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Biomedical subjects

B J Clark

Publications and source records attributed to B J Clark.

At least 91 records · Page 5Linked to original sources
PubMed1996

Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis.

The rate-limiting, hormone-regulated, enzymatic step in steroidogenesis is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme system (CSCC), which is located on the matrix side of the inner mitochondrial membrane. However, it has long been observed that hydrophilic cholesterol-like substrates capable of traversing the mitochondrial membranes are cleaved to pregnenolone by the CSCC in the absence of any hormone stimulation. Therefore, the true regulated step in the acute response of steroidogenic cells to hormone stimulation is the delivery of cholesterol to the inner mitochondrial membrane and the CSCC. It has been known for greater than three decades that transfer of cholesterol requires de novo protein synthesis; however, prior to this time the regulatory protein(s) had yet to be identified conclusively. It is the purpose of this commentary to briefly review a number of the candidates that have been proposed as the acute regulatory protein. As such, we have summarized the available information that describes the roles of transcription, translation, and phosphorylation in this regulation, and have also reviewed the supporting cases that have been made for several of the proteins put forth as the acute regulator. We close with a comprehensive description of the Steroidogenic Acute Regulatory protein (StAR) that we and others have identified and characterized as a family of proteins that are synthesized and imported into the mitochondria in response to hormone stimulation, and for which strong evidence exists indicating that it is the long sought acute regulatory protein.

Adrenal Hyperplasia, Congenital↗
PubMed1996

Metastatic choroidal melanoma to the contralateral orbit 40 years after enucleation.

Metastatic uveal melanoma to the contralateral orbit is extremely rare, with only eight cases in the literature having been reported. We reviewed the clinical presentation, the histologic findings of the primary and secondary tumors, and the clinical outcome of these cases and studied an additional case of metastatic choroidal melanoma to the contralateral orbit that occurred 40 years after enucleation for the primary tumor. The long interval between recognition of the primary tumor and the appearance of clinically manifest metastatic disease in the contralateral orbit is the longest reported in the literature to date. The histologic features of the tumor cells of the enucleation specimen and those of the orbital metastasis were similar, consisting of spindle-cell type with minimal atypia. Combined use of immuno-phenotyping and electron microscopy substantiated the diagnosis of melanoma.

Aged↗
PubMed1996

Recommendations for protein and amino acid intake in phenylketonuric patients.

The methods for the determination of protein requirements are reviewed and the difficulties in achieving the recommendations of the dietary management of phenylketonuria proposed by a Medical Research Council Working Party on Phenylketonuria using currently available low phenylalanine (Phe) protein substitutes and low protein foods are examined. These recommendations are that all infants whose blood Phe concentrations exceed 600 mumol/l in the presence of a normal or low plasma tyrosine and an otherwise normal plasma amino acid profile while receiving a normal protein intake (2-3 g/kg/day), should start a low Phe diet immediately. Infants whose blood Phe concentrations remain persistently between 400 and 600 mumol/l for more than a few days should also start treatment. The diet should contain a protein substitute which is Phe free (or at least very low in Phe) and otherwise nutritionally complete with a composition sufficient to provide 100-120 mg/kg per day of tyrosine and a total amino acid intake of at least 3 g/kg per day in children under 2 years of age. In children over 2 years the intake of amino acids should be maintained at a level of 2 g/kg per day. The protein substitute should be spread as evenly as possible through the 24 h. Blood Phe concentrations should be maintained between 120 and 360 mumol/l. In children aged over 10 years it is suggested that the protein substitute should supply the protein reference nutrient intake + 50%. An upper blood Phe limit of 480 mumol/l rather than 360 mumol/l may be acceptable in school age children. Adults and adolescents should continue treatment with the aim to maintain blood Phe concentrations no higher than 700 mumol/l. During the period before conception and during pregnancy women should aim to have plasma Phe concentrations between 60-250 mumol/l.

Adolescent↗
PubMed1996

Development and validation of a capillary zone electrophoretic method for the determination of atenolol in presence of its related substances in bulk and tablet dosage form.

