Mouse Adrenal Gland Research Articles

Efficient transfection of dissociated mouse chromaffin cells using small-volume electroporation

We have developed an improved procedure for isolating and transfecting a chromaffin cell-enriched population of primary cells from adult mouse adrenal glands. Significantly, the parameters of a novel electroporation transfection technique were optimized to achieve an average transfection efficiency of 45% on the small number of cells derived from the mouse glands. Such transfection efficiency was previously unachievable with the electroporation protocols conventionally used with bovine chromaffin cells, even with use of large cell numbers. Our small scale technique now makes feasible the use of genetically homogenous inbred mouse models for investigations on the exocytotic pathway without the time, expense, and cellular changes associated with viral approaches. High fidelity co-expression of multiple plasmids in individual cells is a further advantage of the procedure. To assess whether the biophysical characteristics of mouse adrenal chromaffin cells were altered by this process, we examined structural integrity using immunocytochemistry and functional response to stimuli using calcium imaging, amperometry, and whole-cell capacitance and current clamp recordings. We conclude these parameters are minimally affected. Finally, we demonstrate that high transfection efficiency makes possible the use of primary mouse adrenal chromaffin cells, rather than a cell line, in human growth hormone secretion assays for high throughput evaluation of secretion.

Kcne3 deletion initiates extracardiac arrhythmogenesis in mice

Mutations in the human KCNE3 potassium channel ancillary subunit gene are associated with life-threatening ventricular arrhythmias. Most genes underlying inherited cardiac arrhythmias, including KCNE3, are not exclusively expressed in the heart, suggesting potentially complex disease etiologies. Here we investigated mechanisms of KCNE3-linked arrhythmogenesis in Kcne3(-/-) mice using real-time qPCR, echo- and electrocardiography, ventricular myocyte patch-clamp, coronary artery ligation/reperfusion, blood analysis, cardiac synaptosome exocytosis, microarray and pathway analysis, and multitissue histology. Kcne3 transcript was undetectable in adult mouse atria, ventricles, and adrenal glands, but Kcne3(-/-) mice exhibited 2.3-fold elevated serum aldosterone (P=0.003) and differentially expressed gene networks consistent with an adrenal-targeted autoimmune response. Furthermore, 8/8 Kcne3(-/-) mice vs. 0/8 Kcne3(+/+) mice exhibited an activated-lymphocyte adrenal infiltration (P=0.0002). Kcne3 deletion also caused aldosterone-dependent ventricular repolarization delay (19.6% mean QTc prolongation in females; P<0.05) and aldosterone-dependent predisposition to postischemia arrhythmogenesis. Thus, 5/11 Kcne3(-/-) mice vs. 0/10 Kcne3(+/+) mice exhibited sustained ventricular tachycardia during reperfusion (P<0.05). Kcne3 deletion is therefore arrhythmogenic by a novel mechanism in which secondary hyperaldosteronism, associated with an adrenal-specific lymphocyte infiltration, impairs ventricular repolarization. The findings highlight the importance of considering extracardiac pathogenesis when investigating arrhythmogenic mechanisms, even in inherited, monogenic channelopathies.

Neurosteroid dehydroepiandrosterone and brain function

For last 30 years it became clear that DHEA and DHEAS are synthesized de novo in brain. Steroids synthesized in brain structures, were received the name "neurosteroids". In the review are submitted data on a biosynthesis and metabolism of DHEA(S) including its metabolism in fatty tissue where it serves as substrate for intracellular formation of its biologically active metabolites--estradiol and testosterone. The role of a sulfatase and sulfotransferase in mutual transformations of DHEA and DHEA-sulfate are analysed. Specific differences in DHEA synthesis in adrenals are surveyed. The adrenals of primates, both human beings and monkeys, produce free DHEA and DHEA-sulphate in large quantity. Their synthesis proceeds on Δ5-pathways: cholesterol-pregnenolone-17-hydroxypregnenolone-DHEA. Adrenals of other animas species, including rats and mice, don't synthesize DHEA. From the authors point of view, process of DHEAS penetration in brain structures include two mechanisms: transformation under steroid sulfatase action DHEAS in DHEA which freely gets through a blood-brain barrier and DHEAS passing through a hypothalamus which isn't protected by a blood-brain barrier. Results of researches on clinical application of DHEA as neurosteroid, with the analysis of its role in a course of Alzheimer's disease, distrurbances of cognitive function and other disorders of a CBS are presented also. The main neurobiological effects of DHEA(S) on brain structures which are studied on various models of animals include: neuroprotection, neurogenesis and neuronal survival, apoptosis, catecholamine synthesis and secretion. Neurosteroids have also antioxidant, anti-inflammatory and anti-glucocorticoid effects.

