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Biomedical subjects

A J Clark

Publications and source records attributed to A J Clark.

At least 163 records · Page 9Linked to original sources
PubMed1994

The Anaesthetic Services Programme Encompassing Nova Scotia (ASPENS)

A comprehensive peer review programme for anaesthesia departments in Nova Scotia was implemented in 1987 by the anaesthesia community. Departments are reviewed by peers at the request of the Head, Department of Anaesthesia, and the President of the Medical Staff. A confidential report with recommendations is written. Twenty-six reviews have been completed (to June 1993) and have most often documented deficiencies in: design of the anaesthesia record, anaesthesia staff recruitment, inadequate documentation of policies for anaesthesia and post-anaesthesia care, peer review/quality assurance processes, organizational structure of hospital medical staffs and operating room committees, and use of anaesthesia equipment that does not meet current (at date of review) CSA standards. In the four hospitals that have been reviewed twice, 24 of the 30 deficiencies noted on the first review had been corrected before the second review which occurred three to five years later. Nine new deficiencies were noted. Departments are encouraged to request a review every four or five years.

Anesthesia Department, Hospital↗
PubMed1994

Nucleotide sequence between recA and alaSp in E. coli K12 and the sequence change in four recA mutations.

The sequence of 366 nucleotides between the C-terminal trailer region of recA and the N-terminal leader region of alaS is presented. This sequence reveals an open reading frame of 166 codons we have named oraA. An NdeI restriction nuclease cleavage site also revealed by the sequence was used to clone, map and sequence three recA mutations: recA11, recA12 and recA52. A mutation in recA (recA946), was discovered in strains originally reported to contain recH166. The relation between recA946 and recH166 is unclear.

Amino Acid Sequence↗
PubMed1994

Chromosomal position effects and the modulation of transgene expression.

Chromosomal position effects can influence strongly the transcription of foreign genes in transgenic animals. This results in low frequencies and levels of gene expression and, in some cases, in aberrant patterns of expression. Strategies for overcoming these effects are described with particular reference to their application in embryonic stem cells.

Animals↗
PubMed1994

Mutations of the ACTH receptor gene are only one cause of familial glucocorticoid deficiency.

Familial glucocorticoid deficiency (FGD) is an autosomal recessive syndrome of failure of adrenal cortisol responsiveness to adrenocorticotropin (ACTH). Defects in the ACTH receptor have been suggested as a possible cause, and we have previously reported a point mutation in this gene in a family with FGD. Investigation of seven additional families has revealed a number of novel mutations in the ACTH receptor in some, but a normal gene in others suggesting that the aetiology of FGD may be heterogeneous. Using a highly polymorphic CA repeat marker (D18S40) closely linked to the ACTH receptor locus, we are now able to confirm that some cases of FGD result from defects at another locus. FGD provides an example of a single relatively homogeneous clinical syndrome resulting from two different molecular aetiologies.

Adrenal Insufficiency↗
PubMed1994

RecOR suppression of recF mutant phenotypes in Escherichia coli K-12.

The recF, recO, and recR genes form the recFOR epistasis group for DNA repair. recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response. Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above. We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes. Expression of the chromosomal recO+ gene is essential for the recR+ suppression. Hence we call this RecOR suppression of recF mutant phenotypes. RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome. This is not true for RecOR suppression of UV sensitivity (the two are equal). Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+. We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.

Bacterial Proteins↗
PubMed1994

Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression.

The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF. In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence. Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation. When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally. We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible. In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene.

Bacterial Proteins↗
PubMed1994

Transcription of the Escherichia coli recE gene from a promoter in Tn5 and IS50.

Six sbc::Tn5 insertions and one sbc::IS50 insertion, which cause recE expression in Escherichia coli, have been cloned, and their DNA sequences have been determined. The sites of insertion are found at three positions in a 10-bp region: 58, 63, and 68 bp upstream of recE. Primer extension experiments with the cloned Tn5 insertions demonstrate that recE transcripts start adjacent to the insertion elements of five of these mutations and both adjacent and one nucleotide within the insertion element for the sixth mutation. This supports the hypothesis that these mutations have inserted a promoter, and PCR analysis reveals an outward promoter within the distal 69 nucleotides of Tn5. Primer extension analysis of RNA from the uncloned Tn5 and IS50 mutants reveals three additional insertion sites close to the others. Because all the insertions lie in the spacer region between racC and recE, transcribed in sbcA6 and sbc-23 strains, we propose that these insertions be renamed recEs::Tn5 and recEs::IS50.

Base Sequence↗
PubMed1994

Human small cell lung cancer cell lines expressing the proopiomelanocortin gene have aberrant glucocorticoid receptor function.

