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Biomedical subjects

J Frost

Publications and source records attributed to J Frost.

At least 19 recordsLinked to original sources
PubMed1992

Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity.

c-Jun, a major component of the inducible transcription factor AP-1, is a phosphoprotein. In nonstimulated fibroblasts and epithelial cells, c-Jun is phosphorylated on a cluster of two to three sites abutting its DNA-binding domain. Phosphorylation of these sites inhibits DNA binding, and their dephosphorylation correlates with increased AP-1 activity. We show that two of these sites, Thr-231 and Ser-249, are phosphorylated by casein kinase II (CKII). Substitution of the third site, Ser-243, by Phe interferes with phosphorylation of the inhibitory sites in vivo and by purified CKII in vitro. Microinjection into living cells of synthetic peptides that are specific competitive substrates or inhibitors of CKII results in induction of AP-1 activity and c-Jun expression. Microinjection of CKII suppresses induction of AP-1 by either phorbol ester or an inhibitory peptide. These results suggest that one of the roles of CKII, a major nuclear protein kinase with no known functions, is to attenuate AP-1 activity through phosphorylation of c-Jun.

Amino Acid Sequence↗
PubMed1988

The stability in vitro of bioactive and immunoreactive LH in human blood and plasma.

The degree of stability in vitro of bioactive and immunoreactive LH in human blood, plasma and serum was examined. Bioactivity and immunoreactivity of LH were assayed by the dispersed mouse Leydig cell assay and by standard radioimmunoassay respectively, using the same reference preparation (first international reference preparation for human pituitary LH 68/40 for immunoassay). Bioactive and immunoreactive LH were stable in blood and plasma at 22 degrees C for up to 4 and 24 h respectively, and in blood at 4 degrees C for up to 24 h. There was no loss of biological or immunological LH activity in plasma which had been either snap-frozen and stored at -70 degrees C, allowed to freeze at -20 degrees C and stored at that temperature or kept at 4 degrees C for 24 h and then stored at -70 degrees C. Likewise, the levels of LH in plasma and serum which had been stored at either -20 or -70 degrees C and then thawed and refrozen up to four times remained unchanged. In addition, the biological and immunological activity of LH was not affected after vortexing samples of plasma or serum for up to 60 s. Bioactive LH was also stable in plasma samples after prolonged storage (up to 9 months) at either -70 or -20 degrees C. We conclude that LH bioactivity and immunoreactivity are stable in blood and plasma following a variety of treatments commonly experienced during normal handling of a blood sample after venepuncture.

Biological Assay↗
PubMed1988

Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. I. Evidence for efficacy in models of focal cerebral ischemia.

Recent studies have strongly implicated the excitatory neurotransmitter glutamate in the cascade of pathological mechanisms that cause neuronal loss after certain types of brain ischemia. The neurotoxic effects of glutamate are mediated, at least in global ischemia, via NMDA receptors. In the present study we have examined the effects of compounds that possess NMDA receptor antagonist properties (ifenprodil, SL 82.0715 [(+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl)methyl]- 1-piperidineethanol] and 1-[1-(2-thienyl)cyclohexyl]piperidine) on the histological consequences of focal, as opposed to global, cerebral ischemia in both the rat and the cat. Ifenprodil (0.3-3 mg/kg i.v.) administered as a perfusion over 3 hr after occlusion of the feline middle cerebral artery reduced the volume of infarcted tissue (measured 4 days after occlusion) in a dose-related manner. At the highest dose a 42% reduction of infarcted volume was noted, essentially in cortical tissue. In an identical protocol, a derivative of ifenprodil, SL 82.0715, reduced the volume of infarction in a manner comparable to that described for ifenprodil. As SL 82.0715 possesses better p.o. bioavailability, this compound was also evaluated in the rat, again after middle cerebral artery occlusion. First administered 30 min after the induction of ischemia, SL 82.0715 (1 and 10 mg/kg p.o.) reduced infarction volume by 34 and 48%, respectively. The quantitative histology was performed 2 days after middle cerebral artery occlusion. The noncompetitive receptor antagonist, 1-[1-(2-thienyl)cyclohexyl]piperidine, administered (1 mg/kg i.p.) before the induction of focal ischemia, similarly and significantly decreased the final volume of infarction. As both ifenprodil and SL 82.0715 are noncompetitive antagonists of the NMDA receptor, two conclusions may be drawn from the present investigation. First, NMDA antagonism by ifenprodil and its derivative is an effective approach for tissue sparing in animal models of stroke and brain infarction. Second, these pharmacological observations provide evidence for the involvement of excitatory amino-acid induced-neurotoxicity in the evolution and consequences of focal cerebral ischemia.