Preimplantation genetic diagnosis (PGD) influences adrenal development and response to cold stress in resulting mice

Preimplantation genetic diagnosis (PGD) has gained widespread application in clinical medicine and hence the health of PGD offspring needs to be systematically assessed. Given the critical role of the stress response in growth and health, assessments of the development and function of the stress system might help to clarify the health outcomes of PGD. In this study, we constructed a PGD-conceived mouse model and used naturally conceived mice as controls; we used this model to evaluate the potential effect of PGD procedures on the stress system of the offspring. Serum and tissues of stress organs, namely the hypothalamus, locus coeruleus and adrenal gland, were collected from 5-week-old mice in the basal state or after cold stress. The serum levels of stress-related hormones and the structural and functional indices of the stress organs were then examined. In the basal state, ultrastructural abnormalities and low expression of genes involved in steroid hormone synthesis were found in the adrenals of the PGD mice, which had low corticosterone and high epinephrine levels compared with those of control mice. After acute cold stress, the PGD mice continued to show structural and glucocorticoid secretion abnormalities resulting in a late response to the environmental change. Thus, our study indicates that PGD manipulations affect adrenal development, result in structural and functional abnormalities of the adrenals in the offspring and influence their reactivity and adaptability to cold stress.

Quantitative Detection of Vibrio cholera Toxin by Real-Time and Dynamic Cytotoxicity Monitoring

We report here the quantitative detection of Vibrio cholerae toxin (CT) in isolates and stool specimens by dynamic monitoring of the full course of CT-mediated cytotoxicity in a real-time cell analysis (RTCA) system. Four cell lines, including Y-1 mouse adrenal tumor cells, Chinese hamster ovary (CHO) cells, small intestine epithelial (FHs74Int) cells, and mouse adrenal gland (PC12-Adh) cells, were evaluated for their suitability for CT-induced cytotoxicity testing. Among them, the Y-1 line was demonstrated to be the most sensitive for CT-mediated cytotoxicity, with limits of detection of 7.0 pg/ml for purified CT and 0.11 ng/ml for spiked CT in pooled negative stool specimens. No CT-mediated cytotoxicity was observed for nontoxigenic V. cholerae, non-V. cholerae species, or non-V. cholerae enterotoxins. The CT-RTCA assay was further validated with 100 stool specimens consecutively collected from patients with diarrhea and 200 V. cholerae isolates recovered from patients and the environment, in comparison to a reference using three detection methods. The CT-RTCA assay had sensitivities and specificities of 97.5% and 100.0%, respectively, for V. cholerae isolates and 90.0% and 97.2% for stool specimens. For stool specimens spiked with CT concentrations ranging from 3.5 pg/ml to 1.8 ng/ml, the inoculation-to-detection time was 1.12 ± 0.38 h, and the values were inversely correlated with CT concentrations (ρ = -1; P = 0.01). The results indicate that the CT-RTCA assay with the Y-1 cell line provides a rapid and sensitive tool for the quantitative detection of CT activities in clinical specimens.