The development of a capillary zone electrophoresis method for determination of the drug atenolol in the presence of its related substances in bulk and in a tablet dosage form is described. The method was fully validated in terms of repeatability (RSDs for migration time and peak area of atenolol at 0.05 mg ml-1 were 0.25% and 0.52%, n = 10, respectively), reproducibility (RSD of peak area 0.84%, n = 5), linearity at two ranges of atenolol concentration, limits of detection and quantitation, ruggedness and robustness. The method was applied to the determination of the drug in a commercial tablet preparation (recovery 99.4%, m/m). The method proved to be fast and reliable for the quantitative analysis of atenolol in the presence of its related substances in bulk and pharmaceutical forms.

Adrenergic beta-Antagonists↗
PubMed1996

Role of calmodulin-dependent protein kinase II in the acute stimulation of aldosterone production.

Acute aldosterone production in adrenocortical cells is highly dependent on calcium (Ca2+) and calmodulin (CaM) activation. To determine the role of calmodulin-dependent protein kinase II (CaM kinase II) in human adrenal aldosterone production, the action of KN93 (a specific CaM kinase II inhibitor) on human adrenocortical H295R cells was examined. The stimulation of aldosterone, production by angiotensin II (Ang II) and potassium (K+) were inhibited by KN93 in a concentration-dependent manner with an IC50 of approximately 0.9 and approximately 0.5 microM, respectively. Aldosterone production was also stimulated by treatment with the calcium channel activator Bay K 8644 (Bay K) (1 microM). This production was inhibited in a concentration-dependent manner by KN93 with an IC50 of between 1 and 3 microM. No inhibition by KN93 (0.3-3 microM) or by the calmodulin inhibitor calmidazolium (0.03-0.3 microM) was observed for 22R-hydroxycholesterol (22R-OHChol) stimulation of aldosterone production. Because 22R-OHChol is a substrate for the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and does not require active transport to the mitochondria, these results indicate that KN93 does not directly inhibit P450scc or later steps leading to aldosterone synthesis. To investigate the site of KN93 action further we examined its effect on agonists induction of steroidogenic acute regulatory (StAR) protein, which was recently shown to regulate the movement of cholesterol from the outer to the inner mitochondrial membranes. Induction of StAR protein in H295R cells by Ang II, or Bay K was not affected by co-treatment with KN93 at concentration which blocked steroidogenesis by 60-80%. These results indicate a direct role of CaM kinase II in Ang II and K+ simulation of aldosterone production and support the hypothesis that CaM kinase II may be involved in the process of cholesterol mobilization to the mitochondria.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Safety and feasibility of liver-directed ex vivo gene therapy for homozygous familial hypercholesterolemia.

OBJECTIVE: The purpose of this report was to provide detailed information on the safety and feasibility of surgical procedures associated with the first ex vivo liver-directed gene therapy trial for the treatment of vivo gene therapy for homozygous familial hypercholesterolemia (FH). SUMMARY BACKGROUND DATA: Familial hypercholesterolemia is an autosomal dominant disease in which the gene encoding the low density lipoprotein receptor is defective. Patients homozygous for this mutation have extraordinarily high levels of cholesterol and accelerated atherosclerosis and die prematurely of myocardial infarction. The concept of liver-directed gene therapy was based on the report of normalization of cholesterol levels by orthotopic cardiac/liver transplant in a child with homozygous FH. METHODS: Five patients with homozygous FH were selected for inclusion in this trial. The patients underwent hepatic resection and placement of a portal venous catheter. Primary hepatocytes cultures were prepared from the resected liver and transduced with a recombinant retrovirus encoding the gene for the human low density lipoprotein receptor. The genetically modified cells were then transplanted into the liver through the portal venous catheter. RESULTS: Numerous clinical, laboratory, and radiologic parameters were analyzed. Elevations of the hepatic transaminases and leukocyte counts and a decline in hematocrit count were noted. Transient elevations of the portal pressure were observed during cell infusion. No major perioperative morbidity--specifically, myocardial infarct, perioperative hemorrhage, or portal vein thrombosis--or death occurred as a result of this protocol. CONCLUSION: Liver-directed ex vivo gene therapy can be accomplished safely in humans and is appropriate for selected patients.

Adult↗
PubMed1996

Home enteral feeding of children in the west of Scotland.

