Biomedical subjects

J P Raynaud

Publications and source records attributed to J P Raynaud.

At least 37 records · Page 2Linked to original sources

PubMed1994

[Sleep disorders in the elderly].

Insomnia dominates the sleep disorders of the elderly. Sleep apnea, the restless legs syndrome and nocturnal agitation represent other facets. After eliminating the possibility of painful or degenerative organic disease, iatrogenic cause and mode of life, other symptoms should be sought, namely mental and personality-related. Investigation requires polysomnogram recording, supported by careful history-taking, including objective and subjective factors. Treatment includes notions of diet, biological rhythm and desynchronisation. It should take into consideration the deleterious effects of prolonged use of certain psychotherapeutic agents.

Aged↗

PubMed1994

Clinical prophylactic activity of melarsomine dihydrochloride (RM 340) against Dirofilaria immitis in heartworm-naive beagles exposed to natural infection in three southeastern states.

Melarsomine dihydrochloride (RM 340), a drug being developed as an adulticide for treatment of heartworm (Dirofilaria immitis) infection in dogs, was safe and highly effective as a clinical prophylactic agent against naturally acquired infections using Strategic and Tactical Treatment Programs. The Strategic Program involved treatment every 4 months (three series of treatments per year), disregarding the mosquito season (MS), to clear the existing infection at each treatment. The Tactical Program consisted of two series of treatments per year, 4 months apart, with the first one given about the middle of the MS (August) and the second one given after the end of the MS (December). Melarsomine was administered as two i.m. injections (lumbar muscles) of 2.2 mg kg-1 given 3 h apart. A total of 90 heartworm-naive beagles and a number of microfilaremic 'seed' dogs were used. Three similar experiments (30 beagles per experiment) were conducted at selected areas (Georgia, Florida, Louisiana) known to be enzootic for heartworm. At each site, 30 beagles were allocated to six groups of five dogs each, and four of these groups were placed outdoors in April of 1988. Two groups (control and treated) were exposed for 12 months, and the treated group was given melarsomine at 4, 8, and 12 months after exposure was started (Strategic Program). Another group was exposed for 8 months and treated with melarsomine at 4 and 8 months (Tactical Program). One group of tracer (sentinel) beagles was exposed from April to August 1988, one group from August to December 1988, and another from December 1988 to April 1989. April-August and August-December tracers served as controls for the tactically treated dogs. After exposure, all dogs were held indoors for 5 months before necropsy. Blood was collected at 4-5 month intervals and examined for microfilariae (MF) and adult heartworm antigen (enzyme-linked immunosorbent assay, ELISA). Treatment by the Strategic Program was 99% effective, with only one of the total of 15 treated dogs harboring any worms (a single female) at necropsy. Thirteen of the 14 control dogs (93%) exposed for 12 months became infected, with average worm recoveries of 6.8, 5.4, and 25.2 (range 1-45) for the Georgia, Florida, and Louisiana sites, respectively. All of the 13 heartworm-infected control dogs were antigen-positive, and 12 of these were also MF-positive, while none of the strategically treated dogs was either antigen- or MF-positive at necropsy. Tactical treatment of the total of 14 dogs twice per year was 100% effective.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Use of melarsomine dihydrochloride (RM 340) for adulticidal treatment of dogs with naturally acquired infections of Dirofilaria immitis and for clinical prophylaxis during reexposure for 1 year.

