O dealkylation and aliphatic and aromatic hydroxylation of 3-methoxy-17 beta-estradiol by Aspergillus alliaceus

Aspergillus alliaceus UI 315 was examined for its ability to metabolize 3-methoxy-17 beta-estradiol. Preparative-scale incubations with this substrate afforded good yields of 6 beta-hydroxy-17 beta-estradiol, 4-hydroxy-17 beta-estradiol, and 4,6 beta-dihydroxy-17 beta-estradiol, which were identified by high-pressure liquid chromatography, 1H and 13C nuclear magnetic resonance, and high-resolution mass spectrometry.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

Lipid metabolism during the initiation of lactation in the rat. The effects of starvation and tumour growth.

1. The effects of starvation post partum (24 h) and tumour growth pre partum on the initiation of lactation in the rat were studied. 2. Tumour growth decreased food intake at 24 h, but not at 2 days post partum. 3. Pup growth rate increased with hyperphagia; starvation and tumour burden decreased pup growth, and starvation decreased maternal body weight. 4. Starvation decreased gastrointestinal-tract mass; tumour growth decreased gastrointestinal-tract and mammary-gland mass. 5. Mammary-gland DNA-synthesis rate was high immediately post partum, but decreased by day 3 of lactation; starvation and tumour burden decreased this rate, and also decreased gastrointestinal-tract DNA-synthesis rate. 6. Arteriovenous differences for glucose and lactate across the mammary gland did not change with time, nor were they affected by the tumour. Starvation decreased arterial glucose and lactate, and the gland extracted less glucose but produced lactate. 7. Mammary-gland lipogenesis was sensitive to starvation and to tumour growth. 8. In contrast with the gradual development of mammary-gland lipogenic enzyme activities, lipoprotein lipase activity was high in the gland by 2 days post partum; starvation or tumour burden decreased the activity. 9. The mammary gland is sensitive post partum to decreased food intake, and to tumour presence. The effects of the latter are apparently independent of hypophagia.     

Comparative effects of tumour necrosis factor-alpha (cachectin), interleukin-1-beta and tumour growth on amino acid metabolism in the rat in vivo. Absorption and tissue uptake of alpha-amino[1-14C]isobutyrate.

Incubation of a membrane preparation enriched in Photosystem Two (PSII) at alkaline pH inhibited the water-splitting reactions in two distinct steps. Up to pH 8.5 the inhibition was reversible, whereas at higher alkalinities it was irreversible. It was shown that the reversible phase correlated with loss and rebinding of the 23 kDa extrinsic polypeptide. However, after mild alkaline treatments a partial recovery was possible without the binding of the 23 kDa polypeptide when the assay was at the optimal pH of 6.5 and in a medium containing excess Cl-. The irreversible phase was found to be closely linked with the removal of the 33 kDa extrinsic protein of PSII. Treatments with pH values above 8.5 not only caused the 33 kDa protein to be displaced from the PSII-enriched membranes, but also resulted in an irreversible modification of the binding sites such that the extrinsic 33 kDa protein could not reassociate with PSII when the pH was lowered to 6.5. The results obtained with these more extreme alkaline pH treatments support the notion that the 23 kDa protein cannot bind to PSII unless the 33 kDa protein is already bound. The differential effect of pH on the removal of the 23 kDa and 33 kDa proteins contrasted with the data of Kuwabara & Murata [(1983) Plant Cell Physiol. 24, 741-747], but this discrepancy was accounted for by the use of glycerol in the incubation media.     

Polymyxin B diminishes blood flow to brown adipose tissue and lactating mammary gland in the rat. Possible mechanism of its action to decrease the stimulation of lipogenesis on refeeding.

