L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide): a novel thromboxane-prostaglandin endoperoxide antagonist

The effects of L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide) have been studied on pulmonary and other smooth muscle preparations in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-641,933 produced significant shifts in the dose-response curves to the prostaglandin endoperoxide analogues, U-44069 (pA2 7.06) and U-46619 (pA2 7.14), and prostaglandin (PG) F2 alpha (pA2 6.33) had minimal activity against contractions induced by histamine (pA2 4.38), 5-hydroxytryptamine (pA2 4.63), and acetylcholine (pA2 4.56) and slightly enhanced relaxation induced by PGE2. When tested on the guinea-pig gall bladder strip in vitro, L-641,953 antagonized contractions induced by U-44069 (pA2 7.03) but was less active against those induced by PGF2 alpha (pA2 6.03), PGE1 (pA2 5.62), and histamine (pA2 4.84). When tested in vitro on the guinea-pig pulmonary artery, L-651-953 significantly antagonized contractions induced by U-44069 (pA2 7.04), U-46619 (pA2 7.14), and PGF2 alpha (pA2 7.16) but was less effective against contractions induced by histamine (pA2 4.19). Schild analysis indicated that L-641,953 was fully competitive against contractions of either the guinea-pig tracheal chain induced by U-46619 or the guinea-pig pulmonary artery induced by U-44069 and U-46619. When tested on human platelets in vitro L-641,953 inhibited aggregation induced by U-44069 (IC50 1.3 X 10(-6) M) but not ADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of the messenger RNA encoding the B880 holochrome protein of Rhodospirillum rubrum

The B880 holochrome messenger RNA was extracted from cultures of the photosynthetic bacterium Rhodospirillum rubrum. It was purified by chromatography on Sepharose 4B followed by sucrose density gradient centrifugation. The purified fractions were shown to program an Escherichia coli cell-free system into synthesizing both the alpha and the beta polypeptides of the holochrome. The translation products were identified by immunoprecipitation with specific antibodies raised against these polypeptides. The latter are effective competitors with the translation products for antigen-antibody complex formation. The purest mRNA preparations contained approximately 33% holochrome messenger RNA activity. Its most probable size, as determined by agarose gel electrophoresis in the presence of 6 M urea or methylmercuric hydroxide, is approximately 620 nucleotides. Since the combined sizes of the alpha and beta polypeptides add up to only 106 amino acid residues, we conclude that the holochrome mRNA is most probably polycistronic.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Lipoproteins: carriers of dehydroepiandrosterone fatty acid esters in human serum

The association between lipoproteins, cholesterol and cholesteryl esters is very well known to facilitate both the transport in plasma and the entry of these non-polar compounds into the cellular compartment. However, recent observations suggest that in addition to cholesterol, lipoproteins contain several other steroids in their lipoidal metabolite forms which may be transported in the very low, low and high density lipoproteins in human serum. Using the important androgen and oestrogen precursor, dehydroepiandrosterone (DHEA), the biosynthetic formation of lipoidal DHEA was demonstrated in human serum. Serum was also fractionated into its lipoprotein components during the course of its incubation with tritiated DHEA. A progressive movement of the label from the fraction containing the conventional steroid binding-proteins to the lipoproteins was observed with the fraction containing the low density lipoproteins demonstrating the greatest incorporation of the label. This displacement occurred simultaneous to an extensive esterification of the labelled DHEA in serum. After 6 h of incubation, approx. 90% of the radioactivity in all the lipoprotein fractions was in the lipoidal form. Very little labelled lipoidal DHEA was associated with the serum protein fraction throughout the duration of incubation. These data suggest that lipoproteins act as the carriers of lipoidal DHEA following its formation from the non-conjugate parent steroid in serum.     

Formation of lipoidal steroids in follicular fluid

The presence of high levels of lipoidal pregnenolone in follicular fluid has recently been established although no evidence has been presented concerning its possible origin. The following investigation focuses on the enzymatic conversion of non-conjugated steroids into their lipoidal derivatives in preovulatory follicular fluid obtained from women undergoing in vitro fertilization. Our observations indicated that pregnenolone, an important precursor steroid, was acylated at a similar rate as cholesterol in follicular fluid. Similar studies were subsequently conducted with serum obtained from a pool of normal women and women undergoing follicular stimulation which showed little difference to the results obtained in follicular fluid. Further studies using dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, estradiol and dihydrotestosterone were were also performed to monitor their respective lipoidal conversion percentages in follicular fluid which revealed a marked difference of conversion rates between steroids. The indirect identification of the lipoidal pregnenolone derivatives formed in follicular fluid was also conducted by incubating radiolabelled pregnenolone in follicular fluid. The fatty acid components of the resulting lipoidal pregnenolone derivatives showed a marked resemblance to those of cholesteryl esters formed in plasma by the enzymatic activity of lecithin:cholesterol acyltransferase. The pregnenolone derivatives were comprised predominantly of unsaturated fatty acids such as linoleate, palmitoleate, oleate, linolenate and arachidonate while saturated fatty acids, namely palmitate, constituted 20% of the total lipoidal pregnenolone.     