Heartworm-infected dogs were treated therapeutically with a new heartworm adulticide (melarsomine dihydrochloride, RM 340) and then put on a Strategic Program with treatment every 4 months for clinical prophylaxis to take advantage of the drug's potent activity against 4-month-old immature as well as adult Dirofilaria immitis. Ten random-source dogs with naturally acquired heartworm infections (microfilariae- and antigen-positive) were given melarsomine (2.2 mg kg-1 twice 3 h apart) by i.m. injection in the lumbar muscles to clear their existing infections. They were then placed outdoors (August 1988) in a high-risk area in Georgia (USA) for heartworm transmission and given melarsomine at the same posology every 4 months (Strategic Program) for 12 months as a clinical prophylactic measure. Five nontreated heartworm-naive beagles placed at this site during the same period served as 'controls' to monitor heartworm transmission. After exposure for 12 months, the ten treated and five 'control' dogs were taken indoors and held for 5 months. Microfilaremia and antigenemia levels were monitored in both groups by testing at 4-5 month intervals throughout the study and the intensity of infection was determined at necropsy. Microfilaremia levels in treated dogs dropped dramatically following the initial therapeutic treatment and remained either negative or low. Only two of the five 'control' dogs became microfilaremic, and this occurred near the end of the study. Nine of the ten treated dogs were antigen-negative 4 months after the initial therapeutic treatment, and all of them were antigen-negative at all bleedings thereafter. Four of the five 'control' dogs were antigen-positive at necropsy, and only one of these was positive 4 months earlier. Based on these antigen data, the initial treatment cleared 90% of the dogs of worms, and no worms were detected in any of the treated dogs thereafter. However, it is possible that undetectable immature heartworms were present. Although all of the treated dogs were antigen-negative at necropsy, three of them had a total of eight heartworms, seven of which were clearly immature, as determined by worm length measurements, and the remaining worm was a young adult female that was probably too young to be detected. All of the five 'control' dogs had heartworms (average 7.4; range 1-16), and about half of these worms were clearly immatures. Therapeutic treatment followed by strategic treatment with melarsomine every 4 months during reexposure was at least 89.2% effective overall.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Inhibition of the activity of 'basic' 5 alpha-reductase (type 1) detected in DU 145 cells and expressed in insect cells.

The purpose of this study was 2-fold: (1) to identify the 5 alpha-reductase (5 alpha-R) isozyme(s) present in DU 145 cells, a human cell-line of low androgen sensitivity derived from a cerebral metastasis of an epithelial prostate cancer; and (2) to compare the inhibitory potencies of three compounds on the 'basic' 5 alpha-R isozyme expressed in a baculovirus-directed insect cell system. Conversion of testosterone (T) into 5 alpha-dihydrotestosterone (DHT) in DU 145 cells was measured by HPLC coupled to a Flo-one HP radioactivity detector. DU 145 cells exhibited 5 alpha-R activity (21 pmol DHT/min/mg protein) at pH 7.4 which disappeared at pH 5.5 suggesting that, of the two genomically distinct human isozymes identified so far, type 1 5 alpha-R is expressed in DU 145 cells. This was confirmed by at least two observations: first, 5 alpha-R activity in DU 145 cells was inhibited with much higher potency by 4-MA than by finasteride which is known to be a very poor competitor of the 'basic' enzyme (IC50s = 2.8 +/- 0.2 and 264 +/- 55 nM, respectively). Second, only the type 1 5 alpha-R cDNA and not type 2 5 alpha-R cDNA hybridized with DU 145 RNA. A high potency differential was also recorded for the inhibition of 'basic' type 1 5 alpha-R expressed in a baculovirus-directed-insect cell system by these two compounds, 4-MA being considerably more active than finasteride (Ki = 8.4 +/- 2.3 and 330 +/- 9 nM, respectively). This inhibition was competitive. On the other hand, inhibition by an n-hexane lipid/sterol extract of Serenoa repens (LSESr) was non-competitive and, when expressed in terms of recommended therapeutic doses, was 3-fold greater for LSESr than for finasteride. These studies suggest that LSESr might exert a regulatory inhibitory activity due to its specific lipid/sterol composition.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1994

Affiliations among steroid receptors as revealed by multivariate analysis of steroid binding data.

