Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.     

Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.     

Cloning and sequence of the gene encoding the major structural component of mannose-resistant fimbriae of Serratia marcescens.

Serratia marcescens US46, a human urinary tract isolate, exhibits mannose-resistant hemagglutination and agglutinates yeast cells, thereby indicating that it has two types of adhesins. We constructed a cosmid library for the DNA of this organism and isolated DNA clones carrying genes for mannose-sensitive (MS) and mannose-resistant (MR) fimbriae. On introduction of the cloned genes into Escherichia coli K-12, MS and MR fimbriae were formed. These fimbriae were functionally and morphologically indistinguishable from those of S. marcescens. Subcloning of these gene clusters revealed that the genes encoding MS fimbriae reside on a 9-kilobase (kb) DNA fragment, while those encoding MR fimbriae are present on a 12-kb fragment. Transposon insertion and maxicell analyses revealed that formation of MR fimbriae is controlled by several genes which reside on the 9-kb fragment. The nucleotide sequence of smfA, the gene encoding the major structural component of MR fimbriae, revealed that this gene encodes a 174-amino-acid polypeptide with a typical procaryotic signal peptide. The primary structure of the smfA product showed significant homology with the primary structure of the E. coli fimbrial subunit.     

A self-correcting indirect calorimeter system for the measurement of energy balance in small animals.

Indirect calorimetry involves measurement of CO(2) produced and O(2) consumed by an organism. These measurements are then used to calculate energy output, metabolic rate (MR), and respiratory quotient (RQ), a relative assessment of carbohydrate and lipid oxidation. By far the most difficult aspect of indirect calorimetry is measurement of O(2). Moreover, the abundance of O(2) (20.95%) relative to CO(2) (0.03%) in ambient conditions dictates that measurement errors of O(2) have greater implications on calculations of MR and RQ. Because compressed air is not feasible for use with animals in long-term experiments, changes in ambient conditions are nearly unavoidable. A self-correcting indirect calorimetry system was designed and constructed utilizing differential O(2) and CO(2) analyzers and a blank cage to monitor ambient conditions periodically. The system was validated by changing ambient O(2) and CO(2) concentrations by infusing N(2) into the system during a test butane burn. MR and RQ were largely unaffected by these changes in ambient conditions, and inclusion of a blank cage in the system accounted for slight calibration offsets. MR and RQ were measured in mice (n = 95) with and without correction for any small changes in ambient conditions measured in the blank cage. Coefficients of variation for MR and RQ were significantly decreased by taking into account ambient conditions measured in the blank cage (P < 0.001), which resulted in a 2.3% increase in precision for measurement of MR. This system will be used to more accurately assess long-term measurements of energy balance in the many murine models of leanness and obesity to gain better insights into pathophysiology and treatment of human obesity.     

Physiological MR signal variations within the brain at 3 T.

The echoplanar technique in magnetic resonance (MR) imaging allows the acquisition of a series of images from a selected slice with a temporal resolution of 10/s. Simultaneous recording of physiological information on pulse and respiration allows correlation of the MR signal intensity with physiological signals, which can be obtained for each pixel examined. Such correlations can be found within the cerebrospinal fluid (CSF) spaces and within vessels if a flow-sensitive MR measurement technique is used. The use of an MR scanner with a field strength of 3 T improves the signal/noise ratio, but there is a stronger signal decay due to local magnetic inhomogeneities. This study shows that 3-T systems can be used for correlation of MR and physiological signals and that clear differentiation between signals from CSF and from vessels can be obtained due to their strongly different signal decays.     

High-frequency antigens of human erythrocyte membrane sialoglycoproteins. VI. Monoclonal antibodies reacting with the N-terminal domain of glycophorin C