Pre-inoculation of Ri T-DNA-transformed pea roots with Pseudomonas fluorescens inhibits colonization by Pythium ultimum Trow: an ultrastructural and cytochemical study

The influence exerted by Pseudomonas fluorescens, strain 63-28R, in stimulating plant defense reactions was investigated using an in-vitro system in which Ri T-DNA-transformed pea (Pisum sativum L.) roots were subsequently infected with Pythium ultimum. Cytological investigations of samples from P. fluorescens-inoculated roots revealed that the bacteria multiplied abundantly at the root surface and colonized a small number of epidermal and cortical cells. Penetration of the epidermis occurred through the openings made by the disruption of the fibrillar network at the junction of adjacent epidermal cell walls. Direct cell wall penetration was never observed and bacterial ingress into the root tissues proceeded via an intercellular route. Striking differences in the extent of fungal colonization were observed between bacterized and non-bacterized pea roots following inoculation with P. ultimum. In non-bacterized roots, the pathogen multiplied abundantly through most of the tissues while in bacterized roots, pathogen growth was restricted to the epidermis and the outer cortex. At the root surface, the bacteria interacted with the pathogen, in a way similar to that observed in dual culture tests. Most Pythium cells were severely damaged but fungal penetration by the bacteria was never observed. Droplets of the amorphous material formed upon interaction between the bacteria and the host root were frequently found at the fungal cell surface. Incubation of sections with a -1,4-exoglucanase-gold complex revealed that the cell wall of markedly altered Pythium hyphae was structurally preserved. Successful penetration of the root epidermis was achieved by the few hyphae of P. ultimum that could escape the first defensive line in the rhizosphere. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. The unusual occurrence of polymorphic wall appositions along the host epidermal cells was an indication that the host plant was signalled to defend itself through the elaboration of physical barriers.Abbreviations AGL Aplysia gonad lectin - PGPR plant growth-promoting rhizobacteria The authors wish to thank Sylvain Noël for excellent technical assistance. This study was supported by grants from the Fonds Québécois pour la formation de chercheurs et l'Aide à la Recherche (FCAR), the Natural Sciences and Engineering Council of Canada (NSERC) and the Ministère de l'Industrie, du Commerce, de la Science et de la Technologie (SYNERGIE).     

Adrenal steroidogenesis in the guinea pig: effects of androgens.

In humans, the onset of adrenache has been found to occur with the appearance of the zona reticularis, the inner zone of the adrenal cortex. Since an increase in the volume of adrenal cortex during maturation in the guinea pig has been associated with the growth of the zona reticularis, we were interested in investigating the changes in adrenal steroidogenesis during maturation in this species. In addition, the effect of androgens on adrenal steroidogenesis was studied. We demonstrated that between 1 and 10 weeks of age, a period of maximal growth of the adrenals in the guinea pig, there is a decrease in the concentrations of adrenal pregnenolone, cortisol, dehydroepiandrosterone, testosterone, androstenedione, and 11 beta-hydroxyandrostenedione, suggesting lower steroid production by the guinea pig adrenals. In plasma, we observed that the concentration of 11 beta-hydroxyandrostenedione (the sole C19 steroid present after castration) remained unchanged during maturation, while cortisol and corticosterone were lower between 1 and 4 weeks of age. Although castration as well as the administration of the antiandrogen flutamide had no effect on adrenal steroidogenesis, dihydrotestosterone caused an inhibition of cortisol and corticosterone levels in the adrenals while the concentrations of progestins (namely, pregnenolone, 17-hydroxypregnenolone, progesterone, and 17-hydroxyprogesterone) tended to increase in the adrenals, thus suggesting that dihydrotestosterone induces a blockade in the steroidogenic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

A physiological dose of estradiol with progesterone affects striatum biogenic amines

The acute effect of estradiol and progesterone on dopamine and serotonin metabolism in rat striatum was studied. One subcutaneous injection of 17 beta-estradiol (300 ng) and progesterone (150 micrograms) into intact male rats increased plasma levels of these steroids, while testosterone, corticosterone, and estrone remained unchanged. Dehydroepiandrosterone, androstane-3 beta, 17 beta-diol and dihydrotestosterone remained undetectably low. Prolactin decreased and androstane-3 alpha, 17 beta-diol, and 17-OH progesterone increased, but less than estradiol and progesterone. Peak levels of striatal dopamine, dihydroxyphenylacetic acid, and homovanillic acid were observed 15-45 min after steroid injection with a return to control values after 45-60 min, while serotonin and 5-hydroxyindoleacetic acid levels were slightly decreased. An injection of estradiol (70 ng) with progesterone (70 micrograms) to ovariectomized female rats left plasma prolactin levels unchanged, while striatum dopamine and serotonin as well as their metabolite concentrations peaked 15-60 min after steroid injection and returned to control values after 45-75 min. To allow for a better comparison of the action of these steroids, the effect of estradiol or progesterone alone and in combination on the brain of ovariectomized rats was compared in the same experiment. A similar increase in metabolites of dopamine levels was observed after these steroids alone or in combination, while dopamine levels were increased only after progesterone alone or in combination with estradiol. An injection of estradiol or progesterone to ovariectomized rats led to peak steroid concentrations at approximately the same time in the brain and plasma. In addition, plasma and brain steroid levels were significantly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colorimetric determination of N-hydroxylated metabolites in microsomal studies

A colorimetric method is described which can be used for the routine determination of primary and secondary N-hydroxylamino compounds in drug metabolism studies using microsomal preparations. The assay is carried out on the supernatant obtained after protein precipitation of the incubation mixture. The method is based on the reduction of ferric ion by the hydroxylamino moiety with the resulting ferrous ion being quantitated by coupling with 2,4,6-tripyridyl-s-triazine to form a purple color with a maximum absorbance at 595 nm. The method is specific to primary and secondary hydroxylamino compounds and calibration curves can be obtained in the range of concentrations of 1 to 20 μg/ml. Hydroxamic acid, amido, amino, phenolic, nitro, nitroso, oxime, nitrone, aldehyde, and ketone compounds do not produce any color reaction when analyzed by the same method. The method is simple, rapid, and many samples can be analyzed in a short period of time.