Sex differences in the regulation of embryonic brain aromatase

Oestrogen formed from androgen by aromatization plays a critical role in the sexual differentiation of the male brain and behaviour. A question which has still to be answered is what regulates the gender-specific changes in aromatase activity forming oestrogen during sensitive periods of brain growth. Using a primary cell culture technique and sexed embryos, we have shown that in the fetal mouse brain, oestrogen formation in the male is neuronal rather than glial and aromatase activity is regionally localized, being higher in the hypothalamus than in the cortex. The aromatase activity measured from cells in culture has the same enzyme binding affinity (apparent Km approximately 40 nM) as intact brain samples. Neurones developing in the embryonic male brain (embryonic day (ED) 15) contain higher aromatase activity (Vmax, 895 fmol/h/mg protein) than the female (Vmax, 604). Although a sex difference exists at early stages of embryonic development (ED 13), the embryonic aromatase system is regulated by steroids later in fetal development. The developing aromatase-containing neuroblasts probably form processes which connect to other aromatase neurones. Immunoreactive staining with an aromatase polyclonal antibody identifies an increase in numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies following testosterone treatment. Testosterone treatment also causes both stimulation of neurite growth and branching as well as functional maturation of aromatase neurones. In particular, there is an increase in aromatase activity per neurone as well as a dramatic increase in the number of neurones expressing the enzyme. Both the functional and morphological changes depend on androgen receptor stimulation for several days in vitro. This conclusion is supported by colocalization studies which reveal a high number of fetal hypothalamic aromatase neurones co-expressing androgen receptor. We conclude that testosterone influences the growth of male hypothalamic neurones containing aromatase at a sensitive period of brain development. Endogenous steroid inhibitors of aromatase, probably formed within the neuroglia, also play a role in the control of oestrogen production. An endogenous 5alpha-reduced metabolite of testosterone, 5alpha-androstanedione, is almost as potent in inhibiting neuronal hypothalamic aromatase activity (Ki = 23 nM) as the synthetic non-steroidal inhibitors such as the imidazole, fadrozole, and the triazoles, arimidex and letrozole. It is clear that the oestrogen-forming capacity of the male hypothalamus has the special characteristics and plasticity of regulation which could affect brain differentiation at specific steroid-sensitive stages in ontogeny.     

New insights into p53 function from structural studies

Recent structural analysis of p53 has greatly enhanced our understanding of the biochemical activities of this protein by presenting us with a detailed picture of the chemical groups in the protein that are involved in protein stability, conformation and functional interactions. The current structures form the basis for the design of potential therapeutics which could, for example, revert a DNA-binding mutant back to a DNA-binding competent conformation. The structure of the tet domain forms the basis for designing an active therapeutic p53 with an oligomerization domain which would not cross react with a DNA-binding mutant p53. However, as useful as these structures have been in providing insight into the structure/function relationship for p53, a complete understanding of this protein awaits more detailed information on the full-length protein. In this respect, one of the most useful roles for future structural studies will be to help identify the nature of the conformational transition between latent and active p53, and how it can be modulated.     

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and the sexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.     

New insights from spectroscopy into the structure/function relationships of lipoxygenases

Spectroscopic properties of the redox-active iron in the active site of plant and mammalian lipoxygenases can now be combined with recent crystal structure determinations to obtain new insights into lipoxygenase reaction mechanisms.     

New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes

Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.     

New insights into proteasome function: from archaebacteria to drug development

The proteasome is not simply a 'garbage disposal unit' but also has functions in the control of the cell cycle and immune responses. The structure of an archaebacterial proteasome has recently been determined to high resolution, and provides insight into the unusual mechanism of proteolytic cleavage by the proteasome.     

New insights into the negative regulation of hematopoiesis by chemokine platelet factor 4 and related peptides

Platelet factor 4 (PF4) has been recognized as an inhibitor of myeloid progenitors. However, the mechanism of action of this chemokine remains poorly understood. The present study was designed to determine its structure/function relationship. A series of peptides overlapping the C-terminal and central regions of PF4 were analyzed in vitro for their action on murine hematopoietic progenitor growth to assess the minimal sequence length required for activity. The peptides p17-58 and p34-58 possessed an increased hematopoietic inhibitory activity when compared with PF4, whereas the shorter peptides p47-58 and p47-70 were equivalent to the native molecule and the peptide p58-70 was inactive. The PF4 functional motif DLQ located in 54-56 was required for the activity of these peptides. The peptide p34-58 impaired to a similar extent the growth of colony-forming unit-megakaryocyte (CFU-MK) as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte-macrophage (CFU-GM), whereas PF4 was more active on CFU-MK. In the experiments using purified murine CD34(+) marrow cells, statistically significant inhibition induced by p34-58 was shown at concentrations of 2.2 nmol/L or greater for progenitors of the three lineages, whereas that induced by PF4 was seen at 130 nmol/L for CFU-MK and 650 nmol/L for CFU-GM and BFU-E, indicating that the p34-58 acts directly on hematopoietic progenitors and its activity is approximately 60- to 300-fold higher than PF4. The p34-58, unlike PF4, lacked affinity for heparin and its inhibitory activity could not be abrogated by the addition of heparin. In addition, an antibody recognizing p34-58 neutralized the activity of p34-58 but not whole PF4 molecule. These results demonstrate that PF4 contains a functional domain in its central region, which is independent of the heparin binding properties, and provide evidence for a model of heparin-dependent and independent pathways of PF4 in inhibiting hematopoiesis.     