Home enteral feeding (HEF) is increasingly used to achieve positive energy and nitrogen balance in children with chronic disease outside hospital. It should be considered when oral feeding cannot or should not be used, and when nutritional support is the sole reason for hospitalisation. In the West of Scotland 156 children (80 male, 76 female) cared for by the Royal Hospital for Sick Children in Glasgow received HEF between 1st September 1994 and 1995. They ranged in age from 0 months to 23 years. Indications for HEF were cystic fibrosis (21), cerebral palsy (17), other neurological disease (29), congenital/metabolic abnormalities (23), chronic renal failure (17), neoplastic disease (17), gastrointestinal/hepatic' disease (12), failure-to-thrive (10), and cardiorespiratory disease (10). Most children received feeds by nasogastric tube (59%) or gastrostomy (41%). One child had a jejunostomy. Feeding regimens were continuous infusion (52%), bolus feeding along (26%), and a combination of bolus and infusion (22%). 73% of patients had infusion pumps. Duration of HEF ranged from one month to 12 years. 78% of children received standard infant or enteral feeds, some receiving energy supplements. The remainder received 'disease-specific' or other special feeds. There was a 23% increase in the number of children receiving HEF during the year of the study. HEF represents an affective way of improving the nutritional status of children with chronic disease at home. It frees hospital beds and nursing resources, is well accepted by children and their families and is growing rapidly as an alternative to hospitalisation.

Adolescent↗
PubMed1996

Papular mucinosis: is the inflammatory cell infiltrate neoplastic? The presence of a monotypic plasma cell population demonstrated by in situ hybridization.

Papular mucinosis is a condition reported to be associated with abnormal serum paraproteins and plasma cell dyscrasias. We report a patient with papular mucinosis, without a serum paraprotein or bone marrow plasmacytosis, in whom the affected skin contained a prominent perivascular plasma cell infiltrate. Using in situ hybridization, for kappa and lambda light chain mRNA, these plasma cells were demonstrably monotypic for lambda light chain and, therefore, presumably monoclonal and putatively neoplastic. We suggest that the absence of a serum paraprotein and marrow plasmacytosis does not exclude the existence of a plasma cell neoplasm in patients with papular mucinosis. Such plasma cell populations may exist in the affected skin, although their true nature and behaviour remains to be determined.

Female↗
PubMed1995

The steroidogenic acute regulatory protein is induced by angiotensin II and K+ in H295R adrenocortical cells.

Adrenal steroid hormone biosynthesis can be activated by the protein kinase A pathway by ACTH, the protein kinase C pathway by angiotensin II (AII), or by increasing intracellular Ca2+ levels by AII or K+. Although their mechanisms of action are not known, each of these pathways is dependent upon the de novo synthesis of a protein that is required for the acute production of steroids. We have recently proposed the steroidogenic acute regulatory (StAR) protein as this required protein, therefore, we examined the effect of different agonists on StAR's expression in H295R human adrenocortical carcinoma cells. (Bu)2cAMP, AII, K+, BAYK8644 (a calcium channel agonist) and TPA are all shown to induce StAR. Aldosterone synthesis was stimulated by all the agonists with the exception of TPA, indicating that AII-stimulated steroid production is mediated by increases in intracellular calcium. Thus, these data suggest that regulation of StAR expression may represent a common mechanism for divergent pathways to acutely control adrenal steroidogenesis.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13.

Steroidogenic acute regulatory protein (StAR) appears to mediate the rapid increase in pregnenolone synthesis stimulated by tropic hormones. cDNAs encoding StAR were isolated from a human adrenal cortex library. Human StAR, coexpressed in COS-1 cells with cytochrome P450scc and adrenodoxin, increased pregnenolone synthesis > 4-fold. A major StAR transcript of 1.6 kb and less abundant transcripts of 4.4 and 7.5 kb were detected in ovary and testis. Kidney had a lower amount of the 1.6-kb message. StAR mRNA was not detected in other tissues including placenta. Treatment of granulosa cells with 8-bromo-adenosine 3',5'-cyclic monophosphate for 24 hr increased StAR mRNA 3-fold or more. The structural gene encoding StAR was mapped using somatic cell hybrid mapping panels to chromosome 8p. Fluorescence in situ hybridization placed the StAR locus in the region 8p11.2. A StAR pseudogene was mapped to chromosome 13. We conclude that StAR expression is restricted to tissues that carry out mitochondrial sterol oxidations subject to acute regulation by cAMP and that StAR mRNA levels are regulated by cAMP.

