PubMed Health⌕ Search

Biomedical subjects

B J Clark

Publications and source records attributed to B J Clark.

At least 19 recordsLinked to original sources
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

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↗
PubMed1995

Differential effects of dimethylsulfoxide on steroidogenesis in mouse MA-10 and rat R2C Leydig tumor cells.

The effects of dimethylsulfoxide (DMSO) on steroidogenesis in MA-10 mouse Leydig tumor cells and R2C rat Leydig tumor cells were compared. MA-10 cells produce low basal levels of progesterone (as the major steroid synthesized), which increase several hundred-fold when stimulated with tropic hormone or the cAMP analog (Bu)2cAMP. R2C cells, on the other hand, constitutively produce high levels of progesterone in the absence of hormone stimulation of any kind. When incubated in the presence of 5% DMSO, MA-10 cells demonstrated an almost complete inhibition of progesterone production, whereas the synthesis of this steroid was virtually unaffected in R2C cells. The inhibition of steroid production in the MA-10 cell could not be attributed to an effect on protein synthesis, because this was unaffected by DMSO during the course of the incubations. The activity of the cholesterol side-chain cleavage enzyme was also unaffected by DMSO, as demonstrated by incubation of the cells with 22R-hydroxycholesterol. The production of cAMP in response to tropic hormone (hCG) and forskolin stimulation was significantly inhibited in MA-10 cells, but was much less affected in R2C cells in response to forskolin treatment. However, DMSO appeared to have no effect on the overall phosphorylation of proteins when tested either in a completely in vitro system or when MA-10 cell homogenates were used as a source of exogenous protein. Strikingly, DMSO treatment resulted in the highly specific inhibition of a series of 30-kilodalton mitochondrial proteins (named StAR for Steroidogenic Acute Regulatory protein), which we have recently shown to be indispensable for the production of steroids in MA-10 cells. The synthesis of these same proteins was much less affected in R2C cells. Although the mechanism of action by which DMSO inhibits steroidogenesis remains unknown, these results show that its action results in the complete cessation of synthesis of the StAR protein, which is required for the acute regulation of steroidogenesis in MA-10 cells.

Animals↗
PubMed1994

The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR).

The acute response of steroidogenic cells to trophic hormone stimulation is the mobilization of cholesterol from cellular stores to the mitochondrial outer membrane and the transfer of this cholesterol to the mitochondrial inner membrane where the first enzymatic step in steroidogenesis occurs. The transfer of cholesterol across the mitochondrial membranes is dependent upon de novo protein synthesis, and this is the regulated step in the process. Although the newly synthesized regulatory protein(s) have yet to be identified, we previously have proposed a candidate protein which we identified in MA-10 cells that is synthesized in response to luteinizing hormone stimulation and that is localized to the mitochondria. In the present study, we report the isolation of a cDNA that encodes this luteinizing hormone-induced protein. Analysis of the cDNA and protein sequences reveals this is a novel protein. Importantly, we demonstrate for the first time that expression of the protein in MA-10 cells in the absence of hormone stimulation is sufficient to induce steroid production. We conclude that this protein is required in the acute regulation of steroidogenesis and propose to call this protein the Steroidogenic Acute Regulatory protein (StAR).

Amino Acid Sequence↗
PubMed1992

Functional expression of bovine 17 alpha-hydroxylase in COS 1 cells is dependent upon the presence of an amino-terminal signal anchor sequence.

Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.

Amino Acid Sequence↗
PubMed1992

The assay and resolution of the beta-blocker atenolol from its related impurities in a tablet pharmaceutical dosage form.

For the complete resolution and determination of the beta-blocker atenolol and its manufacturing impurities a high-performance liquid chromatographic method is developed using structured optimization techniques. The method utilizes a 300 x 3.9 mm i.d. stainless steel column packed with mu Bondapak C18 and a mobile phase containing methanol-25 mM potassium dihydrogen orthophosphate containing 0.06% (v/v) dibutylamine (20:80, v/v) (pH 3.0). The flow rate of 1.0 ml min-1 is used and a detection wavelength of 226 nm. The linearity and repeatability are good for the present compound over the range 1.5-510.0 micrograms ml-1 (r greater than 0.99 and RSD 0.27%, n = 10). Application of the method to 50 and 100 mg tablets gave recoveries of 99% (w/w) and reproducibilities of (RSD) 1.1 and 0.52%, respectively (n = 5). The manufacturing impurities are found to be greater than 0.5% (w/w) of the atenolol peak.

