Constituents of human meconium--I. Identification of 3-hydroxy-etianic acids

The monohydroxylated fraction of bile acids of human meconium was analyzed by capillary GC-MS. In the sulfate-glucuronide fraction three saturated, and one unsaturated C20 steroidal acids were found. These acids were identified as 3 alpha-hydroxy-5 alpha-, 3 alpha-hydroxy-5 beta-,3 beta-hydroxy-5 alpha-androstane-17 beta-carboxylic, and 3 beta-hydroxyandrost-5-ene-17 beta-carboxylic based on the unequivocal GC-MS comparison with standards of all possible epimers at C-3, 5 and 17. The amount of the major C20 acid, 3 alpha-hydroxy-5 alpha-androstane-17 beta-carboxylic, in meconium was 0.2 nmol/g, i.e. 5 to 10 times the amount of lithocholic acid. To prevent the oxidation of 21-hydroxy-20-oxopregnanes to C20 acids meconium was extracted in the presence of sodium borohydride. In the absence of this reducing agent the amount of 3 beta-hydroxyandrost-5-ene-17 beta-carboxylic acid was increased and its 17 alpha-epimer could be detected. This indicates partial artifactual formation of this C20 acid from 21-hydroxypregnenolone, which is known to be present in human meconium. The amount of the saturated C20 acids was unaffected by the presence of sodium borohydride in the extraction medium, and their native occurence in human meconium was further confirmed by the absence of their 17 alpha-epimers in extracts obtained both with and without borohydride. The probable metabolic origin of C20 acids in the fetal-placental-maternal unit is discussed.     

Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.     

Peripheral benzodiazepine binding sites: Effect of PK 11195, 1-(2-chlorophenyl)-n-methyl-n-(1-methylpropyl)-3-isoquinolinecarboxamide: I. In vitro studies

[³H] R05-4864 binding sites have been characterized in kidney, heart, brain, adrenals and platelets in the rat. In all these organs the following order of potency in the R05-4864 displacement was found : R05-4864 > diazepam > clonazepam indicating that they correspond to the “peripheral type” of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [³H] R05-4864 from its binding sites in all the organs. PK 11195 was as potent as R05-4864 in the platelets, heart, adrenals, kidney and several brain regions (midbrain, hypothalamus, medulla + pons and hippocampus. However it was 5 to 10 times more effective in cortex and striatum. In conclusion PK 11195 might represent a new tool to elucidate the physiological relevance of “peripheral type” benzodiazepine binding sites and might help to discriminate the hypothetical subclasses of these binding sites.     

The effect of some environmental factors on the production of L-DOPA by alginate-entrapped cells of Mucuna pruriens

In-vitro-grown cells of Mucuna pruriens, immobilized in calcium-alginate gels, were able to transform the precursor L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). After the immobilization in alginate the plant cells released 90% of the produced L-DOPA into the medium; supplementation of the medium with calcium inhibited both the transformation of L-tyrosine into L-DOPA and the release of L-DOPA into the medium. Continuous illumination of the beads had a slight beneficial effect on the synthesis of L-DOPA. A simple production medium for the transformation of L-tyrosine into L-DOPA was designed. This medium contained only sucrose and sodium chloride as osmotic stabilizers, a low concentration of calcium chloride for stabilization of the alginate beads, and L-tyrosine as the precursor.     

Biochemical parameters to assess cell differentiation of Bouvardia ternifolia Schlecht callus

Calli derived from leaves and radicles of B. ternifolia were grown on Murashige and Skoog (MS) basal medium, and the effects of different nitrogen sources on the rate of callus growth and on the enzymes related to nitrogen assimilation were studied. Ammonium alone did not support callus growth unless a Krebs-cycle intermediate was added to the medium. The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (EC 1.4.1.2) were measured in homogenates of callus grown on media supplied with different nitrogen sources. The results indicate that leaf and root calli have similar levels of these enzymes when grown on MS medium (Murashige and Skoog 1962. Physiol. Plant. 15, 473–497). However, when the calli were supplied with glutamine as the sole nitrogen source, the activity of glutamate synthase increased in leaf callus but was almost completely inhibited in root callus. The results indicate that calli originated from different B. ternifolia tissues do not have the same biochemical dedifferentiated state.     

The effect of calmodulin and far-red light on the kinetic properties of the mitochondrial and microsomal calcium-ion transport system from corn

The kinetic properties of active Ca²⁺ transport into mitochondria and microsomal membrane vesicles prepared from coleoptiles of dark-and light-grown corn seedlings have been studied. The apparent values for K _m and V _max for Ca²⁺ of the mitochondrial transport system from dark-grown plants are about one order of magnitude higher than those from the microsomal transport system. Calmodulin has no effect on the Ca²⁺ accumulation into mitochondria whereas the apparent maximum transport velocity and affinity for Ca²⁺ of the microsomal Ca²⁺-transport system are both increased by calmodulin. When intact corn seedlings are irradiated with far-red light, the calmodulin-induced increase of the apparent maximum transport velocity and affinity for Ca²⁺ can no longer be observed. From these data it can be concluded that the low cytoplasmic Ca²⁺ concentration in the cytoplasm of coleoptile cells from dark-grown corn is maintained by a calmodulin-regulated Ca²⁺ pump. Irradiation with photomorphogenically active far-red light lowers the Ca²⁺-transport activity and thus causes an increase of the cytoplasmic, free-Ca²⁺ concentration. The physiological implications will be discussed.     

Bisphosphoglycerate synthase activity in cells of the erythrocytic line of rat

The specific activity of bisphosphoglycerate synthase (EC 2.7.5.4) is significantly higher in rat mature erythrocytes than in reticulocytes from phenylhydrazine treated animals, while 2,3-bisphosphoglycerate levels do not differ between them. The enzyme specific activity and the concentration of 2,3-bisphosphoglycerate are both negligible in the erythroid cell pool from the bone marrow of anaemic animals. Thus, a cellular specialization in the last stages of erythropoiesis is confirmed. Human bisphosphoglycerate synthase shows a lower specific activity than that of rat without a parallel decrease in 2,3-bisphosphoglycerate levels.     

Metabolism of 3H-estradiol-17 beta by cultures of isolated rat Sertoli cells and the effect of FSH: presence of 16 alpha-hydroxylase

The ability of Sertoli cells to metabolize 3H-estradiol-17 beta was investigated utilizing Sertoli cell cultures isolated from 18d rat testes. The Sertoli cells converted estradiol-17 beta to estriol as shown by recrystallization of estriol from samples containing cells and media but not from cell-free control media. The effect of FSH treatment on such metabolism was investigated and was shown to be similar to nontreated samples. This is the first demonstration that 16 alpha-hydroxylase is present in Sertoli cells and that this enzyme activity is not under the influence of FSH.