Animals↗
PubMed1995

Resolution of the enantiomers of oxamniquine by capillary electrophoresis and high-performance liquid chromatography with cyclodextrins and heparin as chiral selectors.

The methods of separation of the enantiomers of the chiral drug oxamniquine are compared, between HPLC with either cyclodextrins and their related derivatives as chiral selectors in the mobile phase or immobilised in a chiral stationary phase (as Cyclobond I and II) and between capillary zone electrophoresis (CZE) where the cyclodextrins are added to the buffer solution. The HPLC experiments, which included structured method optimisation were largely unsuccessful in resolving the enantiomers, with the exception of when a Chiral-AGP protein stationary phase was introduced into the programme. However although this chiral stationary phase provided baseline resolution of the enantiomers the stability of the method was suspect to small changes in the pH (0.2 units). In contrast the CZE method developed for both cyclodextrins and their derivatives gave good resolution of the enantiomers and method stability (R.S.D. < 1%, n = 10 on precision). The basis of the interaction mechanism between selector and selectand was shown as a 1:2 relationship of cyclodextrin to analyte by NMR. In addition the polysaccharide, heparin was investigated as a chiral additive and excellent resolution of the oxamniquine was achieved with 3 mM heparin in 50 mM sodium dihydrogenphosphate (pH 3.0) as buffer in CZE, which also gave a stable procedure. This method allowed the detection of each of the enantiomers in the presence of the other down to 0.23% (m/m). The overall composition of the heparin material from different sources can however be slightly variable and this can result in small differences in resolution capability.

Capillary Action↗
PubMed1995

Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis.

Congenital lipoid adrenal hyperplasia is an autosomal recessive disorder that is characterized by impaired synthesis of all adrenal and gonadal steroid hormones. In three unrelated individuals with this disorder, steroidogenic acute regulatory protein, which enhances the mitochondrial conversion of cholesterol into pregnenolone, was mutated and nonfunctional, providing genetic evidence that this protein is indispensable normal adrenal and gonadal steroidogenesis.

Adrenal Glands↗
PubMed1995

Abnormal extracellular material in the levator palpebrae superioris complex in congenital ptosis.

OBJECTIVE: To determine the pathologic abnormalities of the levator palpebrae superioris in congenital ptosis. METHODS: By means of light and electron microscopy and immunohistochemistry, anterior levator tissue specimen from 15 patients with congenital ptosis excised during routine levator resections were examined. RESULTS: All specimens showed lack of muscle fibers with endomysial and perimysial fibrosis and thickening of the aponeurosis. In addition, in four of the 15 patients, an abnormal extracellular material was present. By light microscopy this appeared as an unusual amorphous material arranged in clumps and bands, and electron microscopy showed it to consist of parallel coarse bundles of fibrillogranular material. Collagen type III and fibronectin were identified within this material by immunohistochemistry. There was no detectable collagen types I, II, IV, V, VI, or VII or laminin, and the material did not stain for actin, myosin, myoglobin, amyloid P component, or amyloid A. CONCLUSIONS: In four of 15 samples of levator palpebrae superioris from patients with congenital ptosis, we identified an unusual amorphous extracellular material that stained positively for collagen type III and fibronectin on immunohistochemistry. This novel material, which we call "amorphocollagenoid," may represent a product of dysgenesis of the levator tissues. The source and full composition of this material merit further study.

Adolescent↗
PubMed1995

Application of chemometric experimental designs in capillary electrophoresis: a review.

Various chemometric experimental designs have been employed for the optimisation of capillary electrophoresis (CE) methods. Similar designs have been utilised in the assessment of the robustness of CE methods. The designs employed include central composites, fractional factorials, Plackett-Burman, simplex and overlapping-resolution mapping. Optimisation studies have largely concentrated on the use of these designs on selection of the optimal electrolyte composition. The robustness testing studies performed have involved the use of screening designs to identify the critical parameters affecting responses such as migration times and resolution. Further designs such as central composites have then been employed to set method limits following robustness studies. It is concluded that the use of experimental designs and statistical data evaluation in conjunction with personal computer-controlled CE autosamplers and instruments are of great benefit in the optimisation and robustness evaluation of CE methods.