To illustrate the informative value of descriptive multivariate analysis in biochemical screening, we have analyzed several data matrices relating to the binding of steroids to the estrogen, progestin, androgen, glucocorticoid and mineralocorticoid receptors in different organs and species. We first compared dendrograms of steroid hormone receptors, that were obtained by an automatic hierarchical classification analysis of the binding data, to published phylogenetic trees of nuclear receptors based on amino-acid sequence analysis. The former classification describes the affiliations among the receptors as given by the binding specificity of a population of 187 steroids in a traditional cytosol binding assay (an indirect comparison of ligand binding sites); the latter describes the affiliations among the receptors as given by a comparison of selected primary sequences involved in ligand-dependent regulation of transactivation and dimerization. A similar hierarchical classification was also performed on the binding data of 62 steroids to myometrium cytosol from different species in order to show to what extent the progesterone-binding proteins in these species are affiliated. Hierarchical clustering methods classify each type of variable (receptor or steroid) independently. In order to be able to correlate both types of variable (receptors and steroids) on single-display graphs, it is necessary to resort to correspondence factorial analysis (CFA). CFA ranks the information content within the experimental system, highlighting major correlations and disclosing secondary correlations by eliminating redundant information and background noise. This multivariate method, applied to the analysis of published data, illustrated the particular specificity of estrogen binding in human vagina and raised the question of the nature of the binding protein in this tissue. Our examples are based on small data tables that can and have been analyzed de visu. However, it is certain that such descriptive multivariate techniques are indispensable for the analysis of large data banks not only to define structure-activity relationships but to estimate the degrees of affiliation among the biological variables being measured. Knowledge of such affiliations will help to organize available information in a context where the complexity of the biological systems under study is becoming increasingly apparent.

Animals↗

PubMed1993

Multivariate analysis of plasma hormones in patients with metastatic prostate cancer receiving combined LHRH-analog and antiandrogen therapy.

The following hormones, the plasma protein SHBG, and the tumor markers prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were assayed at 18 time-points over 1 month during a double-blind randomized study in 36 stage D2 cancer patients receiving either "buserelin+placebo" or "buserelin+nilutamide (Anandron)": LH, FSH, estradiol, testosterone, dihydrotestosterone, androstenedione, 5-androstene-3,17 beta-diol, dehydroepiandrosterone and its sulfate, cortisol, 17 alpha-hydroxyprogesterone, pregnenolone, 17 alpha-hydroxypregnenolone, and 3 alpha-androstanediol glucuronide. Multivariate analysis of the treatment values (over 10,000 assays) by two complementary methods, correspondence factorial analysis (CFA) and the minimum spanning tree (MST) method, identified those variables within the pathways of androgen metabolism that were correlated over time and, in a comparison of the two treatment groups, identified the enzyme targets of nilutamide action in humans. Whereas nilutamide tended to decrease androstenedione slightly, it affected no other variables including cortisol, except for pregnenolone, 17 alpha-OH-pregnenolone, and 17 alpha-OH-progesterone, which were increased. These increases are indicative of weak inhibition of C17,20 lyase by nilutamide, but, according to the multivariate analysis, are insufficient to account for the more marked and rapid fall in PAP and PSA noted on addition of nilutamide to buserelin that must therefore be explained by another mechanism such as androgen receptor blockade by nilutamide.

Aged↗

PubMed1993

Relative involvement of protein kinase C and of the estrogen receptor in the cytotoxic action of a population of triphenylethylenes on MCF7 cells as revealed by correspondence factorial (CF) analysis.

