Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages. Results We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers. Conclusions Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines

Glucocorticoids are steroid hormones with important functions in development, immune regulation, and glucose metabolism. The adrenal glands are the predominant source of glucocorticoids; however, there is increasing evidence for extraadrenal glucocorticoid synthesis in thymus, brain, skin, and vascular endothelium. We recently identified intestinal epithelial cells as an important source of glucocorticoids, which regulate the activation of local intestinal immune cells. The molecular regulation of intestinal glucocorticoid synthesis is currently unexplored. In this study we investigated the transcriptional regulation of the steroidogenic enzymes P450 side-chain cleavage enzyme and 11beta-hydroxylase, and the production of corticosterone in the murine intestinal epithelial cell line mICcl2 and compared it with that in the adrenocortical cell line Y1. Surprisingly, we observed a reciprocal stimulation pattern in these two cell lines. Elevation of intracellular cAMP induced the expression of steroidogenic enzymes in Y1 cells, whereas it inhibited steroidogenesis in mICcl2 cells. In contrast, phorbol ester induced steroidogenic enzymes in intestinal epithelial cells, which was synergistically enhanced upon transfection of cells with the nuclear receptors steroidogenic factor-1 (NR5A1) and liver receptor homolog-1 (NR5A2). Finally, we observed that basal and liver receptor homolog-1/phorbol ester-induced expression of steroidogenic enzymes in mICcl2 cells was inhibited by the antagonistic nuclear receptor small heterodimer partner. We conclude that the molecular basis of glucocorticoid synthesis in intestinal epithelial cells is distinct from that in adrenal cells, most likely representing an adaptation to the local environment and different requirements.

Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole-organ intervertebral disc model

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted up-regulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was down-regulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity.

Low social support and poor emotional regulation are associated with increased stress hormone reactivity to mental stress in systemic hypertension

CONTEXT: There is strong evidence for a physiological hyperreactivity to stress in systemic hypertension, but data on associated or potentially moderating psychological factors are scarce. OBJECTIVE: The objective of the study was to identify psychological correlates of physiological stress reactivity in systemic hypertension. DESIGN: This was a cross-sectional, quasi-experimentally controlled study. Study participants underwent an acute standardized psychosocial stress task combining public speaking and mental arithmetic in front of an audience. SETTING: The study was conducted in the population in the state of Zurich, Switzerland. SUBJECTS: Subjects included 22 hypertensive and 26 normotensive men (mean +/- sem 44 +/- 2 yr). MAIN OUTCOME MEASURES: We assessed the psychological measures social support, emotional regulation, and cognitive appraisal of the stressful situation. Moreover, we measured salivary cortisol and plasma epinephrine and norepinephrine before and after stress and several times up to 60 min thereafter as well as blood pressure and heart rate. RESULTS: We found poorer hedonistic emotional regulation (HER) and lower perceived social support in hypertensives, compared with normotensives (P < 0.01). Compared with normotensives, hypertensives showed higher cortisol, epinephrine, and norepinephrine secretions after stress (P < 0.038) as well as higher systolic and diastolic blood pressure (P < 0.001). Cortisol reactivity and norepinephrine secretion were highest in hypertensive men with low HER (P < 0.05). In contrast, hypertensives with high HER did not significantly differ from normotensives in both cortisol and norepinephrine secretion after stress. Epinephrine secretion was highest in hypertensives with low social support but was not different between hypertensives with high social support and normotensives. CONCLUSIONS: The findings suggest that both low social support and low HER are associated with elevated stress hormone reactivity in systemic hypertension.