Animals↗
PubMed1986

On the assessment of the in vitro biopotency and site(s) of action of drugs affecting adrenal steroidogenesis.

Dispersed guinea-pig adrenal cells have been employed in the in vitro estimation of the biological potency and sites of action of drugs acting against the adrenal. The effect of 12 drugs on cortisol secretion from cells stimulated with adrenocorticotrophin (ACTH, 50 ng/L, a 95% saturating dose) has been tested. All the drugs depressed cortisol output in a dose-related fashion. The concentration of drug which inhibited secretion by 50% was (mumol/L, mean +/- SEM): etomidate 0.1 +/- 0.002; epostane 0.44 +/- 0.02: 17-ketotrilostane 0.55 +/- 0.04: trilostane 1.3 +/- 0.1: metyrapone 3.5 +/- 0.6: cyproterone acetate 4.6 +/- 0.2: megestrol acetate 11 +/- 2: danazol 22 +/- 2: aminoglutethimide 41 +/- 5: stanozolol 50 +/- 4: thiopentone 160 +/- 18: propofol 170 +/- 18. The sites of the anti-steroidogenic effect of seven of these drugs have also been established using a method based upon the sequential stimulation by the exogenous precursor steroids of the various steps leading to the biosynthesis of cortisol by adrenal cells. Propofol acts between ACTH binding and pregnenolone production, trilostane, megestrol acetate and cyproterone acetate are 3 beta-hydroxysteroid dehydrogenase inhibitors whereas metyrapone, etomidate and thiopentone act at 11 beta-hydroxylase.

Adrenal Cortex↗
PubMed1986

Effect of adrenocorticotrophin on cortisol and androstenedione secretion from dispersed cells of guinea-pig adrenal zonae fasciculata and reticularis.

We have studied cortisol and androstenedione secretion by dispersed cells of the outer zona fasciculata (ZF) plus zona glomerulosa, and the inner zona reticularis (ZR) plus medulla of the guinea-pig adrenal. The ZF and ZR were microdissected apart, the cells dispersed and incubated (200 000 cells/ml) for 90 min in the presence of adrenocorticotrophin (ACTH; 500 ng/l), dibutyryl cyclic AMP (dbcAMP; 1 mmol/l), pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol. The steroid concentrations were 5-25 mumol/l. Cortisol secretion was assayed by radioimmunoassay. There was no detectable cortisol secretion (less than 50 nmol/l) from the ZR in the controls (no additive) or after dbcAMP stimulation. Adrenocorticotrophin-stimulated cortisol secretion was also low (range less than 50-340 nmol/l). In contrast the ZF secreted 177-379 (control), 828-2052 (dbcAMP) and 2863-9735 (ACTH) nmol cortisol/l. There was no detectable (i.e. less than 2 nmol/l) cAMP production by ZR or ZF either basally (no ACTH) or after ACTH stimulation (500 ng/l). Challenge of the ZR cells with each cortisol precursor steroid (5 mumol/l) increased (P less than 0.05) cortisol secretion over that seen with the corresponding basal and ACTH-stimulated controls. Thus pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol (converted directly to cortisol by 21-hydroxylase) gave rise to (mean +/- S.D., n = 4) 406 +/- 86, 680 +/- 180, 1307 +/- 111, 1141 +/- 234 and 3160 +/- 419 nmol cortisol/l respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

17-alpha-Hydroxypregnenolone↗
PubMed1985

A simple in vitro approach to the estimation of the biopotency of drugs affecting adrenal steroidogenesis.