Atenolol↗
PubMed1991

Reversed-phase and chiral high-performance liquid chromatographic assay of bupivacaine and its enantiomers in clinical samples after continuous extraplural infusion.

A previously unreported coupled achiral-chiral high-performance liquid chromatographic method has been developed to assay the levels of bupivacaine and its enantiomers in plasma samples, after the local anaesthetic had been given as a continuous extrapleural intercostal nerve block for 4 days, to relieve postoperative pain following thoracotomy. The method has been used to determine maximum, individual enantiomer and steady state levels in conjunction with an assessment of whether accumulation of bupivacaine occurs. An off-line extraction sample preparation is involved before determination of the "total" levels and final sample clean-up on a cyanopropyl silica column prior to "heart cutting" of the bupivacaine peak to a Chiral-AGP column for assay of the enantiomeric ratio. For an initial 5 patient number the mean maximum level was 5.43 micrograms/ml against a reported toxic level of around 5 micrograms/ml, which was reached in 72 h and the S-enantiomer gave slightly increased concentrations over the R-enantiomer for which there is some evidence of higher toxicity.

Adult↗
PubMed1991

The hydrophobic amino-terminal sequence of bovine 17 alpha-hydroxylase is required for the expression of a functional hemoprotein in COS 1 cells.

The endoplasmic reticulum is a major site of localization for eukaryotic cytochrome P-450 mixed-function oxidase complexes. Previous studies have shown that the microsomal forms of P-450 insert into the membrane via their hydrophobic amino terminus through the signal recognition particle-dependent pathway. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the endoplasmic reticulum of COS 1 cells to evaluate the functional role of its hydrophobic amino-terminal sequence and membrane insertion. An NH2-terminal truncated protein, P450 delta 2-17, which lacked amino acids 2-17 was expressed in COS 1 cells, subcellular fractions were isolated, and P450 delta 2-17 was localized by immunoblot analysis. Compared to the full-length P45017 alpha, the NH2-terminal truncation resulted in a 2.5-fold decrease in P45017 alpha protein recovered with the microsomal fraction, 50% of which was an integral membrane protein as defined by resistance to Na2CO3 extraction. Despite correct membrane localization, P450 delta 2-17 was not a functional enzyme in COS 1 cells. A CO difference spectrum of microsomes containing P450 delta 2-17 did not give a typical 450 nm absorbance. We conclude that the hydrophobic amino terminus is required for the expression of a functionally competent P45017 alpha in COS 1 cells and suggest that the insertion of the amino terminus into the membrane is necessary for the folding of this protein into its correct structural form.

Amino Acid Sequence↗
PubMed1991

Longitudinal spin-order-based pulse sequence for lactate editing.

A new pulse sequence which edits proton spectra of lactate with full signal return and gives good suppression of water and fat signals is described. This sequence exploits longitudinal spin-order from lactate to edit lactate from fat. Experimental results from phantoms and excised pig heart are presented.

Animals↗
PubMed1991

X-linked dilated cardiomyopathy with neutropenia, growth retardation, and 3-methylglutaconic aciduria.

Seven boys with an apparently X-linked syndrome of dilated cardiomyopathy, growth retardation, neutropenia, and persistently elevated urinary levels of 3-methylglutaconate, 3-methylglutarate, and 2-ethylhydracrylate were studied. The natural history of the disorder was characterized by severe or lethal cardiac disease and recurrent infections during infancy and early childhood but relative improvement in later childhood. The initial presentation of the syndrome varied from congenital dilated cardiomyopathy to infantile congestive heart failure to isolated neutropenia without clinical evidence of heart disease. The excretion of 3-methylglutaconate and 3-methylglutarate appeared to be independent of the metabolism of leucine, the presumed precursor of these organic acids in humans. Although the cause of the organic aciduria remains obscure, the constellation of biochemical and clinical abnormalities forms a distinct syndrome that may be a relatively common cause of dilated cardiomyopathy or neutropenia in boys during infancy and childhood.

Adult↗