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Biomedical subjects

D K Scott

Publications and source records attributed to D K Scott.

35 records · Page 2Linked to original sources
PubMed1992

Activation of the phosphoenolpyruvate carboxykinase gene retinoic acid response element is dependent on a retinoic acid receptor/coregulator complex.

The accessory factor 1 (AF1) element is an upstream transcriptional control region that plays a role in the response of the phosphoenolpyruvate carboxykinase (PEPCK) gene to both glucocorticoids and retinoic acid. We demonstrate here that retinoic acid receptor alpha (RAR alpha) binds to a sequence within the AF1 element, TGACCT (site B), that is a consensus retinoic acid response element (RARE) half-site. A similar DNA sequence, TGGCCG (site C), located 1 bp downstream of site B, is not involved in the binding of RAR alpha monomers or dimers but is required for the constitution of a functional RARE. Site C is also required for the formation of a complex involving RAR alpha and a liver nuclear factor designated CR, for coregulator. Mutational analysis of the AF1 element shows that the RAR alpha/CR complex is the trans-acting unit that mediates the retinoic acid response of the PEPCK gene. Another member of the retinoid receptor family, retinoid X receptor alpha (RXR alpha), can also form a complex with RAR alpha and the AF1 element. Several observations, including the observation that RXR alpha antibody interacts with CR, indicate that RXR alpha and CR are identical or closely related proteins. Through RXR alpha forms a complex with RAR alpha and the AF1 element, we demonstrate that the AF1 element is functionally distinguishable from a retinoid X response element. Taken together, our results show that the AF1 element contains an RARE that mediates a retinoic acid response by binding an RAR alpha/coregulator complex; this coregulator is presumably RXR alpha.

Base Sequence↗
PubMed1992

Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells.

PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis.

Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells.

The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells.

We have isolated clones for rat collagenase from a rat osteoblastic cell cDNA library. These clones have been sequenced and the amino acids deduced. The calculated molecular weight is 51,352 for the proenzyme and 42,229 for the active enzyme. The deduced amino acid sequence was compared to those previously reported for: 1) human collagenase, 2) rat transin 1 (stromelysin), 3) human stromelysin, and 4) rabbit collagenase. The number of amino acids conserved was 47, 47, 50, and 47%, respectively. We also compared the collagenase mRNA and protein in different rat cells (osteoblast, uterine smooth muscle, synovial fibroblast) and determined that in rat uterine cells the message is slightly larger, although collagenase protein in all three cell types was identical in size. Parathyroid hormone dramatically induces the 2.9-kilobase collagenase mRNA in the rat osteoblastic cells, UMR 106-01. Nuclear run-on studies in UMR 106-01 cells demonstrated a 4-8-fold induction in the rate of synthesis of collagenase mRNA at 2 and 4 after parathyroid hormone treatment, with steady state levels of mRNA increased 100-fold at 4 h. Thus, parathyroid hormone regulation of the collagenase gene in UMR 106-01 cells is in part transcriptional.

Amino Acid Sequence↗
PubMed1990

Stability of doxorubicin, daunorubicin and epirubicin in plastic syringes and minibags.

The shelf lives of doxorubicin, daunorubicin and epirubicin in infusion fluids were studied using high-performance liquid chromatographic (HPLC) methods. Doxorubicin and epirubicin were stable (loss in potency of less than 10%) for 24 and 20 days respectively, when dissolved in sodium chloride solution (0.9%; pH, 6.47) at 25 degrees C and stored in polyvinyl chloride (PVC) minibags, while daunorubicin was stable for at least 43 days. All three drugs were stable for at least 43 days in sodium chloride (0.9%; pH 6.47 and 5.20) and dextrose (5%; pH 4.36) at 4 and -20 degrees C. Repeated thawing and re-freezing of these solutions at ambient temperature did not cause degradation. All three drugs were stable for at least 43 days when reconstituted with Water-for-Injections BP and stored in polypropylene syringes at 4 degrees C.

Chromatography, High Pressure Liquid↗
PubMed1990

Photodegradation of doxorubicin, daunorubicin and epirubicin measured by high-performance liquid chromatography.

The degradation kinetics of doxorubicin, daunorubicin and epirubicin in aqueous solution under fluorescent light and sunlight were studied using high-performance liquid chromatographic (HPLC) methods. The rates of photodegradation of all three drugs were similar, they were inversely proportional to the drug concentration and were accelerated by an increase in the pH of the vehicle. Photodegradation followed first-order kinetics. At concentrations greater than or equal to 500 micrograms/ml no special precautions appeared to be necessary to protect freshly prepared solutions of these agents from light. Photolysis was very rapid, however, at concentrations in the low microgram range therefore, when these solutions are used for in-vitro work or stability studies, they should be protected from light at all times. In addition, adsorptive losses, which may also be pronounced in low concentration solutions, should be prevented by storage in polypropylene containers.

Analysis of Variance↗
PubMed1989

Regulation of adenylate cyclase by cannabinoid drugs. Insights based on thermodynamic studies.