A multivariate statistical method, correspondence factorial (CF) analysis, was used to examine the correlations among the protein binding and cell proliferation effects of a series of 36 di- and triphenylethylenes (DPEs and TPEs). The analysis was applied to a study which measured their competition for estradiol binding to cytosol estrogen receptor (ER), their influence on protein kinase C (PKC) activity under different conditions of enzyme activation, their ability to promote the growth of a breast cancer cell line and to inhibit growth at high concentrations (cytotoxicity). The CF analysis revealed several levels of correlation. First, it distinguished those molecules within the population that stimulated rather than inhibited PKC activity. Second, it made apparent a strong correlation between cytotoxicity and inhibition of Ca++ and phosphatidylserine-dependent PKC activity, which was most marked when the enzyme had been activated by diacylglycerol indicating that PKC inhibition under physiological conditions might contribute to the overall cytotoxicity of these compounds. Third, a lower level of correlation was established between competition for ER binding and cytotoxicity. Taken together, the results suggest that MCF7 cells might be most sensitive to a cytotoxic effect of TPEs (via PKC and other targets) when they at the same time decrease estrogen-stimulated proliferation via an ER-mediated antiestrogenic effect.

Animals↗

PubMed1993

A multivariate approach to the description of patient populations. An example of the analysis of the hormone profiles of patients with advanced prostate cancer.

In view of the multifactorial nature of the endocrine dysfunctions that may develop during prostate cancer and the unsuitability of the most widely used statistical methods to study such dysfunction, we have in the present study examined the relationships among 17 biological variables in 26 patients with advanced prostate cancer by two complementary multivariate methods, correspondence factorial analysis (CFA) and a hierarchical automatic classification procedure. The 17 variables included 14 hormones, their precursors or metabolites [LH, FSH, estradiol (E2), testosterone, dihydrotestosterone (DHT), androstenedione (A), androstenediol (Ediol), dehydroepiandrosterone (DHA), DHA-sulphate (DS), cortisol (CORT), 17 alpha-hydroxyprogesterone (17-OH-PROG), pregnenolone (PREG), 17 alpha-hydroxypregnenolone (17-OH-PREG), and androstanediol glucuronide (ADG)], one plasma binding protein, namely, sex-hormone-binding protein (SHBG) and two tumour markers, prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA). The originality of these multivariate methods is that they do not preselect a dependent variable nor perform two-by-two correlations as in stepwise multiple regression analysis but describe the patient population by extracting layers of correlations (from strong to weak) from amid confounding variables. Compared to principal component analysis which is based on covariance, CFA, based on the chi 2-metric, enables the licit representation of both tests and patients on the same factorial maps. From an examination of proximity among variables, it is possible to deduce which tests are related, which groups of patients have similar hormone profiles, and which tests vary most in which patients. The most discriminant factors in this particular population of patients were PSA and PAP levels, which were, however, not strongly correlated and were apparently selectively associated with certain hormones. PAP seemed the more pathological marker; PSA was somewhat anticorrelated to the adrenal androgen (DHA and DS) and PREG levels. The hormones with the lowest variance were A, Ediol and CORT reflecting their key roles in metabolism. A number of patients were hypogonadic. SHBG levels were not closely related to total T levels but anticorrelated with ADG suggesting that, in the patients concerned, SHBG decreases the bioavailable T fraction. There was no correlation between ADG and precursor hormones (PREG, DHA, DS) but a slight anticorrelation between these precursors and DHT. Therefore the source of ADG in these patients does not seem to be increased levels of precursor hormones nor of DHT but increased peripheral tissue metabolism of androgens. In future, descriptive multivariate analyses of large patient cohorts should help to define subpopulations with distinctive hormone profiles for prospective clinical studies.

Hormones↗

PubMed1993

Pulmonary thromboembolism and hypertension after thiacetarsamide vs melarsomine dihydrochloride treatment of Dirofilaria immitis infection in dogs.

