in 11 families we have investigated. Patients in these families present the typical clinical features of FGD, but no mutation was found in the coding exon of the ACTH receptor. To determine whether mutations on MC2R gene, but outside the coding region, may be involved in FGD in these families, we have performed a linkage analysis. Using three markers flanking MC2R gene on chromosome 18, we were able to exclude linkage in a region of 12 centimorgans around the gene. This result clearly indicates that FGD is genetically heterogeneous. Defects in gene(s) different from MC2R gene are implicated in this syndrome.

Adrenocorticotropic Hormone↗
PubMed1998

Gene expression in the mammary glands of transgenic animals.

The gene encoding the milk protein beta-lactoglobulin in sheep is expressed in the mammary gland in a tissue-specific manner during pregnancy and lactation. The unmodified sheep gene behaves appropriately in transgenic mice, and we have shown that many of the cis-acting elements that mediate this pattern of expression are located in the proximal 400 bp of the promotor. Using a combination of approaches we have identified a number of discrete cis-acting elements and their corresponding trans-acting factors that control the responsiveness of this gene in vivo. The beta-lactoglobulin promoter elements can be used to target the expression of foreign genes to the mammary gland in transgenic mice. We have used this approach in basic studies of mammary gland biology and for the production of therapeutic proteins in the milk of transgenic animals. In these circumstances, however, the promoter rarely functions optimally, and it may even be silenced; consequently, we have had to develop a number of strategies to overcome this problem. Nevertheless, foreign proteins do appear to be appropriately post-translationally modified when they are expressed in the mammary gland.

Animals↗
PubMed1997

Co-integration and expression of bacterial and genomic transgenes in the pancreatic and intestinal tissues of transgenic mice.

Previous studies in the mammary gland have reported the 'rescue' of poorly expressed cDNA transgenes by their co-integration with a genomic sequence specifically expressed in the mammary tissue. To determine whether a highly expressed genomic sequence co-integrated with a cDNA sequence can rescue expression in other tissues, the expression of a bacterial gene, celE', encoding endoglucanase E' (EGE'), was investigated in the pancreatic and intestinal epithelia of transgenic mice. To rescue pancreatic expression, the human growth hormone genomic sequence was co-integrated with the bacterial gene, whereas to rescue intestinal expression, the genomic sequence encoding the intestinal fatty acid binding protein was used. In both studies the number of transgenics expressing celE' was significantly increased (60%) by the use of a genomic sequence, but only in the intestinal tissues was the level of celE' expression improved. However, this improvement was modest, representing at maximum only a doubling in the levels of EGE'. Thus permissive integration or rescue may be general, but the overall level of rescue is often insubstantial compared to the endogenous expression of the transgene genomic DNA.

Animals↗
PubMed1997

Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein-Barr nuclear antigen-1 protein to neomycin resistance.

Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are 'permissive' for replication of an oriP-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 gene product can be toxic in some cell types and may be selected against. In this paper, we describe a gene construct that overcomes this limitation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein-Barr nuclear antigen-1 and G-418 resistance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely integrated into human cell lines. The presence of EBNA-1 protein was reflected by a large increase in transfection frequencies (1000-fold) using an oriP-based vector which was shown to replicate stably in these cells with no apparent gross rearrangements detected after 8 weeks in culture. Using this system, G-418 resistance should directly reflect integration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector systems and should result in its increased use.

Binding Sites↗
PubMed1997

Intronic sequences modulate the sensitivity of beta-lactoglobulin transgenes to position effects.

We have analysed the expression of beta-lactoglobulin (BLG) gene constructs with combinations of introns deleted to further define the role of intronic regions in directing position-independent mammary expression of BLG transgenes. Intron removal had no obvious effect on hormonal induction of BLG expression in vitro but dramatically reduced expression in vivo, in that removal of intron pairs always resulted in a proportion of the transgenic lines generated failing to express the transgene in the mammary gland. Position-dependent expression was seen for all intron-deleted transgenes regardless of which introns were removed and the ability of the intron-deleted transgenes to be expressed bore no relationship to transgene copy number. Thus, intron removal per se increases the sensitivity of BLG transgenes to position effects.

Animals↗
PubMed1997

Mammalian cDNA and prokaryotic reporter sequences silence adjacent transgenes in transgenic mice.

The ovine beta-lactoglobulin gene is expressed efficiently and at high levels in the mammary gland of transgenic mice. In contrast, when this gene is linked to a second gene construct comprising a mammalian cDNA or a CAT reporter sequence it fails to be expressed in the majority of transgenic lines generated. We suggest that mammalian cDNAs and prokaryotic reporter sequences can serve as active foci for gene silencing in the mammalian genome.

Animals↗
PubMed1997

Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms.