Hexose-6-phosphate dehydrogenase modulates 11beta-hydroxysteroid dehydrogenase type 1-dependent metabolism of 7-keto- and 7beta-hydroxy-neurosteroids

BACKGROUND: The role of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in the regulation of energy metabolism and immune system by locally reactivating glucocorticoids has been extensively studied. Experiments determining initial rates of enzyme activity revealed that 11beta-HSD1 can catalyze both the reductase and the dehydrogenase reaction in cell lysates, whereas it predominantly catalyzes the reduction of cortisone to cortisol in intact cells that also express hexose-6-phosphate dehydrogenase (H6PDH), which provides cofactor NADPH. Besides its role in glucocorticoid metabolism, there is evidence that 11beta-HSD1 is involved in the metabolism of 7-keto- and 7-hydroxy-steroids; however the impact of H6PDH on this alternative function of 11beta-HSD1 has not been assessed. METHODOLOGY: We investigated the 11beta-HSD1-dependent metabolism of the neurosteroids 7-keto-, 7alpha-hydroxy- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) and 7-keto- and 7beta-hydroxy-pregnenolone, respectively, in the absence or presence of H6PDH in intact cells. 3D-structural modeling was applied to study the binding of ligands in 11beta-HSD1. PRINCIPAL FINDINGS: We demonstrated that 11beta-HSD1 functions in a reversible way and efficiently catalyzed the interconversion of these 7-keto- and 7-hydroxy-neurosteroids in intact cells. In the presence of H6PDH, 11beta-HSD1 predominantly converted 7-keto-DHEA and 7-ketopregnenolone into their corresponding 7beta-hydroxy metabolites, indicating a role for H6PDH and 11beta-HSD1 in the local generation of 7beta-hydroxy-neurosteroids. 3D-structural modeling offered an explanation for the preferred formation of 7beta-hydroxy-neurosteroids. CONCLUSIONS: Our results from experiments determining the steady state concentrations of glucocorticoids or 7-oxygenated neurosteroids suggested that the equilibrium between cortisone and cortisol and between 7-keto- and 7-hydroxy-neurosteroids is regulated by 11beta-HSD1 and greatly depends on the coexpression with H6PDH. Thus, the impact of H6PDH on 11beta-HSD1 activity has to be considered for understanding both glucocorticoid and neurosteroid action in different tissues.

Basement membrane remodeling in skeletal muscles of patients with limb ischemia involves regulation of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases

BACKGROUND/AIM: Because the pericapillary basement membrane in skeletal muscles of patients with chronic critical limb ischemia (CLI) is thickened, we determined the expression patterns of genes involved in collagen metabolism, using samples from 9 CLI patients, 4 patients with acute limb ischemia and 4 healthy controls. METHODS: Gene array analysis, quantitative RT-PCR and semiquantitative grading of immunohistochemical reactivity were performed to determine mRNA/cDNA and protein concentrations. RESULTS: In CLI patients compared to controls, cDNA levels of matrix metalloproteinase (MMP)-9 and MMP-19 were higher, collagen type IV chains A1 and A2, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 were similar and MMP-2 were lower. On the protein level, MMP-2, MMP-9, MMP-19 and TIMP-1 were more abundantly expressed. In skeletal muscles from patients with acute limb ischemia, cDNA and protein levels of MMP-9, MMP-19, collagen type IV chains, TIMP-1 and TIMP-2 were high. MMP-2 was elevated at the protein but decreased on the cDNA level. CONCLUSION: Expression of basement membrane components in skeletal muscles of CLI and acute limb ischemia patients is altered, possibly contributing to the pathogenesis of peripheral arterial disease.

Regulation of fetal growth: consequences and impact of being born small

The first trimester of pregnancy is the time during which organogenesis takes place and tissue patterns and organ systems are established. In the second trimester the fetus undergoes major cellular adaptation and an increase in body size, and in the third trimester organ systems mature ready for extrauterine life. In addition, during that very last period of intrauterine life there is a significant increase in body weight. In contrast to the postnatal endocrine control of growth, where the principal hormones directly influencing growth are growth hormone (GH) and the insulin-like growth factors (IGFs) via the GH-IGF axis, fetal growth throughout gestation is constrained by maternal factors and placental function and is coordinated by growth factors. In general, growth disorders only become apparent postnatally, but they may well be related to fetal life. Thus, fetal growth always needs to be considered in the overall picture of human growth as well as in its metabolic development.