The severity of pulmonary thromboembolism and pulmonary hypertension induced by heartworms dying after administration of 2 adulticides was evaluated. Because melarsomine dihydrochloride (RM340) has been shown to be more effective in killing Dirofilaria immitis (heartworms) than the traditional approved adulticide, thiacetarsamide, an attempt was made to determine whether this new adulticide induced more severe lung disease. Before adulticide treatment, 32 dogs with naturally acquired heartworm infections received physical examinations, semiquantitative antigen concentration tests, CBC, platelet counts, serum biochemistry analyses, arterial blood gas determinations, thoracic radiography, pulmonary arteriography, and pulmonary hemodynamic tests. Eight dogs with a low burden and 9 dogs with a high burden of heartworms were treated with thiacetarsamide, and 7 dogs with a low burden and 8 dogs with a high burden were treated with RM340. Except for the heartworm-burden test, tests were repeated at regular intervals during the first 7 weeks after treatment. None of the dogs coughed or had dyspnea after treatment. Six of 9 dogs with high worm burdens and 4 of 8 dogs with low worm burdens had surviving heartworms after thiacetarsamide treatment, in contrast to only 3 of 15 RM340-treated dogs. Differences between the 2 adulticide treatments were minimal as determined by thoracic radiography, pulmonary hemodynamic tests, clinical laboratory analyses, pulmonary arteriography, or necropsy. The RM340 treatment was a more effective adulticide, but it did not increase the severity of hypertension and thromboembolism.

Animals↗

PubMed1992

Multivariate analysis by the minimum spanning tree method of the structural determinants of diphenylethylenes and triphenylacrylonitriles implicated in estrogen receptor binding, protein kinase C activity, and MCF7 cell proliferation.

The response profiles of 36 para-substituted diphenylethylenes (DPEs) and triphenylacrylonitriles (TPEs) have been compared by multivariate analysis. The responses measured were (a) relative binding affinity (RBA) for the cytosol estrogen receptor (ER), (b) ability to promote the growth of the human MCF7 breast cancer cell-line, (c) cytotoxicity in MCF7 cells, and (d) ability to stimulate or inhibit protein kinase C (PKC) III activity under three different conditions of enzyme activation. The prime object of the analysis was to observe the simultaneous influence of diverse combinations of substituents on all these in vitro responses. To do this, the minimum spanning tree (MST) method was used to organize the molecules into a network in which proximate molecules are closely related with regard to their responses whereas remote molecules are distinct. The MST of this population of molecules had four main branches. E2 and its TPE mime were located in a central position within the trunk whereas the tips of the branches tended toward molecules of different specificity, i.e., cytotoxic molecules that bind to ER and interfere with PKC, noncytotoxic molecules that also bind to ER and interfere with PKC but promote cell growth, molecules only active on PKC, and molecules active on all parameters except PKC stimulation. A parallel MST analysis of the relationships among the response parameters themselves confirmed previous conclusions: For this population of molecules, RBAs for ER are fairly closely related to ability to promote MCF7 cell growth and only little to cytotoxicity (Bignon et al. J. Med. Chem. 1989, 32, 2092). Cytotoxicity is much more clearly correlated with inhibition of diacylglycerol-stimulated PKC activity than with RBAs for ER. PKC inhibition differs substantially depending upon whether the substrate is H1 histone or protamine sulfate.

Acrylonitrile↗

PubMed1992

A model for the determination of the 3D-spatial distribution of the functions of the hormone-binding domain of receptors that bind 3-keto-4-ene steroids.

A method of comparing the hydrophobic clusters of proteins (hydrophobic cluster analysis, HCA) has revealed that the 3D-folding pattern of the hormone-binding domain (HBD) of steroid hormone receptors (SHRs) may have an unexpectedly high degree of analogy with the known 3D-crystal structures of proteins belonging to the serine proteinase inhibitor (SERPIN) superfamily, e.g. alpha 1-antitrypsin and ovalbumin. The present paper briefly reviews some of the biochemical evidence that supports the structural validity of the SERPIN model and shows how the model can be used to establish hypothetical 3D-locations for functions attributed to different amino-acids or peptide sequences of the HBD: i.e. heat-shock protein binding, transcription activation, phosphorylation, steroid binding, but also ATP-binding. Indeed, the model has enabled the identification of a Rossmann-fold in SHRs that might bind ATP. Visualization of all these functions should help to interpret the chain of concerted events induced by steroid binding.