The epitopes of seven mouse monoclonal antibodies which are related to the Gerbich blood group system were investigated. BRIC4, BRIC10, GERO and MR4-130 have been published earlier. The three others (APO1, APO2, APO3) were prepared by immunization with normal human erythrocytes and detected by screening with red blood cells that lack glycophorins C and D. Using immunoblotting and hemagglutination inhibition assays, the epitopes for all antibodies were found to be located on glycophorin C. Hemagglutination inhibition experiments with peptides and chemically modified glycophorins revealed that MR4-130, GERO, BRIC10 and APO2 are all directed against identical or rather similar epitopes comprising the N-terminal three or four residues of glycophorin C. Modification of the N-terminal methionine residue or release of sialic acid attached to oligosaccharide(s) at the third and/or fourth position(s) destroyed all these antigens. The epitope of APO3 was found to comprise glutamic acid17 and/or aspartic acid19 as well as the oligosaccharide attached to serine15. The antigens of BRIC4 and APO1 were found to be located within the residues 2-21 and to comprise sialic acid attached to O-glycosidically linked oligosaccharide(s). However, these epitopes could not be elucidated further. Radio-iodinated MR4-130 bound to 39,000 receptor sites per normal red blood cell. Binding of the labelled antibody was completely inhibited by unlabelled MR4-130, BRIC10, APO2 and GERO. APO1 caused partial inhibition suggesting that it is directed against an adjacent site. BRIC4, APO3 and anti-Ge3 did not inhibit the binding of labelled MR4-130 to any significant extent.     

Histochemical profiles of rat soleus intrafusal fibres after chronic exercise.

Intrafusal fibres from the rat soleus were investigated for representative histochemical profiles in sedentary animals and animals chronically exercised for 17 weeks on a treadmill. The pattern of myosin adenosine triphosphatase (ATPase) activity in the polar region revealed three intrafusal fibre types: (1) myosin ATPase-dark (MD) fibres, alkali- and acid-stabile; (2) myosin ATPase-light (ML) fibres, alkali- and acid-labile; and (3) myosin ATPase-reversible (MR) fibres, alkali-stabile and acid-labile. The three fibre types were correlated with the level of reduced NADH diaphorase activity, with MR, ML and MD fibres staining dark, moderate and light, respectively. In the equatorial region the morphological features of representative ML and MD fibres revealed that they were nuclear bag fibres, while representative MR fibres were identified as nuclear chain fibres. The MR fibres in the exercised animals had higher levels of myosin ATPase alkaline stability and acid lability than MR fibres in the sedentary animals, suggesting the MR fibre profiles are selectively influenced by chronic exercise. The mean cross-sectional area of MR fibres from the exercised animals was significantly less than the MR fibres from the sedentary animals. In contrast to the effect of endurance training on NADH diaphorase activity in extrafusal muscle fibres, there was evidence of less activity in the MD fibres of the exercised animals.     

Regulated expression of the trophoblast alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Differentiation and cAMP modulate protein and mRNA levels.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) has several ligands including activated alpha 2-macroglobulin, pregnancy zone protein, and very low density lipoproteins enriched with apolipoprotein E. The diversity of ligands suggests a role for the alpha 2MR/LRP in a variety of processes including tissue remodeling and lipoprotein metabolism. We examined alpha 2MR/LRP in placental trophoblasts, invasive cells that also function in lipid transport and cholesterol metabolism. alpha 2MR/LRP protein was localized by immunohistochemistry in the syncytiotrophoblast of term placenta. Cytotrophoblasts did not stain prominently. alpha 2MR/LRP (protein and message) in primary cultures of human trophoblast cells increased as cytotrophoblasts differentiated into syncytiotrophoblast. 8-Bromo-cAMP prevented this increase and suppressed alpha 2MR/LRP expression. The cyclic nucleotide had similar suppressive effects on alpha 2MR/LRP in BeWo choriocarcinoma cells. In contrast, low density lipoprotein receptor gene expression was increased. We conclude that: 1) there is a differentiation-dependent pattern of alpha 2MR/LRP expression in the human trophoblast; 2) cAMP negatively regulates alpha 2MR/LRP; 3) there is an inverse relationship between alpha 2MR/LRP and low density lipoprotein receptor gene expression in trophoblast cells.     

The load of genetic and partially genetic diseases in man. III. Mental retardation

This paper summarizes estimates of detriment associated with different etiologic categories of mental retardation (MR) in Hungary. The basic data derive from an earlier study carried out in Budapest on 1276 school-age mentally retarded children (with some etiologic reclassification based on recent studies). Detriment associated with these different categories of MR is expressed in terms of years of lost and impaired life. About 30 per 10(3) school-age children in Hungary are mentally retarded (mild + severe MR), one-tenth of whom have severe MR (IQ less than or equal to 50); 50% of the latter are institutionalized. The breakdown on the basis of etiology is as follows: gene mutations and chromosomal abnormalities, about 4 per 10(3); 'familial' (multifactorial) causes, 12 per 10(3); adverse pre-, peri- and post-natal causes, 11 per 10(3); and 'causes as yet unknown', the remainder. The estimates of mean number of years of lost life range from 42 to 68 (depending on the etiologic category), with an overall mean of 58. The total number of years of lost life is about 36,000 per 10(4) live births of which over 70% is due to pre-, peri- and post-natal causes, 18% due to 'familial' causes and the remainder due to Mendelian and chromosomal diseases. The total number of years of impaired life is about 7300 per 10(4) livebirths, 50% of which is due to 'familial' causes. While admittedly approximate, these estimates suggest that detriment associated with MR-related causes is not inconsiderable. Additionally, they provide some indication of causes of MR which are minimizable.     