The importance of growth hormone in the regulation of erythropoiesis, red cell mass, and plasma volume in adults with growth hormone deficiency

Total body water (TBW) is reduced in adult GH deficiency (GHD) largely due to a reduction of extracellular water. It is unknown whether total blood volume (TBV) contributes to the reduced extracellular water in GHD. GH and insulin-like growth factor I (IGF-I) have been demonstrated to stimulate erythropoiesis in vitro, in animal models, and in growing children. Whether GH has a regulatory effect on red cell mass (RCM) in adults is not known. We analyzed body composition by bioelectrical impedance and used standard radionuclide dilution methods to measure RCM and plasma volume (PV) along with measuring full blood count, ferritin, vitamin B12, red cell folate, IGF-I, IGF-binding protein-3, and erythropoietin in 13 adult patients with GHD as part of a 3-month, double blind, placebo-controlled trial of GH (0.036 U/kg.day). TBW and lean body mass significantly increased by 2.5 +/- 0.53 kg (mean +/- SEM; P < 0.004) and 3.4 +/- 0.73 kg (P < 0.004), respectively, and fat mass significantly decreased by 2.4 +/- 0.32 kg (P < 0.001) in the GH-treated group. The baseline RCM of all patients with GHD was lower than the predicted normal values (1635 +/- 108 vs. 1850 +/- 104 mL; P < 0.002). GH significantly increased RCM, PV, and TBV by 183 +/- 43 (P < 0.006), 350 +/- 117 (P < 0.03), and 515 +/- 109 (P < 0.004) mL, respectively. The red cell count increased by 0.36 +/- 0.116 x 10(12)/L (P < 0.03) with a decrease in ferritin levels by 39.1 +/- 4.84 micrograms/L (P < 0.001) after GH treatment. Serum IGF-I and IGF-binding protein-3 concentrations increased by 3.0 +/- 0.43 (P < 0.001) and 1.3 +/- 0.15 (P < 0.001) SD, respectively, but the erythropoietin concentration was unchanged after GH treatment. No significant changes in body composition or blood volume were recorded in the placebo group. Significant positive correlations could be established between changes in TBW and TBV, lean body mass and TBV (r = 0.78; P < 0.04 and r = 0.77; P < 0.04, respectively), and a significant negative correlation existed between changes in fat mass and changes in TBV in the GH-treated group (r = -0.95; P < 0.02). We conclude that 1) erythropoiesis is impaired in GHD; 2) GH stimulates erythropoiesis in adult GHD; and 3) GH increases PV and TBV, which may contribute to the increased exercise performance seen in these patients.

Abnormalities of very low density lipoprotein apolipoprotein B-100 metabolism contribute to the dyslipidaemia of adult growth hormone deficiency

Increased cardiovascular mortality in adult growth hormone deficiency (GHD) may be, in part, explained by the dyslipidaemia associated with this condition. It is possible that abnormalities of very low density lipoprotein apolipoprotein B-100 (VLDL apoB) metabolism contribute to this dyslipidaemia. To test this hypothesis, we measured VLDL apoB kinetics in adult GH deficient patients (4 females, 3 males; age 50.1 +/- 4.7 yr (mean +/- SEM); BMI 28.2 +/- 1.1 kg/m2; total cholesterol (TC) 6.6 +/- 0.3 mmol/l; triglyceride (TG) 2.8 +/- 0.6 mmol/l; HDL cholesterol 1.1 +/- 0.1 mmol/l) and in control subjects (4 females, 3 male; age 47.0 +/- 4.7 yr; BMI 27.0 +/- 2.6 kg/m2; TC 5.0 +/- 0.4 mmol/l; TG 0.9 +/- 0.2 mmol/l; HDL cholesterol 1.4 +/- 0.1 mmol/l). [1-(13)C] leucine was administered by a primed (1 mg/kg), constant intravenous infusion (1 mg/kg/hr) and VLDL apoB enrichment with 13C leucine was determined using gas-chromatography mass-spectrometry. The GHD patients had a significantly higher hepatic secretion rate of VLDL apoB (15.5 +/- 1.8 mg/kg/day vs 9.4 +/- 0.6 mg/kg/day p = 0.007) and reduced catabolism ofVLDL apoB (metabolic clearance rate; 12.3 +/- 1.7 ml/min vs 24.3 +/- 4.8 ml/min p < 0.05) compared with control subjects. These findings suggest that GH is integrally involved in the regulation of VLDL apoB metabolism.

Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae

Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH(2). The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.

Brain Regulation of Growth in Beef Calves

Small peptide hormones produced in the lower part of the brain (hypothalamus) regulate episodic and basal secretion of hormones from the anterior pituitary gland that affect metabolism and growth in cattle. This study focused on long-term growth in young calves subjected to hypophysectomy (HYPOX), hypophyseal stalk transection (HST), and sham operation control (SOC). Crossbred (Hereford x Aberdeen Angus) and Hereford, and Aberdeen Angus calves were HYPOX (n = 5), HST (n = 5), or SOC (n = 8) at 146 days of age, whereas another group was HST (n = 5) or SOC (n = 7) at 273 days of age. Body weight was determined every 21 days from birth to 1008 days of age. From day 146-1008, growth was arrested (P < 0.001) in HYPOX (0.06 kg/day) compared with SOC (0.50 kg/day) calves. Growth continued but at a significantly lower rate (P < 0.05) in calves HST at 146 days (0.32 kg/day) and 273 days (0.32 kg/day) compared with SOC (0.50 kg/day). Although episodic growth hormone (GH) secretion was abolished and peripheral blood serum GH concentration remained consistently lower in HST calves (2.4 ng/ml) than in the SOC (5.5 ng/ml; P < 0.01), the calves continued to grow throughout 1008 days. Peripheral serum thyroid stimulating hormone (TSH) concentration was less (P < 0.05) in HST compared with SOC calves. There was an abrupt decrease (P < 0.001) in serum thyroxine (T4) (4-fold) and triiodothyronine (T3) (3-fold) concentration after surgery that remained to 360 days in HST compared with SOC calves. At sacrifice, pituitary gland weight was markedly reduced (P < 0.001) in HST (0.18 g/100 kg body weight) compared with SOC (0.55 g/100 kg body weight) calves. Histological examination of pituitary glands from HST calves indicated the persistence of secretory GH and TSH cells in the same areas of the anterior pituitary gland as SOC calves. Coronal sections of the gland revealed GH and TSH secreting cells in HST calves that were similar to the controls. These results indicate that long-term growth continues, but at a slower rate, after hypophyseal stalk transection of immature calves in spite of complete abolition of episodic GH secretion and consistently decreased basal secretion of GH, TSH, T4, and T3 compared with sham-operated animals. Growth was abolished after hypophysectomy of immature calves in which circulating GH and TSH was undetectable.

Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows.