Genomic Analysis of Sexual Dimorphism of Gene Expression in the Mouse Adrenal Gland

A relevant gender difference exists in adrenal physiology and propensity to disease. In mice, a remarkable sexual dimorphism is present in several components of the hypothalamic-pituitary-adrenal axis, with females displaying higher adrenal weight, plasma ACTH, corticosterone, and aldosterone levels than males. The molecular bases of this sexual dimorphism are little known. We have compared global gene expression profiles in males vs. female mouse adrenal glands and also studied the effect that testosterone treatment and castration have on adrenal gene expression in female vs. male mice, respectively. Our study evidenced a set of 71 genes that are coordinately modulated according to sex and hormonal treatments and represent the core sexually dimorphic expression program in the mouse adrenal gland. Moreover, we show that some genes involved in steroid metabolism have a remarkable sexual dimorphic expression and identify new potential markers for the adrenal X-zone, a transitory cellular layer in the inner adrenal cortex, which spontaneously regresses at puberty in males and during the first pregnancy in females and has an uncertain physiological role. Finally, sexually dimorphic expression of the transcriptional regulators Nr5a1 and Nr0b1 may explain at least in part the differences in adrenal steroidogenesis between sexes.

Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn't fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.

DNA Microarray Analysis of the Mouse Adrenal Gland for the Detection of Hypothermia Biomarkers: Potential Usefulness for Forensic Investigation

We analyzed the adrenal gland transcriptome of mice killed by hypothermia using DNA microarray technology. A total of 4051 significantly expressed genes were identified; 2015 genes were upregulated and 2036 were downregulated.The FBJ osteosarcoma oncogene was the most upregulated,whereas stearoyl coenzyme A desaturase 3 was the most downregulated. Validation by quantitative polymerase chain reaction revealed that results obtained by both methods were consistent. In the gene set analysis, significant variations were found in nine pathways, and we suggest that transforming growth factor β and tumor necrosis factor α would be involved in the pathogenesis of hypothermia. Gene functional category analysis demonstrated the most overexpressed categories in upregulated and downregulated genes were cellular process in biological process, binding in molecular function, and cell and cell part in cellular component. The present study demonstrated acute adrenal responses in hypothermia, and we suggest that understanding adrenal mRNA expression would be useful for hypothermia diagnosis. Furthermore, the present microarray data may also facilitate development of immunohistochemical analysis of human cases. In forensic practice, the combination of macroscopic and microscopic observations with molecular biological analyses would be conducive to more accurate diagnosis of hypothermia. Although this study is aimed at forensic practice, the present data regarding more than 20,000 genes of the adrenal gland would be beneficial to inform future clinical hypothermia research. From the viewpoint of adrenal gene activity, they could contribute to elucidating the pathophysiology of hypothermia.

Immunohistochemical detection of angiotensin II receptors in mouse cerebellum and adrenal gland using “in vivo cryotechnique”

Angiotensin II (AT) receptors, including AT receptor type 1 (AT1R) and type 2 (AT2R), are expressed in the rodent central nervous system, but their distributions and activation states are still unclear. In this study, we have performed immunohistochemical analyses of AT receptors in mouse cerebellum and adrenal gland using our "in vivo cryotechnique" (IVCT). We used antibodies against amino-terminal domains of AT receptors, which are considered to undergo conformational changes upon the binding of AT. Immunoreactivity of AT1R was detected in mouse cerebellum, and was highest in the outer tissue areas of molecular layers using IVCT. The AT1R immunostaining largely overlapped with glial fibrillary acidic protein (GFAP), a marker of Bergmann glia. Surprisingly, the AT1R immunoreactivity in the cerebellar cortex was remarkably reduced following 5 and 10min of hypoxia or direct administration of an AT1R antagonist, losartan. By contrast, in the adrenal cortex, such AT1R immunoreactivity detected at the zona glomerulosa did not change even after 15min of hypoxia. The correlation of localization with GFAP and also hypoxia-induced decrease of its immunoreactivity were similarly observed by immunostaining of AT2R in the cerebellar specimens. These findings demonstrated that IVCT is useful to reveal dynamically changing immunoreactivities usually affected by receptor-ligand binding as well as hypoxia, and also suggested that functional activities of AT receptors are time-dependently modulated under hypoxia in the central nervous system in comparison with the adrenal glands.