Co-localization of brain corticosteroid receptors in the rat hippocampus.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Ca1 and CA2 and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the excess of mental retardation and/or congenital malformations in apparently balanced reciprocal translocations. A critical review of the Leuven data 1966-1991.

In this report we review 286 reciprocal translocations (rcpt) diagnosed in Leuven in the period 1966, mid 1991. They were selected from a total number of 82,000 patients karyotyped for constitutional reasons. Special attention is paid to: (1) the phenotypic effect of de novo reciprocal chromosomal rearrangements and (2) the incidence of mental retardation/congenital malformations (MR/CM) in familial rcpt. Important conclusions of this study were: 1) The high incidence of MR/CM in de novo rcpt, not only in patients with complex chromosomal rearrangements (greater than 80%) but also in patients with classical two breaks rcpt (greater than 60%). In contrast, the phenotypic effect of normal/mosaic rcpt seems to be minimal. 2) The overall incidence of MR/CM in carriers of familial balanced rcpt was 6.4%. Interestingly, the incidence of MR/CM problems in rcpt carriers from families detected because of reproductive failure was not increased (2.3%). However, the risk to find MR/CM in a rcpt carrier was much higher (12.8%) if he/she belonged to a family in which the rcpt was detected in an index patient with MR/CM.     

A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) consists of two polypeptides, 515 and 85 kDa, that are noncovalently associated. A 39-kDa polypeptide, termed the receptor-associated protein (RAP), interacts with the 515-kDa subunit after biosynthesis of these molecules and remains associated on the cell surface. This molecule regulates ligand binding of alpha 2MR/LRP (Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266, 21232-21238). Titration and binding studies indicate that RAP binds to two equivalent binding sites on alpha 2MR/LRP, with a KD of 14 nM. Heterologous ligand displacement experiments demonstrated that RAP completely inhibits the binding of 125I-activated alpha 2M to human fibroblasts and to the purified alpha 2MR/LRP, with a Ki of 23 and 26 nM, respectively. A direct correlation between the degree of binding of RAP to the receptor and the degree of ligand inhibition was observed, indicating that as the RAP binding sites are saturated, alpha 2MR/LRP loses its ability to bind ligands. Thus, the amount of RAP bound to alpha 2MR/LRP dictates the level of receptor activity. A model is proposed in which alpha 2MR/LRP contains multiple ligand binding sites, each regulated by a separate RAP site.     

The MR P-type transposable elements and the genetic activities of mutagens and carcinogens in Drosophila melanogaster. I. N,N-Dimethylnitrosamine (DMN)

A technique was developed for the assay of the genetic activities of carcinogens in both the soma and germ line in the course of early larval development and to assess the extent of their modification through the introduction into the genome of the MR IInd autosome P-type transposable elements. The influence of MR on genotoxicity for a given treatment was indicated by the relative frequencies of non-MR (Cy) to MR-carrying sibs emerging within the same cultures. The viable genetic changes in the soma were classified as recombinational or mutational events on the basis of the comparative yields of mosaic sectors in females heterozygous for the markers y w sn carried in standard order (XS) or multiply inverted (XIn) X-chromosomes. The results with this technique are here described for the carcinogen DMN. In the absence of MR, the topical application of DMN induced no larval lethality up to the highest tested doses (20 mM), but raised the yields of the somatic sectors for all the test markers in the emerging females in accordance with a linear dose fit. Comparison of the XS and XIn data indicated that somatic mutagenesis by DMN entailed the induction of recombinational and mutational events in roughly equal proportions. The introduction of MR into the genome, whether patro- or matroclinously, resulted in dramatic and proportionately equivalent enhancements in the activities of DMN, both with respect to the induction of larval lethality and somatic sectoring. These activities increased exponentially with dose, following a 4th-degree polynomial course up to 10 mM, when larval lethality approached 100%. At lower dose levels, the yields of all sector types in the viable females also followed comparable polynomial curves at different heights, except for y sn in the XIn series, where sector recovery remained at the control level throughout the examined dose range. Analysis of the sector size distribution for eye and bristle mosaicism in the XS control and DMN-treated series gave statistically comparable mean values, irrespective of the presence or absence of MR, indicating that DMN alone, or in conjunction with the P elements, did not alter the timing of aberrant clone initiation. In contrast, estimates of the genetic induction events in the somatic primordia with 5 mM DMN indicated greatly increased response in the presence of MR, which was in excess of 20-fold for the mutational changes involving the sn locus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of monodehydroascorbate reductase from soybean root nodules.

Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.     

Hyperthermia-induced enhancement of melphalan activity against a melphalan-resistant human rhabdomyosarcoma xenograft.

The effects of regional hyperthermia (42 degrees C for 70 min) on the antitumor activity of melphalan were examined in athymic mice bearing melphalan-resistant human rhabdomyosarcoma (TE-671 MR) xenografts growing in the right hind limb, and results were compared with similar studies of melphalan-sensitive (TE-671) parent xenografts. Melphalan alone at a dose of 36 mg/m2 (0.5 of the 10% lethal dose) produced growth delays of 4.1 to 10.2 days in TE-671 MR xenografts and 21.8 to 28.7 days in TE-671, respectively. Hyperthermia alone produced growth delays of 0.9 days in TE-671 MR xenografts and 0.8 days in TE-671. Combination therapy with melphalan and hyperthermia produced growth delays of 7.2 to 13.3 days in TE-671 MR xenografts and 34.3 to 42.8 days in TE-671, respectively, representing a mean thermal enhancement ratio of 1.7 in TE-671 MR and 1.5 in TE-671. Measurement of glutathione levels in TE-671 MR xenografts following treatment with melphalan, hyperthermia, or melphalan plus hyperthermia revealed significant reductions in glutathione content with the nadir (60% of control values) seen 6 h following treatment. Glutathione levels in TE-671 xenografts following identical therapy revealed no differences from control values. Hyperthermia plus melphalan did not result in a higher tumor-to-plasma melphalan ratio compared with treatment with melphalan alone in either TE-671 MR or TE-671 xenografts. These studies suggest that heat-induced alterations in tumor glutathione or melphalan levels are not responsible for the increase in melphalan activity produced by hyperthermia. Combination therapy with melphalan plus regional hyperthermia offers promise for treatment of melphalan-resistant neoplasms.     

Characterization of mineralocorticoid and glucocorticoid receptors in pigs: comparison of Meishan and Large White breeds.

Corticosteroids receptors were characterized and compared in central and peripheral tissues of two pig breeds, the Meishan (MS) and the Large White (LW) pigs, that display differences in the basal activity and stress-induced reactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In vitro kinetic experiments on kidney and liver cytosols from adrenalectomized pigs allowed to identify two distinct corticosteroid receptors referred to as mineralocorticoid (MR) and glucocorticoid (GR) receptors. The binding specificities were determined for kidney and hippocampal MR and for liver and hippocampal GR. In hippocampus and peripheral tissues, cortisol showed a greater affinity for MR than for GR. As already described in the dog, mouse and human, dexamethasone and progesterone display a moderate affinity for MR. Putative differences in corticosteroid receptors binding capacities and affinities were investigated by saturation binding studies in specific regions implicated in the regulation of HPA axis (hippocampus and pituitary). The MS pigs evidenced higher densities of hippocampal MR, while LW pigs had higher densities of pituitary GR. Thus, this study suggests that a difference in the MR/GR balance in hippocampus and pituitary could be implicated in the different HPA activity between MS and LW pigs.     

MR 20492 and MR 20494: two indolizinone derivatives that strongly inhibit human aromatase.