Dispersed guinea-pig adrenal cells can be maximally stimulated to secrete cortisol by adrenocorticotrophin (ACTH greater than 50 ng/l). Further, this stimulation appears to be specific to ACTH alone, with other naturally occurring chemicals (e.g. steroids, protein hormones) at supra-physiological concentrations being without effect on cortisol production. The effect of drugs of differing structure and therapeutic function (aminogluthethimide, metyrapone, trilostane, 17-ketotrilostane, danazol, epostane, megestrol acetate, stanozolol and etomidate) on ACTH-stimulated (50 ng/l) cortisol production has been tested in this system. All the drugs depressed steroid output in a similar dose-related fashion. The concentration of drug which inhibited cortisol output by 50% was (mumol/l, mean +/- SEM): etomidate 0.097 +/- 0.002: epostane 0.44 +/- 0.02: 17-ketotrilostane 0.55 +/- 0.04: trilostane 1.3 +/- 0.1: metyrapone 3.5 +/- 0.6: megestrol acetate, 11 +/- 2: danazol 22 +/- 2: aminogluthethimide 41 +/- 5: stanozolol 50 +/- 4. Thus, etomidate, an anaesthetic, is more potent than the established anti-steroidogenic drugs metyrapone, aminogluthethimide and trilostane. Further, direct anti-steroidogenic effects have been demonstrated for megestrol acetate and stanozolol for the first time. We conclude that this technique offers a promising new approach to the assessment of biological potency of drugs affecting endocrine tissues.

Adrenal Glands↗
PubMed1985

On the stability in vitro of bioactive human adrenocorticotrophin in blood and plasma.

The disappearance in vitro of ACTH from whole human blood and plasma held at 22 degrees C has been monitored using a dispersed adrenal cell bioassay and an unextracted radioimmunoassay. In three normal subjects after a metyrapone test and two patients with Addison's disease, endogenous bioactive ACTH levels were unchanged for at least 1 h in blood and 2 h in plasma. Moreover greater than 50% of the bioactive plasma ACTH was still present in the plasma samples from the patients with Addison's disease after 24 h incubation at ambient temperatures. Human pituitary ACTH (1-39), spiked into plasma from dexamethasone suppressed subjects to give a concentration of 250 ng/l, was stable by bioassay for at least 2 h. No loss of biological activity was observed on subjecting plasma from a patient with Addison's disease or spiked plasma to two cycles of thawing at 37 degrees C and freezing at -70 degrees C or thawing at 20 degrees C and freezing at -20 degrees C. Some loss of bioactivity (20%) occurred on subjecting the patient's, but not ACTH-spiked plasma to four cycles of thawing at 20 degrees C/freezing at -20 degrees C. We conclude that bioactive ACTH (endogenous or exogenous) may be more stable in vitro in human blood and plasma than has been previously thought. If our studies can be confirmed in a larger series then it may be that conditions for handling blood specimens for ACTH assays could be reappraised.

Addison Disease↗
PubMed1985

Guinea pig striatum as a model of human dopamine deamination: the role of monoamine oxidase isozyme ratio, localization, and affinity for substrate in synaptic dopamine metabolism.

The kinetic properties of type A and type B monoamine oxidase (MAO) were examined in guinea pig striatum, rat striatum, and autopsied human caudate nucleus using 3,4-dihydroxyphenylethylamine (dopamine, DA) as the substrate. MAO isozyme ratio in guinea pig striatum (28% type A/72% type B) was similar to that in human caudate nucleus (25% type A/75% type B) but different from that in rat striatum (76% type A/24% type B). Additional similarities between guinea pig striatum and human caudate nucleus were demonstrated for the affinity constants (Km) of each MAO) isozyme toward DA. Endogenous concentrations of DA, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were also measured in guinea pig and rat striatum following selective type A (clorgyline-treated) and type B (deprenyl-treated) MAO inhibition. In guinea pig, DA metabolism was equally but only partially affected by clorgyline or deprenyl alone. Combined treatment with clorgyline and deprenyl was required for maximal alterations in DA metabolism. By contrast, DA metabolism in rat striatum was extensively altered by clorgyline but unaffected by deprenyl alone. Finally, the deamination of DA in synaptosomes from guinea pig striatum was examined following selective MAO isozyme inhibition. Neither clorgyline nor deprenyl alone reduced synaptosomal DA deamination. However, clorgyline and deprenyl together reduced DA deamination by 94%. These results suggest that the isozyme localization and/or isozyme affinity for DA, rather than the absolute isozyme content, determines the relative importance of type A and type B MAO in synaptic DA deamination. Moreover, based on the enzyme kinetic properties of each MAO isozyme, guinea pig striatum may serve as a suitable model of human DA deamination.

Animals↗
PubMed1985

On the biopotency and site of action of drugs affecting endocrine tissues with special reference to the anti-steroidogenic effect of anaesthetic agents.