LCAT deficiency in mice is associated with a diminished adrenal glucocorticoid function

In vitro studies have suggested that HDL and apoB-containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specific contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio (P < 0.001) and complete HDL-cholesteryl ester deficiency. ApoB-containing lipoprotein and associated triglyceride levels are increased in LCAT KO mice as compared with C57BL/6 control mice (44%; P < 0.05). Glucocorticoid-producing adrenocortical cells within the zona fasciculata in LCAT KO mice are devoid of neutral lipids. However, adrenal weights and basal corticosterone levels are not significantly changed in LCAT KO mice. In contrast, adrenals of LCAT KO mice show compensatory up-regulation of genes involved in cholesterol synthesis (HMG-CoA reductase; 516%; P < 0.001) and acquisition (LDL receptor; 385%; P < 0.001) and a marked 40-50% lower glucocorticoid response to adrenocorticotropic hormone exposure, endotoxemia, or fasting (P < 0.001 for all). In conclusion, our studies show that HDL-cholesteryl ester deficiency in LCAT KO mice is associated with a 40-50% lower adrenal glucocorticoid output. These findings further highlight the important novel role for HDL as cholesterol donor for the synthesis of glucocorticoids by the adrenals.

Ontogeny of the Adrenal Gland in the Spiny Mouse, With Particular Reference to Production of the Steroids Cortisol and Dehydroepiandrosterone

Synthesis of the androgen dehydroepiandrosterone (DHEA) by the fetal adrenal gland is important for placental estrogen production and may also be important for modulating the effects of glucocorticoids on the developing brain. The presence of cortisol in spiny mouse (Acomys cahirinus) blood led us to determine whether the adrenal gland of this precocial rodent also synthesized DHEA. Cytochrome P450 enzyme 17α-hydroxylase/17,20-lyase (P450c17), cytochrome-b5 (Cytb5), and 3β-hydroxysteroid dehydrogenase (3βHSD) were detected in the adrenal gland from 30 days gestation (term = 39 days), and DHEA, cortisol, and aldosterone were detected in fetal plasma from this time. Plasma DHEA concentrations increased 4-fold, whereas cortisol concentrations decreased from day 30 of gestation until the day of birth. Explant culture of fetal adrenal tissue showed that DHEA was produced from exogenous pregnenolone, and thus, the DHEA in the fetal circulation is likely to be of fetal origin. Clear zonation of the fetal adrenal cortex was evident by 38 days gestation when expression of Cytb5 was present throughout the cortex, and coexpression of P450c17 and Cytb5 occurred in the zona reticularis and fasciculata. 3βHSD was expressed in the cortex from at least 30 days gestation and decreased as term approached, consistent with the fall of cortisol in late gestation in this species. These results show that the spiny mouse adrenal gland, like that of the human fetus, can synthesize and secrete DHEA from at least 30 days (relative gestation length, 30 days of a 39-day gestation, 0.76) of gestation, and DHEA may have important roles in placental biosynthesis of estrogens and in modulating the actions of glucocorticoids in the developing brain in this species.

Transgenic GATA-4 expression induces adrenocortical tumorigenesis in C57Bl/6 mice.

A link between elevated luteinizing hormone (LH) levels, GATA-4 and LH receptor (LHCGR) expression and gonadotropin-dependent adrenocortical tumorigenesis in humans and mice has been shown. To assess the mechanistic tumorigenic interrelationships between these factors, we transgenically expressed Gata4 under the 21-hydroxylase promoter (Cyp21a1, 21-OH) in C57Bl/6N mice. There was a gradual age-dependent increase of GATA-4 expression only in 21-OH-GATA-4 (TG) female adrenals, in association with slowly progressing neoplasia of non-steroidogenic spindle-shaped A cells in the subcapsular cortex. Gonadectomy (GDX), apparently through direct action of elevated serum LH, markedly enhanced the adrenocortical neoplasia, which now also appeared in GDX TG males. The neoplastic areas of the post-GDX TG adrenals contained, besides A cells, larger lipid-laden, steroidogenically active and LHCGR-positive B cells. Prolonged (>10 months) exposure to elevated post-GDX LH levels resulted in formation of adrenocortical adenomas in the TG mice. Intact and GDX TG mouse adrenals displayed elevated FOG-2 and decreased GATA-6 expression. Additionally, increased expression/activation of components of the Inhbb-Acvr2a-Acvr1c-Smad2/3 signaling system was observed in 12-month-old GDX TG adrenals. Our findings show that two distinct GATA-4-dependent populations of neoplastic adrenocortical cells form: non-steroidogenic LH-independent A cells and steroidogenic LH-dependent B cells.