Some human small cell lung carcinomas (SCLC) secrete proopiomelanocortin (POMC) derived peptides, but in contrast to the pituitary, glucocorticoids fail to inhibit this hormone production. We have previously described an in vitro model using human SCLC cell lines that express POMC and are resistant to glucocorticoids. We have now identified the glucocorticoid receptor (GR) in the SCLC cell line COR L24 using a whole cell ligand binding assay (Kd = 5.7 nM; Bmax = 11 fmol/million cells), while another cell line, DMS 79, lacked significant glucocorticoid binding. To analyze GR function both positive (GMCO) and negative (TRE)3-tkCAT), glucocorticoid-regulated reporter gene constructs were transfected into COR L24 cells. In the SCLC cell line, neither hydrocortisone nor dexamethasone (500-2,000 nM) significantly induced chloramphenicol acetyltransferase expression from GMCO; in addition, they did not suppress chloramphenicol acetyltransferase expression from (TRE)3-tkCAT. Similar results were obtained with two other POMC-expressing SCLC cell lines. Expression of wild type GR in COR L24 cells restored glucocorticoid signaling, with marked induction of GMCO reporter gene expression by dexamethasone (9,100 +/- 910%; n = 3), and an estimated EC50 of 10 nM. This failure of the GR explains the resistance of the POMC gene to glucocorticoid inhibition and may have implications for cell growth in SCLC.

Carcinoma, Small Cell↗
PubMed1994

Regulation of the sheep beta-lactoglobulin gene by lactogenic hormones is mediated by a transcription factor that binds an interferon-gamma activation site-related element.

Polypeptide and steroid hormones regulate the transcription of milk protein genes in the mammary gland. The promoter sequence motifs and factors through which these hormones mediate their effects in vivo are not clearly defined. Milk protein binding factor (MPBF) is a factor that has recognition sites in the promoters of many milk protein genes including three sites in the promoter of the sheep beta-lactoglobulin (BLG) gene. Mutagenesis of these sites reduced expression of the BLG gene in lactating mammary glands of transgenic mice but did not affect the tissue specificity of the transgene. Furthermore, mutation of all three sites abolished the response of the BLG gene to lactogenic hormones in HC11 mammary cells. Together these results indicate that MPBF mediates the effects of lactogenic hormones in the mammary gland but does not play a role in determining mammary specificity. The similarity between the MPBF binding site and the gamma-interferon activating site suggests that MPBF is related to the STAT family of cytokine-induced transcription factors.

Animals↗
PubMed1994

Homologous genetic recombination: the pieces begin to fall into place.

One of the authors (AJC) acknowledges with gratitude the important role Fernando Bastarrachea played in the author's discovery that E. coli could carry out homologous genetic recombination by multiple pathways. This in turn led to the discovery of several genes, including recF, recO, and recR, whose role in recombination would not otherwise have been detected. Subsequent genetic and biochemical studies have led to a general formulation in which there are multiple nucleolytic ways to achieve a presynaptic intermediate bound to RecA protein. Postsynaptic events in the general formulation occur by means of multiple branch migration enzymes to form Holliday DNA structures and a specific nuclease to cleave them. The general formulation is built on synapsis catalyzed by RecA protein. A second RecA-independent synapsis catalyzed by RecT (and RecE?) protein is now under study and a third type independent of both RecA and RecT has apparently been discovered. How these will affect the general formulation remains to be seen. Some proteins, most prominently RecF, RecO, and RecR, have no role in the general formulation. The hypothesis is presented that these proteins act as a switch between replication and recombination by helping to convert replication to recombination intermediates. Universality of the general formulation is supported by the widespread occurrence of recA, recB, recC, and recD genes among bacteria. Recent discovery of recA-like genes in several eukaryotes further supports its universality. We have contributed additional support by sequencing a recA-like gene from an archaeal species, thus making it plausible that the mechanism of synapsis worked out for E. coli RecA protein will hold for all three organismal domains. The boundaries of the puzzle of homologous genetic recombination therefore seem complete and the pieces to the complex picture they encompass are falling into place.

DNA Replication↗
PubMed1994

Assessment of painful late effects of lumbar spinal fusion with SPECT.