Amino Acid Sequence↗

PubMed1992

Thiacetarsamide (adulticide) versus melarsomine (RM 340) developed as macrofilaricide (adulticide and larvicide) to cure canine heartworm infection in dogs.

To implement a new macrofilaricide, treatment of heartworm infection or disease in dogs was checked in all the clinical situations ie from subclinical to severe disease. After preliminary toxicity and efficacy models on experimentally infected dogs, in addition, to the reference posology (2.5 mg of melarsomine (RM 340)/kg twice, 24 h apart by deep IM injection) a more practical program for vet practitioners was suggested (2.2 mg/kg twice, 3 h apart) using modelization of the pharmacokinetic data. The two treatments were equivalent as shown on models with experimental infection of dogs, critical tests on naturally infected dogs and clinical trials in veterinary practice. We advise using specific and well adapted therapeutic programs for each of the clinical classes (class 1: subclinical, class 2: moderate, class 3: severe). The safety margin is respectively x 3 or x 2.5 in contrast with thiacetarsamide which, being hepatotoxic, has no safety margin, and sometimes is nephrotoxic at the recommended dose. RM 340 is fully effective on D immitis adults (even on young ones of 7 months old) and L5 immatures (4 months old) when thiacetarsamide is poorly effective on 7 months or ineffective on 4-month-old parasites. Clinical trials in veterinary practice showed that the programs are well adapted to many clinical situations. The product is effective, relatively safe and easy to handle by IM injection. Preliminary results show its possible use as tactical treatment (2.2 mg/kg twice, 3 h apart) twice a year in mid August and December-January to prevent heartworm disease.

Animals↗

PubMed1991

Influence of di- and tri-phenylethylene estrogen/antiestrogen structure on the mechanisms of protein kinase C inhibition and activation as revealed by a multivariate analysis.

We have performed a systematic study of the interaction of 36 di- and tri-phenylethylene derivatives (DPEs and TPEs) with protein kinase C (PKC). The results were submitted to a multivariate analysis in order to identify the structural features that might be implicated in interference with the activity of three PKC subspecies under three enzyme activation conditions. Four groups of test-compounds, each with common chemical features, could be distinguished clearly. The first group comprised all TPEs substituted with at least one basic dialkylaminoethoxy side-chain. These inhibited type alpha, beta and gamma PKC subspecies activated by Ca2+ and phosphatidylserine (PS) with or without diolein (DO) at micromolar concentrations but did not inhibit protamine sulfate phosphorylation. The other effectors, which all possessed a 1,1-bis-(p-hydroxyphenyl) ethylene moiety, influenced PKC activity at high concentrations (30-200 microM) and could be divided into two groups. One group constituted PKC inhibitors in the TPE series and inhibited PKC activated by Ca2+, PS and DO, as well as protamine sulfate phosphorylation. The other group constituted dual-type inhibitors/activators in the DPE series and stimulated PKC in the presence of Ca2+ and low PS concentrations but inhibited the enzyme in the simultaneous presence of DO. The fourth group of compounds was inactive and had, for the most part, one or two substituents with weak steric hindrance. In agreement with previous data for six lead compounds, this study suggests that, in these chemical series, a basic amino side-chain leads to interaction with phospholipid and the regulatory domain of PKC, whereas a 1,1-bis-(p-hydroxyphenyl) ethylene moiety leads to interaction with the catalytic domain of the enzyme.

Animals↗

PubMed1991

Risk assessment of antibiotic residues of beta-lactams and macrolides in food products with regard to their immuno-allergic potential.