Widespread expression of zinc transporter ZnT (SLC30) family members in mouse endocrine cells

Zinc is abundant in most endocrine cell types, and plays a pivotal role in the synthesis and secretion of many hormones. Recent studies have demonstrated the expression of numerous zinc transporter (ZnT) family members in the pancreas, thyroid, and adrenal glands, suggesting a role for ZnTs in regulating cellular zinc homeostasis in endocrine cells. However, the cellular distribution of ZnTs in the endocrine organs has not been well established. In the present study, the mRNA expression level, cellular distribution of ZnTs as well as liable zinc ions were examined in the mouse pituitary, adrenal glands, thyroid, and pancreas. In general, ZnT1-10 mRNA was expressed to various degrees in the detected endocrine organs, with no detectable ZnT10 mRNA in the pancreas. In the anterior pituitary, both the acidophilic and basophilic cells were immunopositive to ZnT1-5, 7, 8, except for ZnT10. In the adrenal cortex, the immunoreactivity of all the tested ZnTs, including ZnT1-5, 7, 8, 10, was observed in the zona fasciculata, and some ZnTs were detected in the zona glomerulosa, zona reticularis, and the adrenal medulla. Both the follicle epithelial cells and parafollicular cells in the thyroid gland were immunostained with ZnT1-5, 7, 8, but not ZnT10. In the endocrine pancreas, the immunoreactivity of tested ZnTs was observed to various degrees except for ZnT10 in the cytoplasm of islet cells. Furthermore, autometallographic staining showed that liable zinc was observed in the endocrine cells, such as the adrenal cortical cells, thyroid follicle epithelial cells, and the pancreatic islet cells. All together, the wide distribution of liable zinc and the phenomenon that numerous ZnT family members are partially overlapped in a subset of endocrine cells suggest an important role for the ZnT family in controlling cellular zinc levels and subsequently regulating the synthesis and secretion of hormones in the endocrine system.

Bidirectional regulation of adrenal catecholamine release by prostaglandin E2

Catecholamine secretion from adrenal chromaffin cells is elevated in response to adverse physiological conditions. Similarly, systemic immune challenge leads to intra‐adrenal inflammatory signaling and a resulting induction of synthetic enzymes for prostaglandin E2 (PGE2). In the present study we investigated the functional impact of PGE2 on calcium signaling and catecholamine release from mouse adrenal chromaffin cells. PGE2 potently (EC50 = 5.5 nM) inhibited Cav2 voltage‐gated calcium currents (ICa), while having no effect on resting intracellular [Ca2+] or the peak amplitude of nicotinic acetylcholine receptor currents. The inhibition of ICa is mediated by pertussis toxin‐sensitive G proteins, and reversed by a strongly depolarizing voltage step, characteristic of direct G protein βγ subunit binding to the channel. PGE2 acts primarily through a family of cognate GPCRs (EP1‐EP4). We detected mRNA for all four EP receptors in mouse adrenal gland, but using selective pharmacological tools and EP receptor knockout mice we show the inhibition of ICa is mediated by EP3 receptors. Monitoring changes in membrane capacitance in response to brief depolarizations, we show Ca2+‐ dependent exocytosis is reduced in parallel with ICa in wild‐type mice. Conversely, PGE2 potentiates exocytosis in chromaffin cells isolated from EP3 receptor knockout mice, revealing a signaling modality distinct from the inhibitory EP3 pathway. Using carbon fiber amperometry, we find the potentiation of exocytosis is also apparent in wild type cells during sustained depolarizations mimicking acute stress. Our data support a model in which distinct EP receptor subtypes mediate activity‐dependent, bidirectional modulation of catecholamine release by PGE2. This work is supported by the American Heart Association [11GRNT7890031] and the National Institutes of Health [R01 NS052446].

