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Biomedical subjects

B J Clark

Publications and source records attributed to B J Clark.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1995

Hormonal and developmental regulation of the steroidogenic acute regulatory protein.

A crucial event in the acute regulation of steroidogenesis by trophic hormones is the delivery of cholesterol into the mitochondria where it is converted to pregnenolone by the cholesterol side chain cleavage enzyme. Although considerable controversy exists regarding the exact mechanisms that underlie this acute response to hormone stimulation, recent studies suggest that the Steroidogenic Acute Regulatory (StAR) protein, a hormone-induced 30-kilodalton mitochondrial protein, plays an essential role. We now extend these studies by establishing in MA-10 mouse Leydig tumor cells a temporal relationship between levels of StAR expression and steroidogenesis in response to hormone stimulation. These data indicate that trophic hormones regulate StAR mRNA and protein within a time frame concomitant with the acute production of steroid hormones and provide the first evidence implicating changes in StAR transcription and/or mRNA stability in the functional response of steroidogenic cells to hormone action. In addition, in situ hybridization analyses of StAR expression in embryonic and adult mice demonstrated a precise spatial and temporal relationship in vivo between StAR expression and the capacity to produce steroid hormones. These experiments strengthen considerably the evidence that StAR is the key mediator of the acute induction of steroidogenesis and provide new insights into the mechanisms by which trophic hormones activate steroidogenesis in steroidogenic 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↗
PubMed1994

Microbial contamination of cases used for storing contact lenses.

A survey of the hygienic practices of 178 contact lens wearers in west central Scotland was conducted along with assessment of microbial contamination of their lens storage cases. Multivariate analysis indicated that in some subgroups of lens wearers, the method of disinfection used, the use of unmodified tap water and the age of the lens, were significantly associated with microbial contamination of storage cases. In addition, statistical analysis suggested that features other than those commonly considered as important in contact lens hygiene, and therefore included in this study, may be more significant predictors of contamination of cases. Instruction of patients, patient compliance and simplicity of the regimen for the care of contact lenses may be important for achieving their safer use.

Adolescent↗
PubMed1993

Clinical utility and cost-effectiveness of an air suspension bed in the prevention of pressure ulcers.

OBJECTIVE: To determine, in critically ill patients at risk, both the clinical utility and cost-effectiveness of using an air suspension bed in the prevention of pressure ulcers. DESIGN: Randomized, parallel group, controlled clinical trial with accompanying cost-effectiveness analysis. SETTING: 30-bed multidisciplinary intensive care unit. PATIENTS: 100 consecutive patients at risk for the development of pressure ulcers randomly assigned to receive treatment on either an air suspension bed or a standard intensive care unit bed. Patients considered at risk were those at least 17 years of age with an Acute Physiology and Chronic Health Evaluation II (APACHE II) score greater than 15 who had an expected intensive care unit stay of at least 3 days. MAIN OUTCOME MEASURES: The development of pressure ulcers by site and severity and the costs associated with each of the two programs. RESULTS: The air suspension bed was associated with fewer patients developing single, multiple, or severe pressure ulcers. In patients at risk, the use of an air suspension bed in the prevention of pressure ulcers was a cost-effective therapy. CONCLUSIONS: Despite intense nursing care, pressure ulcers are more prevalent in the critically ill patient population than in the general hospital population. Air suspension therapy provides a clinically effective means of preventing pressure ulcers in these patients. In patients at risk, air suspension therapy was a cost-effective means of managing pressure ulcers compared with the standard hospital bed.

Adolescent↗
PubMed1993

LC studies on the potential interaction of paraben preservatives with sorbitol and glycerol.

Pharmaceutical formulations often contain one or more paraben preservatives in conjunction with a polyol such as sorbitol or glycerol. In one of these experimental formulations a number of unknown polar peaks have been detected near the solvent front by reversed-phase LC. These degradation products were not attributable to the active drug component or a hydrolysis product. The possibility of an interaction between the polyols and paraben preservatives has been explored using a three-variable, two-level, factorial design to determine the relative significance of the factors involved in the formation of these unknown peaks. The factors examined were pH, temperature and the ratio of polyol to paraben. This study has shown that pH and temperature are key factors affecting the formation of these unknown peaks. On the basis of these results suitable conditions can be suggested for minimizing the production of these unknown peaks. It seems clear that a number of pharmaceutical formulations containing a polyol and a paraben would present potential problems for assay validation on storage owing to the formation of these degradation products, particularly if the drug component is polar and elutes near the solvent front.

Chromatography, Liquid↗
PubMed1993

The requirement of phosphorylation on a threonine residue in the acute regulation of steroidogenesis in MA-10 mouse Leydig cells.

In the present study we have used several non-phosphorylatable analogs of the amino acids threonine and serine to determine the role of phosphorylation in the acute regulation of steroidogenesis in MA-10 mouse Leydig tumor cells. Our results indicate that substitution of the threonine analog into protein results in a inhibition of hormone stimulated steroid production in these cells while none of the serine analogs employed displayed a similar inhibition. Strikingly, only the threonine analog resulted in the inhibition of the synthesis of several 30 kDa mitochondrial proteins which we have previously shown to be induced by hormone stimulation of MA-10 cells. Thus, it is apparent that phosphorylation of a threonine residue is obligatory for the acute production of steroids in MA-10 Leydig cells and also for the synthesis of a series of previously described mitochondrial proteins. However, a causal relationship between the 30 kDa mitochondrial proteins and steroid regulation cannot be made unequivocally at this time.