Polymyxin B, a cyclic decapeptide antibiotic, increased blood glucose and lactate, and inhibited the stimulation of lipogenesis in interscapular brown adipose tissue and lactating mammary gland of starved-refed virgin and lactating rats respectively. Lipogenesis was not inhibited in white adipose tissue or liver. The antibiotic increased the haematocrit. The relative blood flow to brown adipose tissue and lactating mammary gland was decreased by polymyxin B, and this was accompanied by a decrease in tissue ATP content. In vitro polymyxin B did not affect glucose utilization or conversion into lipid, nor the stimulation by insulin of these processes in brown-adipose-tissue slices. Treatment of rats in vivo with polymyxin B resulted in decreased utilization of glucose in vitro in brown-adipose-tissue slices. Similarly, acini from mammary glands of polymyxin B-treated lactating rats had decreased rates of conversion of [1-14C]glucose to lipid. It is concluded that the effects of polymyxin B may be brought about by decreases in tissue blood flow. The possibility that these effects are secondary to inhibition of glucose utilization cannot be ruled out.     

Re-examination of the putative roles of insulin and prolactin in the regulation of lipid deposition and lipogenesis in vivo in mammary gland and white and brown adipose tissue of lactating rats and litter-removed rats.

1. The effects of various treatments to alter either plasma prolactin (bromocryptine administration or removal of litter) or the metabolic activity of the mammary gland (unilateral or complete teat sealing) on the disposal of oral [14C]lipid between 14CO2 production and [14C]lipid accumulation in tissues of lactating rats were studied. In addition, the rates of lipogenesis in vivo were measured in mammary gland, brown and white adipose tissue and liver. 2. Bromocryptine administration lowered plasma prolactin, but did not alter [14C]lipid accumulation in mammary gland or in white and brown adipose tissue. 3. In contrast, complete sealing of teats results in no change in plasma prolactin, but a 90% decrease in [14C]lipid accumulation in mammary gland and a 4-fold increase in white and brown adipose tissue. The rate of lipogenesis in mammary gland was decreased by 95%, but there was no change in the rate in white and brown adipose tissue. Unilateral sealing of teats resulted in a decrease in [14C]lipid accumulation in white adipose tissue. 4. Removal of the litter for 24 h (low prolactin) produced a similar pattern to complete teat sealing, except that there was a 6-fold increase in lipogenesis in white adipose tissue. Re-suckling for 5 h increased plasma prolactin, but did not alter the response seen in litter-removed lactating rats. 5. Changes in lipoprotein lipase activity and in plasma insulin paralleled the reciprocal changes in [14C]lipid accumulation in white and brown adipose tissue and in mammary gland. 6. It is concluded that the plasma insulin is more important than prolactin in regulating lipid deposition in adipose tissue during lactation, and that any effects of prolactin must be indirect.     

Ethnobotany of purple coneflower (Echinacea angustifolia,Asteraceae) and OtherEchinacea Species

The purple coneflower, Echinacea angustifolia, was the most widely used medicinal plant of the Plains Indians. It was used for a variety of ailments, including toothache, coughs, colds, sore throats, snakebite, and as a painkiller. H. C. F. Meyer used it as a patent medicine in the 1870s and introduced it to the medical profession. Recent scientific research (mostly German) onEchinacea species has shown that they possess immunostimulatory activity. Increased cultivation ofE. purpurea andE. angustifolia may be needed to meet the increased demand for its roots and to alleviate the effects of overharvesting of wild stands.     

Biological degradation of 2,4-dichlorophenoxyacetic acid: chloride mass balance in stirred tank reactors.

A mass balance was developed for the degradation of 2,4-dichlorophenoxyacetic acid by a mixed culture. Batch culture experiments showed the degradation to be an acid-producing step. Inorganic chloride concentration consistently correlated with the expected value and with base consumption to maintain a constant pH.     

O dealkylation and aliphatic and aromatic hydroxylation of 3-methoxy-17 beta-estradiol by Aspergillus alliaceus.

Aspergillus alliaceus UI 315 was examined for its ability to metabolize 3-methoxy-17 beta-estradiol. Preparative-scale incubations with this substrate afforded good yields of 6 beta-hydroxy-17 beta-estradiol, 4-hydroxy-17 beta-estradiol, and 4,6 beta-dihydroxy-17 beta-estradiol, which were identified by high-pressure liquid chromatography, 1H and 13C nuclear magnetic resonance, and high-resolution mass spectrometry.