In human medicine drug allergy is a well-established side-effect of the therapeutic use of antibiotics, especially the beta-lactams. Side-effects caused by macrolides are uncommon and only a very few of these seem to be caused by allergic mechanisms. Clinically, drug allergy is characterized by a spectrum of reactions ranging from mild skin rashes to angio-oedema or life-threatening anaphylaxis. Concern has been expressed that antibiotic residues in meat and other foods might be responsible for similar hypersensitivity reactions in a small number of individuals. This review assesses the potential risk of such reactions in general, but focuses on allergy to penicillin and macrolide residues in particular. In relation to the risk of primary sensitization, it is unlikely that residues could contribute to the overall immune response in view of the very low levels that are likely to be encountered in comparison with the high levels received during therapeutic use. No evidence has been found that any individual has become sensitized by residues of either penicillins or macrolides. Furthermore, the oral route is much less sensitizing than parenteral administration and immunochemical studies with penicillin indicate that hapten-protein complexes formed in vivo are unlikely to be immunogenic because of their low dose, low epitope density and binding to autologous carrier proteins. For performed allergens, the epitope density was also too low to be immunogenic. Because of the ubiquitous nature of penicillin-producing moulds in nature and the extensive use of beta-lactam antibiotics in human medicine, it is unlikely that epidemiological studies could be undertaken that could allow quantification of the minimal risk. The risk of allergic reactions in pre-sensitized individuals can be assessed similarly and again it is concluded that factors such as dose, oral administration and low epitope density make it unlikely that a significantly antigenic derivative could be formed. However, a review of the literature on penicillin hypersensitivity revealed a very small number of previously sensitized individuals from whom there is reasonable clinical and documentary evidence that penicillin residues in milk triggered an allergic reaction, usually a rash. Although these cases are very rare (less than 10 cases reported in the last 25 years), they illustrate the continuing need to control antibiotic residues vigilantly. Animal models have not proved useful for predicting the risk of hypersensitivity reactions to drugs, since allergy in man is determined by genetic and other factors and no validated methods exist to determine a no-effect level.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Hydroxylated triphenylacrylonitriles adopt a unique orientation within the binding site of the estrogen receptor.

The relative binding affinities of a series of twelve para-hydroxylated triphenylethylenes (TPEs) for the estradiol receptor (ER) of calf uterus cytosol were measured by a competition method. The results obtained under equilibrium conditions support the hypothesis of the additivity of the energies corresponding to each of the hydrogen-bond type interactions of di- or tri-hydroxylated TPEs with the estradiol binding site of ER and strongly suggest that, whichever ring is hydroxylated, the orientation of the TPE in the steroid binding site is always the same. A hydroxyl group in a given position always interacts with the same location within the site. Mono-hydroxylation of the highly hydrophobic non-substituted TPE skeleton led to a large increase in relative binding affinity for ER which could be explained by a dual mechanism whereby the interaction specific to the hydroxyl is accompanied by a temperature- or time-dependent binding process that is not related to the hydroxylation position.

Animals↗

PubMed1989

Hydrophobic cluster analysis (HCA) of the hormone-binding domain of receptor proteins.

A new technique of protein sequence analysis, namely, Hydrophobic Cluster Analysis (HCA), has been used to align and compare the sequences of proteins belonging to the receptor superfamily (steroid, thyroid hormone and retinoic acid receptors) and serpin superfamily (corticosteroid binding globulin (CBG) and alpha 1-antitrypsin (alpha 1-AT]. By matching up clusters of hydrophobic amino-acids that oftenmost correspond to identifiable secondary structures (alpha-helices, beta-strands etc.), it has been possible to deduce the following information on the secondary structures of these proteins: CBG is structurally related to alpha 1-AT (HCA score greater than 80%), the structures of the hormone-binding domains of the steroid receptors that bind 3-keto-delta 4-steroids are closely interrelated (greater than 80%) but less closely related to that of the estrogen receptor (ER) (approximately 75%), vitamin D, retinoic acid and thyroid hormone receptors are structurally closely related (greater than or equal to 80%). Their secondary structures are, however, also related to that of the steroid receptors (approximately 70%), and a high degree of analogy exists between the structures of serpins and of the hormone-binding domains of members of the steroid superfamily (60-70%). HCA has clearly shown that a previous local sequence alignment of the estrogen receptor with other steroid receptors and cytochromes P450 has to be reconsidered. The published consensus steroid binding sequence previously identified in cytochromes is in fact 80 amino-acids upstream from its previously defined position. Other regions of contiguous sequence identity have also been identified which may be involved in the hydrophobic core of the protein or in steroid binding. Their positions have been indicated using the crystal structure of alpha 1-AT as a model.