Electrophoresis, Capillary↗
PubMed1995

Thrombocytopenia with absent radii (TAR) syndrome: a new increased cellular radiosensitivity syndrome.

A 70-year-old woman with congenital absence of both radii but preservation of the thumb developed a marked pancytopenia after two i.v. injections of 1 g of 5-fluorouracil (5-FU) 1 week apart. She developed bloody diarrhoea after nine fractions of 2 Gy to parallel opposed 16 x 15 cm abdominal fields. This unusual response prompted an investigation of the radiosensitivity of the patient's cells by the sensitive G2 assay of transformed lymphocytes. The radiosensitivity of the patient's lymphoblastoid line appeared to be intermediate between that of normal individuals and an ataxia telangiectasia line. The clinical response and in vitro radiosensitivity testing suggest that the thrombocytopenia with absent radii (TAR) syndrome appears to be one of the inherited impaired DNA repair syndromes and is a very newly described radiation sensitivity syndrome. The development of three separate primary cancers in this patient (small bowel, ovary and bladder) suggests there is an increased risk of neoplasia in this condition.

Aged↗
PubMed1995

A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia.

The outcome of the first pilot study of liver-directed gene therapy is reported here. Five patients with homozygous familial hypercholesterolaemia (FH) ranging in age from 7 to 41 years were enrolled; each patient tolerated the procedure well without significant complications. Transgene expression was detected in a limited number of hepatocytes of liver tissue harvested four months after gene transfer from all five patients. Significant and prolonged reductions in low density lipoprotein (LDL) cholesterol were demonstrated in three of five patients; in vivo LDL catabolism was increased 53% following gene therapy in a receptor negative patient, who realized a reduction in serum LDL equal to approximately 150 mg dl-1. This study demonstrates the feasibility of engrafting limited numbers of retrovirus-transduced hepatocytes without morbidity and achieving persistent gene expression lasting at least four months after gene therapy. The variable metabolic responses observed following low-level genetic reconstitution in the five patients studied precludes a broader application of liver-directed gene therapy without modifications that consistently effect substantially greater gene transfer.

Adult↗
PubMed1995

Steroid production after in vitro transcription, translation, and mitochondrial processing of protein products of complementary deoxyribonucleic acid for steroidogenic acute regulatory protein.

We have previously demonstrated that steroidogenic acute regulatory protein (StAR) is essential for the rate-limiting step in the acute regulation of steroidogenesis, which is the transport of cholesterol from the outer to the inner mitochondrial membrane. We have hypothesized that this transport occurs as the 37-kilodalton (kDa) precursor form of StAR is imported into the mitochondria and processed to its 30-kDa mature forms. Using an in vitro transcription and translation system in the presence of mitochondria isolated from unstimulated mouse MA-10 Leydig tumor cells, we now directly show that the 37-kDa form is indeed the cytosolic precursor of StAR and can be processed by mitochondria to all four 30-kDa mature forms. To determine the subcellular location of StAR in steroidogenic cells, ultrastructural immunocytochemistry was performed in adrenal zona fasciculata cells using the protein A-gold technique. We show that StAR is associated exclusively with the mitochondria. There, StAR is primarily localized in the intermembrane space and the intermembrane space side of the cristae membrane. StAR was shown to induce steroid production in isolated mitochondria. StAR protein was expressed in COS1 cells and the cell lysate, which was shown to contain abundant levels of StAR by Western blot analysis, was incubated with mitochondria isolated from unstimulated MA-10 cells. In these experiments, StAR increased steroid production by at least 4-fold over control mock-transfected lysate, and this increase was time and dose dependent. Furthermore, the increase in steroid production induced by StAR-containing lysate was not observed when COS1 lysate containing high levels of another mitochondrially imported protein, adrenodoxin, was used. We conclude from these results that in response to tropic hormone stimulation of steroidogenic cells, StAR is synthesized as a 37-kDa precursor, imported into the mitochondria, processed to its 30-kDa mature forms, and localized to the intermembrane space. During import and processing in vitro, StAR induces steroid production in isolated mitochondria in a specific manner.

Animals↗