Protein sequences with similarities to Escherichia coli RecA were compared across the major kingdoms of eubacteria, archaebacteria, and eukaryotes. The archaeal sequences branch monophyletically and are most closely related to the eukaryotic paralogous Rad51 and Dmc1 groups. A multiple alignment of the sequences suggests a modular structure of RecA-like proteins consisting of distinct segments, some of which are conserved only within subgroups of sequences. The eukaryotic and archaeal sequences share an N-terminal domain which may play a role in interactions with other factors and nucleic acids. Several positions in the alignment blocks are highly conserved within the eubacteria as one group and within the eukaryotes and archaebacteria as a second group, but compared between the groups these positions display nonconservative amino acid substitutions. Conservation within the RecA-like core domain identifies possible key residues involved in ATP-induced conformational changes. We propose that RecA-like proteins derive evolutionarily from an assortment of independent domains and that the functional homologs of RecA in noneubacteria comprise an array of RecA-like proteins acting in series or cooperatively.

Amino Acid Sequence↗
PubMed1997

Familial glucocorticoid deficiency: one syndrome, but more than one gene.

Familial glucocorticoid deficiency is a rare autosomal recessive disease characterised by resistance to the action of ACTH. A number of mutations in the ACTH receptor have been demonstrated in patients with this disorder which are likely to lead to loss of receptor function and thus would account for the syndrome. Several patients, however, do not have mutations in the ACTH receptor gene coding region, and it can be demonstrated by segregation analysis that another distant gene must account for the disease in some of these cases. The nature of several candidate genes for this normal receptor form of the disease is discussed.

Animals↗
PubMed1997

Transgene rescue in the mammary gland is associated with transcription but does not require translation of BLG transgenes.

Many transgenes, particularly those comprising cDNA sequences fail to be expressed when they are introduced into transgenic mice. We have previously shown that this problem can be overcome in the mammary gland by co-integrating a poorly expressed cDNA transgene, comprising the sheep beta-lactoglobulin promoter, with the efficiently expressed, unmodified beta-lactoglobulin gene. In this report we demonstrate that the transcription of the beta-lactoglobulin gene is associated with this effect because co-integration with a non-transcribed beta-lactoglobulin gene fails to rescue expression. By contrast, co-integration with a translationally inactivated beta-lactoglobulin transgene does rescue the expression of the second gene, but without the co-production of beta-lactoglobulin protein.

Animals↗
PubMed1997

Selective cell ablation in transgenic mice expression E. coli nitroreductase.

The gene encoding E. coli nitroreductase (NTR) was expressed in the luminal cells of the mammary gland of transgenic mice using the ovine beta-lactoglobulin promoter. Treatment of NTR expressing animals with the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) resulted in a rapid and selective killing of this population of cells whereas the closely associated myoepithelial cells were unaffected. NTR-mediated inducible cell ablation offers a number of advantages over the use of HSV1-tk for the selective killing of cells in vivo.

Animals↗
PubMed1997

Diminished adrenal androgen secretion in familial glucocorticoid deficiency implicates a significant role for ACTH in the induction of adrenarche.

OBJECTIVE: The mechanism of adrenarche is controversial, and there have been competing claims that its origin is in the hypothalamo-pituitary axis or in the adrenal gland itself. ACTH is proposed inducer of adrenarche so patients with ACTH resistance due to be familial glucocorticoid deficiency syndrome provide a model to clarify the degree to which ACTH is involved in the regulation of adrenal androgen secretion during adrenarche. DESIGN: Random analysis of plasma adrenal androgens and urinary adrenal androgen output in treated patients with familial glucocorticoid deficiency. PATIENTS: Eleven patients (6 males and 5 females, aged 6.5-21.6 years, 4 prepubertal, minimum bone age 9 years) with familial glucocorticoid deficiency were studied. In 6, mutations, in the coding region of the ACTH receptor are the cause of their ACTH resistance. In the remaining 5 the receptor was normal. MEASUREMENTS: Physical examination, basal serum cortisol, basal plasma ACTH, serum cortisol after stimulation with 250 micrograms ACTH(1-24), plasma dehydroepiandrosterone-sulphate (DHEAS) and plasma androstenedione (A4), total urinary androgen excretion and single-stranded sequencing of the coding region of the ACTH receptor. RESULTS: DHEAS was undetectable in 8, and detectable but below the age-matched reference values in 3 patients. A4 was measured in 10 patients, and in 3 patients (2 in late puberty) was found to be subnormal whereas in the remaining 7 patients A4 was normal for age and pubertal stage consistent with the gonadal contribution to peripheral A4 levels. Significantly diminished output of urinary adrenal androgen metabolites in 3 patients confirmed the results found in serum. The lack of adrenarche was independent of the presence of a mutation within the ACTH receptor and of the severity of glucocorticoid deficiency. Despite adequate glucocorticoid replacement therapy ACTH levels remained elevated in 10 of the 11 patients. CONCLUSIONS: Although reduced adrenocortical inner zone cell number may contribute to the lack of adrenarche, in some patients there appears to be a discrepancy between partial glucocorticoid deficiency and significantly diminished adrenal androgen secretion. These data imply a significant contribution of ACTH to the regulation of adrenarche in normal children either by having a priming effect on the adrenal gland or by acting in concert with other adrenal androgen stimulating factors.

Adolescent↗