Amino Acid Sequence↗

PubMed1989

Non-michaelian behavior of 5 alpha-reductase in human prostate.

An in-depth analysis of the kinetics of 5 alpha-reductase in human prostatic tissue gave findings inconsistent with the claim that the enzyme is michaelian. In both hyperplastic and malignant tissue, the time-course of the conversion of testosterone (T) into dihydrotestosterone (DHT) was non-linear under conditions ensuring less than 15% conversion of substrate and cofactor. An initial rapid phase of conversion was followed by a long steady-state phase. This time-dependent change in conversion rate was not due to enzyme denaturation, fast inhibition by substrate or product effects. It resulted from a true slow transient kinetic process induced in the reactive enzyme by the substrates. Under our experimental conditions at pH 5.5, 5 alpha-reductase appeared to undergo a conformational change from an initially highly reactive form to a less reactive form. Since this "hysteretic" behavior was correlated with apparently negative cooperativity in enzyme kinetics, we postulate that, as previously described for other key metabolic enzymes, regulation of 5 alpha-reductase activity in the prostate depends on the molecular flexibility of the enzyme and on changes in the cooperativity of different enzyme forms over time. This original non-michaelian behavior may explain the conflicting kinetics reported so far in the literature for this enzyme. The clinical implications of 5 alpha-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1989

Effect of triphenylacrylonitrile derivatives on estradiol-receptor binding and on human breast cancer cell growth.

In a study of a series of 26 triphenylacrylonitrile derivatives (TPEs), we investigated the influence of several possibly interrelated factors on the proliferation of human breast cancer cell lines. (1) Chemical substituents: the test compounds were for the most part para-hydroxylated with increasingly bulky hydrophobic and/or basic side chains [isopropyloxy or (diethylamino)ethoxy] or standard reference compounds. (2) Relative binding affinities (RBAs): they competed diversely for [3H]estradiol (E2) binding to calf uterus cytosol and little, if at all, for binding to the [3H]tamoxifen-labeled antiestrogen binding site (AEBS) in lower speed supernatant. A multiparametric comparison of RBAs recorded for calf, rat, and mouse uterus cytosol estrogen receptor (ER) revealed a possible influence of species-specific receptor conformation and/or environment on binding. (3) Estrogen/antiestrogen potency: their stimulation and inhibition of the proliferation of the ER-positive human breast cancer cell line (MCF7) was measured. Compounds with only hydroxy substituents stimulated proliferation more markedly than methylated derivatives and had a maximum effect at 10(-11)-10(-6) M. Stimulation was related to the RBA for ER. Compounds with isopropyloxy or (diethylamino)ethoxy side chains only weakly stimulated MCF7 cell growth and more powerfully antagonized E2-promoted growth. The extent of inhibition depended upon the bulk of the side chain and could be reversed by 10(-7) M E2. Within the same concentration ranges, the test compounds were without effect on the BT20 ER-negative cell line. (4) Cytostatic and/or cytolytic activity: most compounds could arrest the proliferation of both MCF7 and BT20 cells at concentrations above 3 x 10(-6) M. This activity was thus independent of ER. Nevertheless, those compounds with a charged hydrophobic side chain, which were the most powerful antagonists of E2-promoted cell growth, were also the most cytotoxic. The overall results for all molecules on all parameters were submitted to a multivariate analysis (correspondence analysis) which revealed the progressive influence of increasing substitution by hydroxy and more bulky groups on the generation of antagonist activity and cytotoxicity.

Animals↗