ACTH-Dependent Regulation of MicroRNA As Endogenous Modulators of Glucocorticoid Receptor Expression in the Adrenal Gland

MicroRNA (miR) are a subset of small RNA molecules, which posttranscriptionally modulate target gene expression. Although miR have been demonstrated to impact a number of processes during development and tumorigenesis, little is known about the expression and the role of miR in the adrenal gland. Because tight regulation of steroid synthesis is crucial for maintaining homeostasis upon stressful stimuli, here, we determined the miR expression pattern in mouse adrenal glands under baseline conditions, as well as 10, 30, and 60 min upon ACTH stimulation, using miR microarray. Changes in miR expression levels detected by array analysis were confirmed by real-time PCR and further analyzed by bioinformatic tools to identify miR that putatively target genes involved in adrenal function. After selecting miR, with a significant change in their expression level upon ACTH stimulation, four of the predefined miR (miR-96, miR-101a, miR-142-3p, and miR-433) were found to putatively target the glucocorticoid receptor [nuclear receptor subfamily 3, group C, member 1 (Nr3c1)]. Nr3c1 expression levels were elevated 10 min after ACTH stimulation but decreased after 60 min in comparison with baseline conditions. Modified Nr3c1-3'-untranslated region constructs were further tested by in vitro luciferase assays. Thereby, we could confirm that miR96, miR101a, miR142-3p, and miR433 target the Nr3c1-3'-untranslated region and result in a 20-40% repression of it. Taken together, ACTH stimulation could be demonstrated to acutely influence adrenal miR expression pattern in vivo; thus, potentially modulating adrenal response to acute stressors.

Transition from organogenesis to stem cell maintenance in the mouse adrenal cortex

Mice showing mosaic expression of an appropriate marker gene that is activated during development provide simple tools for investigating cell lineages. We used the mosaic β-galactosidase staining patterns in adrenal cortices of 21OH/ LacZ transgenic mice to study both organogenesis and maintenance of the adult tissue. Randomly orientated mosaic patterns present in embryonic day 14.5 (E14.5) adrenals changed progressively during the perinatal period from discrete spots, via patches and radial arrays, to radial stripes, which first emerged between postnatal days 0 and 7 (P0 and P7). The mosaic radial stripe pattern was fully established by P21 and remained unchanged throughout the adult period (8–52 weeks). The mouse adrenal gland grew continuously between E14.5 and P21, including the period during which stripes emerge. Ki67-positive, proliferative cells in the adrenal cortex were mainly localized to the outer cell layers between E18.5 and P3. By P10, cell proliferation had increased, and the proliferative region had expanded but was still mainly confined to the outer cortex. Correlation of changes in mosaic patterns in 21OH/LacZ adrenal cortices with the locations of adrenocortical cell proliferation suggest that the radial stripes arise by edge-biased growth during the perinatal period, even if they are maintained by stem cells in adults. The stability of the adult stripe pattern suggests that stem cell function is unchanged between 8 and 52 weeks.

Circadian expression of steroidogenic cytochromes P450 in the mouse adrenal gland – involvement of cAMP‐responsive element modulator in epigenetic regulation of Cyp17a1

The cytochrome P450 (CYP) genes Cyp51, Cyp11a1, Cyp17a1, Cyb11b1, Cyp11b2 and Cyp21a1 are involved in the adrenal production of corticosteroids, whose circulating levels are circadian. cAMP signaling plays an important role in adrenal steroidogenesis. By using cAMP responsive element modulator (Crem) knockout mice, we show that CREM isoforms contribute to circadian expression of steroidogenic CYPs in the mouse adrenal gland. Most striking was the CREM-dependent hypomethylation of the Cyp17a1 promoter at zeitgeber time 12, which resulted in higher Cyp17a1 mRNA and protein expression in the knockout adrenal glands. The data indicate that products of the Crem gene control the epigenetic repression of Cyp17 in mouse adrenal glands.