New insights into the genetics of lissencephaly

The cause of chronic inflammatory bowel disease remains unknown. A genetic origin has been suggested by studies in twins. The gene or genes involved in Crohn's disease is probably situated on chromosome 16 and the genes involved in ulcerative colitis on chromosomes 2 and 7. It will undoubtedly take some time before the exact loci are precisely identified, but current research suggests that clinical applications are not far off.     

Subunit epsilon of the Escherichia coli ATP synthase: novel insights into structure and function by analysis of thirteen mutant forms

Structural models of subunit epsilon of the ATP synthase from Escherichia coli have been determined recently by NMR [Wilkens et al. (1995) Nat. Struct. Biol. 2, 961-967] and by X-ray crystallography [Uhlin et al. (1997) Structure 5, 1219-1230], revealing a two-domain protein. In this study, six new epsilon mutants were constructed and analyzed: Y63A, D81A, T82A, and three truncated mutants, tr80(S), tr94(LAS), and tr117(AS). Seven mutants constructed previously were also analyzed: E31A, E59A, S65A, E70A, T77A, R58A, and D81A/R85A. Subunits were purified by isoelectric focusing from extracts of cells that overproduced these 13 mutants. F1 was prepared lacking subunit epsilon by immobilized-Ni affinity chromatography. Three mutants, E70A, S65A, and E31A, showed somewhat higher affinities and extents of inhibition than the wild type. Three mutants, T82A, R85A, and tr94(LAS), showed both lower affinities and extents of inhibition, over the concentration range tested. Two showed no inhibition, D81A and tr80(S). The others, T77A, Y63A, E59A, and tr117(AS), showed lower affinities than wild type, but the extents of inhibition were nearly normal. Results indicate that the C-terminal domain of subunit epsilon contributes to inhibition of ATP hydrolysis, but it is not necessary for ATP-driven proton translocation. Interactions with subunit gamma are likely to involve a surface containing residues S65, E70, T77, D81, and T82, while residues R85 and Y63 are likely to be important in the conformation of subunit epsilon.     

Inhibition of breast cancer tissue aromatase activity and estrogen concentrations by the third-generation aromatase inhibitor vorozole

In about one-third of advanced breast cancers, estrogen deprivation causes tumor regression. Estrogen concentrations in tumor tissue seem to depend largely on local production. The aromatase enzyme complex is thought to be the key enzyme in this respect. In the present study, the effect of the new third-generation nonsteroidal aromatase inhibitor vorozole (Rivizor) on tumor tissue aromatase activity and estrogen concentrations was evaluated. During 7 days preceding mastectomy, 11 postmenopausal breast cancer patients were treated with 2.5 mg of vorozole once daily. Eight patients could be evaluated. Intratumoral aromatase activity and estrone and estradiol levels were measured and compared to the values of nine untreated postmenopausal breast cancer patients. In treated patients, median tissue aromatase activity was 89% lower than that in controls (P < 0.001). Similarly, median tissue estrone and estradiol concentrations were 64 and 80% lower, respectively, in treated patients (P = 0.001 and P < 0.05, respectively). These results support the hypothesis that depleting the tumor of estrogens, thus impairing estrogenic stimulation, is an important mechanism in the antitumor activity of aromatase inhibitors.     

Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9)

Primate lentiviruses encode for an unique nef gene with an essential function in both viral replication and pathogenicity in the host. The molecular basis for this function remains however poorly defined. Several Nef-binding cellular proteins are thought to be instrumental in its function. Indeed, Nef contains a proline-rich motif implicated in the binding to the Src-like tyrosine kinase Hck and also to a Ser/Thr kinase of molecular weight 62 kDa. The disruption of this motif affects the binding to both these kinases as well as viral replication. Whereas Hck is expressed in the myeloid lineage and hence may account for the nef function in infected monocytes, we and others have reported previously that Nef also interacts with the T-lymphocyte Src-kinase Lck, leading to specific cell signaling impairment. This interaction occurs through the binding of Nef to both Lck SH2 and SH3 domains. Both the proline motif and phosphorylation of Nef on tyrosine residue were proposed to account for these interactions. Here, we investigate the mechanism of Lck SH2 binding by HIV-1 Nef. Using recombinant fusion proteins to precipitate lysates, we show that although SH2 binding is dependent on phosphorylation events, it occurs in a tyrosine independent manner because it requires neither tyrosine residues in Nef nor the phosphotyrosine binding pocket from the Lck SH2 domain, hence suggesting a role for a phosphoserine or a phosphothreonine residue. Further, we show that Hck SH2 does not interact with Nef, indicating that Hck SH3 binding is sufficient for Nef binding, whereas Lck SH2 cooperate together with SH3 to allow Nef binding to a level similar to Hck SH3. Together, our results establish different mechanisms for Hck and Lck binding by HIV-1 Nef protein, and identify a novel mechanism for Src-like tyrosine kinase targeting by a viral protein.     