The abilities of lipophilic cannabinoid drugs to regulate adenylate cyclase activity in neuroblastoma cell membranes were analyzed by thermodynamic studies. Arrhenius plots of hormone-stimulated adenylate cyclase activity exhibited a break point at 20 degrees. The break point was reduced to 14 degrees by benzyl alcohol, consistent with results from other laboratories that have correlated this response with the increase in membrane fluidity induced by benzyl alcohol. Because cannabinoid drugs partition into membrane lipids and alter membrane fluidity parameters in a number of model systems, it was of interest to examine the influence of delta 9-tetrahydrocannabinol and cannabidiol on enzyme activity analyzed by the Arrhenius plot. delta 9-Tetrahydrocannabinol, known to inhibit adenylate cyclase, failed to decrease the transition temperature either at 1 microM or at concentrations exceeding its aqueous solubility (30 microM), suggesting that delta 9-tetrahydrocannabinol could not mimic the effects observed with benzyl alcohol. In contrast, 30 microM cannabidiol, which stimulated enzyme activity slightly, decreased the Arrhenius plot break point to 17.5 degrees. The decrease in the transition temperature in response to benzyl alcohol or cannabidiol was not accompanied by a change in activation energies above or below the transition temperature. delta 9-Tetrahydrocannabinol inhibits adenylate cyclase activity via Gi as does the muscarinic agonist carbachol (Howlett et al., Mol Pharmacol 29: 307-313, 1986). Both carbachol and delta 9-tetrahydrocannabinol decreased the enthalpy and entropy of activation. The net free energy of activation at 37 degrees was increased in the presence of both of these inhibitory agonists. These data suggest that, for the entropy-driven hormone-stimulated adenylate cyclase enzyme, less disorder of the system occurs in the presence of regulators that inhibit the enzyme via Gi. In summary, thermodynamic data suggest that cannabidiol can influence adenylate cyclase by increasing membrane fluidity, but that the inhibition of adenylate cyclase by delta 9-tetrahydrocannabinol is not related to membrane fluidization.

Adenylyl Cyclase Inhibitors↗
PubMed1989

Optimization of blood sampling time after intravenous bolus doses of gentamicin.

Comparisons were made, based on a one-compartment model, between predicted serum concentrations obtained at various sampling times after i.v. bolus and i.m. injections of gentamicin. Post-dose concentrations 1 h after i.m. injection were within 3% of the maximum concentration at steady-state, indicating that this was the optimal sampling time. Serum concentrations 1 h post-i.v. bolus were less than 7% different from those 1 h post-i.m. injection. In a clinical trial, blood samples taken earlier during the distribution phase of the drug, after an i.v. bolus, resulted in serum concentrations which were up to 74% higher than concentrations at 1 h. A survey of a sample of U.K. hospitals showed that only 41% were taking samples at 1 h post i.v. bolus of gentamicin. The majority of hospitals recommended a range of post-dose serum concentrations between 4 and 12 mg/l for patients with septicaemia, but when adjusted for errors related to sampling time, 36% of hospitals recommended a minimum which was equivalent to less than 4 mg/l at 1 h post dose. Post-dose concentrations of gentamicin should be measured in samples taken 1 h after either i.v. bolus and i.m. injections.

Gentamicins↗
PubMed1984

Comparison of methods for estimating gentamicin clearance and retrospective analysis of changes in clearance with emphasis on patients with normal renal function.

The subjects were 29 general medical and surgical patients with apparently normal renal function at the start of treatment with gentamicin. Nine methods of estimating gentamicin clearance (CL) were compared as means of calculating gentamicin dosage. In 23 of the 29 patients CL was measured twice during treatment. The first measurement was based on 10 blood samples taken around a dose early in treatment. CL calculated from the area under the serum concentration-time curve using all 10 samples was designated a reference method for comparison with other methods. There was considerable inter-individual variation in CL. Serum creatinine and body weight gave poor estimates of an individual's CL. A good estimate of CL was obtained from four blood samples taken immediately before and 2, 3 and 4 h after dosing. Prediction of serum levels is not a good method for checking CL estimations. A significant decrease in CL was found during treatment. This was probably due to a reduction in glomerular filtration rate.

Adult↗
PubMed1983

Clinical experience with a method for adjusting gentamicin dose from measured drug clearance.

Gentamicin clearance was measured in 26 patients by a method which requires four blood samples. Clearance was used to calculate mean steady state serum concentrations [Css] with the aim of maintaining Css within the range 2.5-3.0 mg/l. All of the patients who failed to respond to antibiotic treatment had adequate Css and peak levels; all of these patients had serious underlying problems to explain their poor response. In contrast some patients responded satisfactorily despite being apparently underdosed. In two patients poor clinical response was associated with low Css levels despite adequate doses and peak levels; both patients responded to increased doses. In patients with stable renal function doses calculated from initial clearance measurements did result in Css within the desired range. In patients with unstable renal function measurement of clearance gave a better indication of the severity of renal impairment than measurement of serum creatinine or peak and trough levels but the bioassay used was too slow to allow adequate dose adjustment.

Adult↗
PubMedDate not supplied

Whatalotwegot--the messages in drug advertisements.

Advertisers are increasingly using symbols to circumvent logical argument when trying to persuade people (the "targets" of the advertisement) to make choices that are not strictly rational. Symbols can convey covert meanings and awaken or exploit subconscious feelings, such as a desire for power or a fear of doing harm. Some of the ways in which pharmaceutical advertisements use these techniques are examined: advertising by contagion; adding to our worries; polarity of choices; teasers; idealisation. Rational prescribing should be based on logic, but advertisements do not depend on logical arguments for their most powerful effects: the advertisers may subvert us by appealing to our unconscious desires.

Advertising↗