Dispersed guinea-pig adrenal cells or mouse Leydig cells were stimulated with a saturating dose of adrenocorticotrophin (ACTH, 50 ng/1) or luteinizing hormone (LH, 5IU/1), respectively. The incubations were performed in the presence of increasing concentrations (10(-9) - 5 X 10(-4)mol/l) of the anaesthetic agents propofol, thiopentone and etomidate. At the end of this stimulation period, cortisol (from the adrenal preparation) or testosterone (from the Leydig cell culture) were assayed by radioimmunoassay. Propofol, thiopentone and etomidate all inhibited ACTH-stimulated cortisol secretion in a dose-related fashion. Similar inhibition of LH-stimulated testosterone output was found with propofol and thiopentone whereas etomidate was without effect at any concentration employed, an observation in accordance with its known site of action, 11 beta-hydroxylase, an enzyme which is not involved in the biosynthesis of testosterone. The concentration (mumol/l) of anaesthetics which gave 50% inhibition (ED50) of ACTH-stimulated cortisol secretion was 0.1 +/- 0.002 (n = 7), 160 +/- 18 (n = 3) and 170 +/- 18 (n = 3) (mean +/- s.e.m.) for etomidate, thiopentone and propofol, respectively. The corresponding values for the LH stimulated testosterone output from the Leydig cell preparations were 186 (thiopentone) and 180 (propofol) mumol/l. In a separate series of experiments adrenal cells were stimulated with (a) the cortisol precursor steroids (all at 10(-5)mol/l) pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone and 11-deoxycortisol, (b) dibutyryl cAMP (10(-3)mol/l) or (c) ACTH (100 ng/l) in the presence and absence of either etomidate (5 X 10(-5)mol/l), propofol (2.5 X 10(-4)mol/l) or thiopentone (5 X 10(-4)mol/l). All the stimulators increased cortisol production by > 7-fold over that seen in their absence. Propofol depressed ACTH and dibutyryl cAMP induced cortisol output by > 60% (P < 0.05) but was without effect when the steroid precursors were used, suggestive of an inhibition between the sequence involving ACTH binding -> pregnenolone production. In contrast, etomidate and thiopentone reduced cortisol secretion by > 40% (P < 0.05) regardless of the stimulator used, indicating that at least one site of action was at the level of the final enzymic step of cortisol synthesis, i.e. 11beta-hydroxylase.

Adrenocorticotropic Hormone↗
PubMed1985

Myocardial infarction and cerebral infarction in a Danish suburban community.

A comparison was made of 485 cases of cerebral infarction (CI), registered prospectively in Frederiksberg, Copenhagen, with 495 cases of myocardial infarction (AMI), recorded retrospectively in the same population. The overall annual incidence of AMI was 6.5 per 1,000 population for males, 3.8 for females; the rates for CI were 2.4 and 2.5, respectively. The age-incidence curves, particularly those for AMI, were significantly steeper in the females than in the males. Correspondingly, the male predominance in the AMI-group subsided with age. Age-specific incidence-rates rose exponentially with advancing age; for each sex and diagnosis the relationship can thus be expressed as a simple mathematical formula, which may facilitate comparisons of incidence patterns in different communities. Theories explaining the similarities and differences of the age-incidence curves for AMI and CI are discussed.

Adult↗
PubMed1984

Cortisol production by dispersed guinea-pig adrenal cells; a specific, sensitive and reproducible response to ACTH....and its fragments.