Alkaline Phosphatase↗
PubMed1993

Microbial contamination of contact lens cases in the west of Scotland.

A cross-sectional study of 178 asymptomatic contact lens wearers attending 10 contact lens practices in the west of Scotland was conducted over a 4 month period. The aims of the study were to identify specific microbial contaminants in lens cases, to determine the rate of contamination of such containers and to assess the value of the steps involved in different lens care regimens in the prevention of case contamination. Microbial contamination affected 53% of lens cases. Cases used with conventional wear and disposable systems were contaminated at similar rates and, therefore, the advantage of regular lens replacement may have been lost if these lenses were stored in contaminated cases. Four percent of lens cases were contaminated with amoebal species and all of these showed concomitant bacterial colonisation. These findings imply that case hygiene is probably as important as lens hygiene if new or disinfected lenses are not to be immediately re-contaminated by storage in dirty cases. Unfortunately simple and effective methods of lens and case disinfection, which would be suitable for use in the average home environment, are not yet available. It follows that frequent and regular disposal of lens cases may prove to be a necessary measure to prevent the build-up of microbial colonisation in such containers.

Adolescent↗
PubMed1993

The effects of hydrogen peroxide on steroidogenesis in mouse Leydig tumor cells.

It has previously been reported that treatment of rat luteal cells and human granulosa luteal cells with hydrogen peroxide (H2O2) results in a significant inhibition of steroid production. The mechanism of inhibition in the former was found to be at the level of cholesterol transport into the mitochondria, whereas in the latter it was found to be a result of inhibition of one or more enzymes in the steroidogenic pathway. In the present study we examined the effects of H2O2 on hormone-stimulated steroid production in another steroidogenic cell type, the Leydig cell. Our results demonstrate that treatment of either LH- or cAMP analog [(Bu)2cAMP]-stimulated MA-10 mouse Leydig tumor cells with H2O2 results in a dose-dependent inhibition of the production of progesterone (the major steroid produced in MA-10 cells). It was also observed that similar concentrations of H2O2 resulted in a significant inhibition of protein synthesis, a finding which could in part be responsible for the observed decrease in steroid production. Furthermore, although H2O2 resulted in a dose-dependent inhibition of (Bu)2cAMP-stimulated pregnenolone production, addition of the hydroxylated intermediate 22R-hydroxycholesterol and inhibitors of further pregnenolone metabolism demonstrated that cholesterol side-chain cleavage complex activity was unaffected by H2O2. Conversely, incubation of H2O2-treated cells in the presence of pregnenolone resulted in a very significant inhibition of progesterone synthesis. These data indicate that H2O2 inhibits steroidogenesis in Leydig tumor cells primarily by inhibiting the activity of the 3 beta-hydroxysteroid dehydrogenase, but that other effects of H2O2 such as inhibition of protein synthesis and the transfer of cholesterol to the cholesterol side-chain cleavage complex may also be involved.

Animals↗
PubMed1993

Does implementing pulse oximetry in a critical care unit result in substantial arterial blood gas savings?

OBJECTIVES: To examine the impact of pulse oximetry on the use of arterial blood gas and other laboratory determinations and to examine predictors of the use of arterial blood gas measurements. DESIGN: Before (preoximetry)/after (postoximetry) study. SETTING: Thirty-bed multidisciplinary critical care unit. PATIENTS: Consecutive admissions of 300 patients (150 before and 150 after oximetry). MEASUREMENTS: For each patient examined, the number of arterial blood gas determinations, serum electrolyte levels, complete blood chemistries, arterial lactate levels, and creatinine samples were recorded for the initial 9 days of the stay in the critical care unit. These data were stratified by nursing shift (day vs night) and by the source of the admission (medical vs surgical). Other information collected included demographic variables, the severity of illness, the length of stay in the critical care unit, and various ventilatory parameters. RESULTS: Introducing pulse oximetry was associated with a marginal (10.3 percent; p < 0.025) reduction in the use of arterial blood gas determinations. This decrease was accounted for by changes occurring on the night shift and in the surgical patient. These findings were also observed for serum electrolyte determinations. No significant differences in the use of arterial blood gas measurements were found for medical patients. No significant differences were found in the use of arterial lactate levels, complete blood chemistries, or creatinine determinations. Significant predictors of arterial blood gas determinations included the number of days intubated, the number of ventilator orders, the number of days on an inspired oxygen content (FIO2) greater than 50 percent, and the acute physiology and chronic health evaluation II (APACHE II) score. CONCLUSIONS: The implementation of pulse oximetry in this manner gives an idea how effective the technology will be in reducing the use of arterial blood gas determinations without guidelines for the use of pulse oximetry. As only a marginal decrease was observed in the total population of medical and surgical patients, and only on the night shift, formal and standardized guidelines for the most efficient use of pulse oximetry should be considered. If these were considered, pulse oximetry may indeed make a significant contribution to improving the efficiency of care services.

Blood Chemical Analysis↗
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↗