In this study, we describe the synthesis of a new family of indolizinone derivatives designed to fit an extrahydrophobic pocket within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the specificity of the inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of human aromatase (Ki/Km: 1.0+/-0.3 and 0.5+/-0.3, respectively). The Ki/Km for MR 20494 is slightly higher than that obtained for fadrozole (0.1+/-0.0) and Ki/Km for both indolizinone derivatives are lower than those obtained for 4-hydroxyandrostenedione (1.9+/-0.8) and MR 20814 (8.1+/-.7). These new compounds are not enzyme inactivators. Moreover, as indicated by the higher Ki/Km values obtained with equine enzyme (9.0+/-0.6 and 6.1+/-1.6 for MR 20492 and MR 20494, respectively), both human and equine aromatase active sites appear to be structurally different. Difference absorption spectra study (350-500 nm) revealed that MR20492 and MR20494 were characterized by a combination of type-I and -II spectra with both enzymes. This result could be due to the isomerization of the molecule in polar solvent (Z and E forms). The evaluation of these new molecules, as well as 4-hydroxyandrostenedione and fadrozole, on aromatase activity in transfected 293 cell cultures evidenced a strong inhibition (IC50: 0.20+/-0.03 microM, 0.20+/-0.02 microM and 0.50+/-0.40 microM for MR 20494, fadrozole and 4-OHA, respectively) except for MR 20492 (3.9+/-0.9 microM) and MR 20814 (10.5+/-0.6 microM). These results proved that these molecules formed part of a promising family of potent inhibitors and that they penetrate 293 cells, without evidencing any cytotoxicity in Hela cells with MTT assay. This is thus encouraging for the development of new drugs for the treatment of estrogen-dependent cancers, these molecules also constitute new tools for understanding the aromatase active site.     

The role of progesterone metabolism and androgen synthesis in renal blood pressure regulation.

11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol into inactive cortisone, conferring specificity onto the human mineralocorticoid receptor (MR). Progesterone binds with even higher affinity to the MR, but acts as an MR antagonist. How aldosterone is able to keep its function as predominant MR ligand in clinical situations with high progesterone concentrations, such as pregnancy, is not clear. We have shown in vitro that the human kidney possesses an effective enzyme system that metabolizes progesterone to inactive metabolites in a process similar to the inactivation of cortisol by 11beta-HSD2. In studies on patients with adrenal insufficiency, we have shown that the in vivo anti-mineralocorticoid activity of progesterone is diminished by inactivating metabolism of progesterone, local formation of the deoxycorticosterone mineralocorticoid from progesterone, and inhibition of 11beta-HSD2 by progesterone and its metabolites resulting in decreased inactivation of cortisol and hence increased MR binding by cortisol. The enzymes involved in progesterone metabolism are also responsible for the capability of the human kidney to convert pregnenolone to DHEA and androstenedione leading to the formation of active androgens, testosterone and 5alpha-DH-testosterone. Locally produced androgens might be responsible for the observed difference in blood pressure between men and women and higher susceptibility to hypertension in men.     

Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant

The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticosteroids, receptors, and the organ-specific functions of 11 beta-hydroxysteroid dehydrogenase.

Reversible oxidation of the biologically active corticosteroids to the inactive 11-dehydrocorticosteroids is catalyzed by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). The properties of the enzyme based on clinical observations of individuals with defective 11 beta HSD expression, and laboratory studies of the properties and behavior of the enzyme, are consistent with separate 11 beta-dehydrogenase and 11-oxoreductase species. However, recombinant enzyme expressed in mammalian cells retain both activities, leading to the conclusion that 11 beta HSD is a unique, reversible enzyme. 11 beta HSD is present in most tissues, but its specific functions in most tissues are unknown. How the enzyme may mediate corticosteroid-receptor interaction is illustrated by studies using kidney, testis, and brain. In kidney, 11 beta HSD prevents glucocorticoids from competing inappropriately with aldosterone for mineralocorticoid receptor (MR). Lack of enzyme in humans due to natural causes or inhibition by pharmacological agents results in maximum activation of MR by glucocorticoids, leading to the clinical symptoms of apparent mineralocorticoid excess. Leydig cells of the testes synthesize testosterone, a process that is suppressed by events initiated by the binding of corticosteroid to glucocorticoid receptors (GR). Depletion of active steroid mediated by 11 beta HSD may initiate testosterone production at puberty and affect testosterone production during adult life, as for example during periods of stress. The heterogeneous distribution of MR and GR in the brain reflects the specific regional effects of glucocorticoids and mineralocorticoids on neural function. Colocalization of 11 beta HSD and corticosteroid receptors in brain may be important in controlling the specificity of corticosteroid interaction with GR and MR. The patterns of 11 beta HSD-steroid-receptor interaction illustrated with these three tissues may provide models applicable to other tissues in which corticosteroid receptors and 11 beta HSD coexist.