Naturally occurring steroids and peptide hormones, tested at supraphysiological concentrations, were without effect on basal and human (h) 1-39 ACTH (NIBSC code 74/555, 25 ng/l (5.5 X 10(-12) mol/l] stimulated cortisol production. Further, low concentrations of angiotensin II, N-pro-opiocortin (N terminal fragment 1-76) and gamma-MSH all of which have been reported to synergise with ACTH with regard to cortisol production, were without significant effect alone or in combination with ACTH over the range 2.2 X 10(-13) to 5.5 X 10(-12) mol/l. The activity of h 1-39 was compared with that of the ACTH related peptides 1-24, 1-18, 1-17, 1-16, 1-13-NH2 (alpha MSH), 1-10 and 4-10. The dose responses were parallel and the same maximal cortisol output was observed with all the peptides except the 1-10 fragment. Half maximal stimulation occurred at 3.1 X 10(-12) (1-24), 4.4 X 10(-12) (h 1-39), 1.5 X 10(-11) (1-39), 3.3 X 10(-10) (1-18), 5 X 10(-9) (1-13-NH2), 8 X 10(-9) (1-17), 2 X 10(-7) (1-16) and 1 X 10(-5) (4-10) mol/l respectively. Interference by the above ACTH-derived peptides in cortisol secretion by the cells in response to 5.5 X 10(-12) mol/l h 1-39 ACTH was minimal over the range 5.2 X 10(-12)-2.2 X 10(-6) mol/l. The sensitivity of the adrenal cells to h 1-39 ACTH was such that 2 ng/l (4.4 X 10(-13) mol/l) provoked cortisol secretion over the control (P less than 0.05, n = 17). The coefficient of variation within assay for each dose on the full standard curve (2.2 X 10(-13)-1.1 X 10(-10) mol/l) was less than 10% (n = 6). Half maximal stimulation was given by 14.5 ng/l (3.2 X 10(-12) mol/l). Between control and 1.1 X 10(-10) mol/l ACTH there was a 32 +/- 8 (mean +/- SD, n = 9) fold change in cortisol production.

Adrenal Glands↗
PubMed1984

Preliminary experiences with a bioassay for adrenocorticotrophin (ACTH) in unextracted human plasma using dispersed guinea-pig adrenal cells.

A simple, accurate and precise dispersed cell bioassay suitable for the assay of ACTH levels greater than 50 ng/1 in unextracted human plasma is described. Isolated adrenal cells were prepared by tryptic digestion of the guinea-pig adrenal gland and cortisol production by these cells was controlled specifically by ACTH. The cortisol response to 2 ng/1 of human pituitary ACTH (1-39) was always significantly (P less than 0.05, n = 17) different from the control. ACTH concentrations between 1 and 20 ng/1 gave a 14 +/- 3 fold (n = 17) increment in cortisol response. Dilutions of test plasmas were parallel with standard ACTH (1-39) when the plasma concentrations were maintained throughout at 4% (1 in 25 dilution) by the addition of dexamethasone-suppressed plasma ('ACTH free'). Using a 25 fold plasma dilution, the limit of detection of ACTH in unextracted plasma was 50 ng/1. Recovery of ACTH (1-39) spiked into dexamethasone suppressed plasma at concentrations of 250 and 100 ng/1 was 97 +/- 10% (n = 17) and 114 +/- 15% (n = 17) respectively. The within assay coefficient of variation (CV) of both quality control and patients' samples never exceeded 9% while between assay variation was less than or equal to 13%. The sample throughput was 30 plasma samples/week/technician.

Addison Disease↗
PubMed1984

On the site of action of the anti-adrenal steroidogenic effect of etomidate and megestrol acetate.

The sites of action of the anti-steroidogenic action of etomidate and megestrol acetate have been established with a novel in vitro approach based upon the inhibition of cortisol (Co) secretion by dispersed guinea-pig adrenal cells. The cells were challenged with the Co precursor steroids (all at 10(-5) mol/l) pregnenolone (Pe), 17-hydroxy-pregnenolone (17-Pe), progesterone (Po), 17-hydroxyprogesterone (17-Po), 21-deoxycortisol (21-DOC) and 11-deoxycortisol (11-DOC), or 1-24 adrenocorticotrophin (ACTH, 100 ng/l) in the presence or absence of either etomidate, megestrol acetate, metyrapone or trilostane (all at 5 X 10(-5) mol/l). In the absence of drug, the steroid precursors or ACTH provoked a cortisol secretion of greater than 14 times that secreted by cells incubated in their absence. ACTH-stimulated Co secretion was inhibited by greater than 85% by all the drugs employed. In the presence of trilostane and megestrol acetate, Co secretion provoked by the delta 4 3-keto steroids (Po, 17-Po, 21-DOC and 11-DOC) was similar to the controls. However, with the delta 5, 3 beta-hydroxy steroids, 17-Pe and Pe, Co secretion was inhibited by greater than 57% in the presence of these drugs. In contrast, etomidate and metyrapone inhibited Co secretion by greater than 60% when 11-deoxycortisol was employed, indicative of a block at 11 beta-hydroxylase, the final step in the cortisol biosynthetic pathway. Similar results were seen with Pe, 17-Pe, Po and 17-Po, all of which are converted to cortisol via a biosynthetic route which includes catalysis by 11 beta-hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