Paraquat Induced Toxicity in Adrenals in Albino Mice

Objectives: The current study aims to observe the histological effects of paraquat on the adrenal Glands of the mice after single exposure to sub-lethal dose of the drug. Materials and Method: Forty male mice, fifteen control (group I) and twenty-five experimental (group II) were used. Each experimental animal (group II) was given a single dose of paraquat i.e 20 mg/kg dissolved in 0.2 ml normal saline as an intraperitoneal injection, while for the control group (I) each animal was given an equal amount of normal saline by the same route. The exposure duration for the study was comprised of ten days. On the tenth day all the animal (control and experimental) were slaughtered. Whole adrenals were removed, fixed in 10 % formal saline for 48 hours and processed for routine histological studies. Results: Histological examination of the experimental group showed that the adrenal cortical sinusoids contained large number of erythrocytes, suggesting congestion of the organ; similar changes were observed in blood vessels of medulla. Conclusion: Findings of the current study demonstrate congestion in the sinusoids. It can be assumed that this congestion was due to the formation of peroxynitrite anion (ONOO - ) which may have major role in the progression of the paraquat toxicity.

Differential regulation of the human CYP11A1 promoter in mouse brain and adrenals

CYP11A1 encodes the first enzyme of steroid biosynthesis, cytochrome P450scc. The expression of CYP11A1 in the nervous system allows neurosteroids to be synthesized de novo. In the classic steroidogenic tissues, adrenals and gonads, the key regulator controlling CYP11A1 expression is steroidogenic factor-1 (SF-1), but the transcriptional regulation of CYP11A1 in the brain is unclear. We recently used the 4.4-kb regulatory region of the human CYP11A1 gene to drive Cre recombinase expression in the diencephalon and midbrain. In this study, we characterized the regional-specific expression of Cre reporter in the SCC-Cre transgenic brain using a transient Cre/ROSA26R transgenic system. Mutation of either the upstream or proximal SF-1 binding site did not affect brain CYP11A1 promoter activity. The upstream SF-1 binding site, however, is required for CYP11A1 promoter function in the embryonic adrenals. The 3.8-kb promoter, like the 4.4-kb length promoter, directed Cre expression in the diencephalon, midbrain and olfactory epithelium, whereas Cre expression controlled by the 2.7-kb promoter was only observed in the caudal part of midbrain. This suggests that the 5'-flanking region between 3.8 and 2.7 kb contains a crucial element for activation of CYP11A1 promoter in the diencephalon, olfactory epithelium and the anterior part of midbrain. Thus we have identified regions of the promoter that control CYP11A1 expression in the brain and embryonic adrenals.

Rapid determination of amino acids in biological samples using a monolithic silica column

A high-performance liquid chromatography method in which fluorescence detection is used for the simultaneous determination of 21 amino acids is proposed. Amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and then separated on a monolithic silica column (MonoClad C18-HS, 150 mm×3 mm i.d.). A mixture of 25 mM citrate buffer containing 25 mM sodium perchlorate (pH 5.5) and acetonitrile was used as the mobile phase. We found that the most significant factor in the separation was temperature, and a linear temperature gradient from 30 to 49°C was used to control the column temperature. The limits of detection and quantification for all amino acids ranged from 3.2 to 57.2 fmol and 10.8 to 191 fmol, respectively. The calibration curves for the NBD-amino acid had good linearity within the range of 40 fmol to 40 pmol when 6-aminocaproic acid was used as an internal standard. Using only conventional instruments, the 21 amino acids could be analyzed within 10 min. This method was found to be suitable for the quantification of the contents of amino acids in mouse plasma and adrenal gland samples.