Fat mobilization to meet energy requirements during early lactation is inevitable because of insufficient feed intake, but differs greatly among high-yielding dairy cows. Therefore, we studied milk production, feed intake, and body condition as well as metabolic and endocrine changes in high-yielding dairy cows to identify variable strategies in metabolic and endocrine adaptation to overcome postpartum metabolic load attributable to milk production. Cows used in this study varied in fat mobilization around calving, as classified by mean total liver fat concentrations (LFC) postpartum. German Holstein cows (n=27) were studied from dry off until d 63 postpartum in their third lactation. All cows were fed the same total mixed rations ad libitum during the dry period and lactation. Plasma concentrations of metabolites and hormones were measured in blood samples taken at d 56, 28, 15, and 5 before expected calving and at d 1 and once weekly up to d 63 postpartum. Liver biopsies were taken on d 56 and 15 before calving, and on d 1, 14, 28, and 49 postpartum to measure LFC and glycogen concentrations. Cows were grouped accordingly to mean total LFC on d 1, 14, and 28 in high, medium, and low fat-mobilizing cows. Mean LFC (±SEM) differed among groups and were 351±14, 250±10, and 159±9 mg/g of dry matter for high, medium, and low fat-mobilizing cows, respectively, whereas hepatic glycogen concentrations postpartum were the highest in low fat-mobilizing cows. Cows in the low group showed the highest dry matter intake and the least negative energy balance postpartum, but energy-corrected milk yield was similar among groups. The decrease in body weight postpartum was greatest in high fat-mobilizing cows, but the decrease in backfat thickness was greatest in medium fat-mobilizing cows. Plasma concentrations of nonesterified fatty acids and β-hydroxybutyrate were highest around calving in high fat-mobilizing cows. Plasma triglycerides were highest in the medium group and plasma cholesterol concentrations were lowest in the high group at calving. During early lactation, the decrease in plasma glucose concentrations was greatest in the high group, and plasma insulin concentrations postpartum were highest in the low group. The revised quantitative insulin sensitivity check index values decreased during the transition period and postpartum, and were highest in the medium group. Plasma cortisol concentrations during the transition period and postpartum period and plasma leptin concentrations were highest in the medium group. In conclusion, cows adapted differently to the metabolic load and used variable strategies for homeorhetic regulation of milk production. Differences in fat mobilization were part of these strategies and contributed to the individual adaptation of energy metabolism to milk production.

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

A novel form of CYP3A from rat brain: cDNA cloning, protein characterization and gene regulation of cytochrome P450 3A9

One full length cDNA clone, designated 3aH15, was isolated from a rat brain cDNA library using a fragment of CYP3A2 cDNA as a probe. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to mouse Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported rat CYP3A sequences. Clone 3aH15 was thus named CYP3A9 by Cytochrome P450 Nomenclature Committee. CYP3A9 seems to the major CYP3A isozyme expressed in rat brain. Sexual dimorphism of the expression of CYP3A9 was shown for the first time in rat brain as well as in rat liver. CYP3A9 appears to be female specific in rat liver based on the standards proposed by Kato and Yamazoe who defined sex specific expression of P450s as being a 10-fold or higher expression level in one sex compared with the other. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Male rats treated with estrogen had a similar expression level of CYP3A9 mRNA both in the liver and brain. Ovariectomy of adult female rats drastically reduced the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. On the other hand, only a two-fold induction of CYP3A9 expression by dexamethasone was observed in male liver and no significant induction of CYP3A9 mRNA was observed in female liver or in the brains. These results suggest that estrogen may play an important role in the female specific expression of the CYP3A9 gene and that CYP3A9 gene expression is regulated differently from other CYP3A isozymes. ^ P450 3A9 recombinant protein was expressed in E. coli using the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity (10.1 nmol P450/mg protein) from solubilized fractions using two chromatographic steps. The purified P450 3A9 protein was active towards the metabolism of many clinically important drugs such as imipramine, erythromycin, benzphetamine, ethylmorphine, chlorzoxazone, cyclosporine, rapamycin, etc. in a reconstituted system containing lipid and rat NADPH-P450 reductase. Although P450 3A9 was active towards the catabolism of testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 17β-estradiol, P450 3A9 preferentially catalyzes the metabolism of progesterone to form four different hydroxylated products. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture and GSH. The possible mechanisms of the stimulatory effects of GSH on P450 3A9 activities are discussed. Sexually dimorphic expression of P450 3A9 in the brain and its involvement in many neuroactive drugs as well as neurosteroids suggest the possible role of P450 3A9 in some mental disorders and brain functions. ^