UNLABELLED: The authors reviewed planar, SPECT and other contemporaneous radiologic images of the spine and the medical records of 33 patients with back pain after lumbar fusion surgery in order to determine the value of SPECT in the assessment of painful late effects of spinal fusion surgery. METHODS: Twenty-one patients had lateral fusion, nine patients had posterior fusion only and three patients had anterior and posterior fusions. There were 24 patients who had surgery more than 4 yr ago (late group, mean 11.8 yr) and 9 patients who had surgery less than 4 yr ago (early group, mean 17.8 mo). RESULTS: The most common SPECT abnormality in patients in the late group were lesions in the vertebral bodies and apophyseal joints in the free motion segments adjacent to the fused segments (62.5% of patients). Such lesions occurred in 46% of patients after lateral fusion, in 87.5% of patients after posterior fusion and in 67% of patients after posterior and anterior fusions. No SPECT abnormalities were detected in the fused segments in patients in the late group with solid lateral fusion but were detected in three patients with solid posterior fusion. These results correlate with biomechanical studies that have shown posterior fusion to produce the largest amount and lateral fusion to produce the least amount of stress in the free segments adjacent to the fusion. Lateral fusion was found to have a more stabilizing effect than posterior fusion. CONCLUSION: In addition to the already established value of SPECT in detecting painful pseudoarthrosis, our results indicate that SPECT is of value in the assessment of painful late effects of fusion.

Adult↗
PubMed1993

Functional characterization of the cloned human ACTH receptor: impaired responsiveness of a mutant receptor in familial glucocorticoid deficiency.

The putative ACTH receptor gene has been identified on the basis of its tissue specific expression, structure, and limited expression data. We have expressed this gene in COS-7 cells and measured cAMP production in response to ACTH. An EC50 of 5.5 x 10(-9) M for ACTH (1-24) was determined. The S74I mutant ACTH receptor gene that associates with the syndrome of familial glucocorticoid deficiency had an EC50 of 67 x 10(-9) M. This discrepancy is consistent with the clinical data, and supports the hypothesis that this point mutation could account for the syndrome.

Adrenocorticotropic Hormone↗
PubMed1993

Comparison of COS cell transfected AT1A and AT1B angiotensin II receptors and angiotensin II receptor isoforms in rat tissues using isoelectric focusing.

Rat adrenal AT1A and AT1B receptors from transfected COS-7 cells were labelled with 125I-Angiotensin II, solubilised, and run on isoelectric focusing gels. Receptors from rat tissues were treated similarly. COS-7 cell-expressed AT1A and AT1B receptors each produced a single peak of specific radioactivity at pI 6.8. Rat liver and rat ovary tissue preparations gave peaks at pI 6.8 and 6.5, respectively. In contrast, rat adrenal tissue preparations gave four peaks at pI 7.0, 6.8, 6.5, and 6.3. The additional isoforms found in the rat adrenal tissue preparations may represent post-translationally modified or novel receptors.

Adrenal Glands↗
PubMed1993

Enhancing the efficiency of transgene expression.

Two strategies for enhancing gene expression in transgenic animals are described with particular reference to targeting expression to the mammary gland. Gene constructs in which the protein-encoding DNA sequences are contained within a genomic segment (comprising most or all of the natural introns of the corresponding gene) are shown to be expressed more efficiently than their intronless counterparts. Secondly, co-integrating an otherwise poorly expressed transgene in the vicinity of an actively expressed transgene can dramatically improve its efficiency of expression.

Animals↗
PubMed1993

Familial glucocorticoid deficiency associated with point mutation in the adrenocorticotropin receptor.

Familial glucocorticoid deficiency is an uncommon disorder that appears to be due to congenital insensitivity or resistance to adrenocorticotropin (ACTH), and is usually inherited in an autosomal recessive pattern. We investigated the DNA base sequence in a family with this condition by polymerase chain reaction amplification of DNA with pairs of primers that span the ACTH-receptor domain. The affected male proband showed a single base mutation, ser74-->ile, in the sequence coding for the second transmembrane domain of the ACTH receptor. A similar defect was found in an affected sister, a normal sequence in an unaffected brother, and both alleles in each parent. This is only the second clinical disorder associated with a GTP-binding-protein-linked hormone-receptor mutation.

Adolescent↗
PubMed1993

Characterization of the ovine LH beta-subunit gene: the promoter directs gonadotrope-specific expression in transgenic mice.

The alpha- and beta-subunits of the gonadotropin hormones are expressed in the gonadotrope cells of the anterior pituitary. There are no adequate in vitro systems for the analysis of beta-subunit gene expression. In this study, therefore, transgenic mice have been used to investigate the regulation of expression of the ovine luteinizing hormone beta-gene (oLH beta) in vivo. oLH beta was isolated, characterized, and 1.9 kb of the promoter fused to the bacterial reporter chloramphenicol acetyl-transferase (CAT). Three lines of transgenic mice were generated. CAT enzyme was detected in the pituitary of two lines, whereas the third line did not express. Measurement of endogenous luteinizing hormone and follicle stimulating hormone levels in both expressing lines revealed small differences when compared to controls, but these did not affect the fertility of the animals. Immunostaining of the anterior pituitary revealed that the oLH beta CAT transgene was expressed specifically in gonadotrope cells.

Amino Acid Sequence↗