New insights into the pathogenesis of distal renal tubular acidosis

The diagnosis and classification of distal renal tubular acidosis (distal RTA) have traditionally been made on the basis of the renal response to physiologic maneuvers, providing only indirect information on the underlying pathophysiology. In the past several years significant advances have been made in our understanding of the molecular basis of distal H+/HCO3- secretion and absorption, at the level of individual transporters. With these advances, a new era of classifying disorders of distal acidification at the molecular level has arrived. In this article we review the cellular and molecular basis of normal acidification mechanisms in the distal nephron. We also review the recent information on the molecular basis of derangements in these mechanisms which lead to distal RTA.     

New insights into the catalytic cycle of flavocytochrome b2

Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction in the mitochondrial intermembrane space. The catalytic cycle for this process can be described in terms of five consecutive electron-transfer events. L-Lactate dehydrogenation results in the two-electron reduction of FMN. The two electrons are individually passed to b2-heme (intramolecular electron transfer) and then onto cytochrome c (intermolecular electron transfer). At 25 degrees C, I 0.10, in the presence of saturating concentrations of ferricytochrome c and L-lactate, the catalytic cycle progresses with rate constant 104 (+/- 5) s-1 [per L-lactate oxidized; Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. Stopped-flow spectrophotometry has been used to show that the major rate-limiting step in the catalytic cycle is electron transfer from flavin semiquinone to b2-heme. This conclusion is based on the observation that pre-steady-state flavin oxidation by ferricytochrome c takes place at 120 s-1. Although flavin oxidation involves several other electron transfer steps, these are considered too fast to contribute significantly to the rate constant. It was also shown that the reaction product, pyruvate, is able to inhibit pre-steady-state flavin oxidation (Ki = 40 +/- 17 mM) consistent with reports that it acts as a noncompetitive inhibitor in the steady state at high concentrations [Ki = 30 mM; Lederer, F. (1978) Eur. J. Biochem, 88, 425-431]. This novel way of measuring the electron transfer rate constant is directly applicable to the catalytic cycle and has enabled us to derive a self-consistent model for it, based also on data collected for enzyme reduction [Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192] and its interaction with cytochrome c [Daff, S., Sharp, R. E., Short, D. M., Bell, C., White, P., Manson, F. D. C., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6351-6357]. Rapid-freezing quenched-flow EPR has been used to confirm the model by demonstrating that during steady-state turnover of the enzyme approximately 75% of the flavin is in the semiquinone oxidation state.     

New insights into the role of follicle-stimulating hormone in reproduction

Follicle-stimulating hormone (FSH) has been considered essential for normal ovarian follicular development and spermatogenesis. Although this concept remains valid, the recent reports of defects in genes encoding FSH and its receptor (FSHR) have altered the concepts, particularly with regard to the role of FSH in spermatogenesis. Complete FSH resistance caused by a mutation of the FSHR gene has demonstrated that normal FSH action is an absolute requirement for female fertility, but spermatogenesis and male fertility are possible without FSH action. Thus, while normal ovarian function is critically dependent on normal FSH action, male fertility is relatively independent of this hormone. The finding contradicts the potential of inhibition of FSH secretion or action as a means of male contraception.     

Identification of aromatase in the reptilian brain

The present study tests the hypothesis that brain aromatase is an "ancient" property of nervous tissue and may be identified in homologues of the limbic system in a non-mammalian vertebrate, the turtle Chrysemys picta. Tissue homogenates (180 mg wet weight/2 ml) were incubated with [7 alpha-3H]androstenedione and cofactors for 60 min at 37 C. Estrone (E1) was isolated and characterized by thin layer chromatography, methylation and recrystallization to constant specific activity. No estradiol-17 beta was detected. Aromatase was found only in the forebrain but was diffusely distributed throughout this major brain division. No other neural or non-neural tissues, including mid- and hindbrain structures, testis, and ovary, synthesized detectable quantities of E1 in our system. The strio-amygdaloid complex of both sexes synthesized more E1 per unit weight than the preoptic-hypothalamic area (POA-HTH) or other forebrain structures. It is possible that the conversion of androgen to estrogen has biological significance in this species since the reaction occurred throughout the physiological temperature range experienced during activity in nature and sex differences in brain aromatase activity during the breeding season were apparent. These experiments in Chrysemys demonstrate that the synthesis of estrogen from androgen by the brain is not limited to mammals, but also occurs at a more primitive level of phylogenetic development. Restriction of aromatase to forebrain structures of the turtle is consistent with the neuroanatomic distribution of enzyme activity in the limbic system of mammals. Estrogen yield from adult turtle brain incubates (3.2-22.6 pmol/g) is more like that reported for fetal (2.7-33 pmol/g) than for adult (0.1-1.9 pmol/g) mammals. We suggest that the in situ synthesis of estrogen by the central nervous system has its orgins early in vertebrate evolution and may be a primitive characteristic of brain-steroid interactions that regulate physiological and behavioral sex in vertebrates.