Estrogen regulation of uterine genes in vivo detected by complementary DNA array.

INTRODUCTION: In the present study, our aim was to identify differentially expressed genes involved in estrogen actions at the endometrium level in rats. METHODS: Thirty adult rats were ovariectomized four days prior to drug administration for 48 days. Rats were divided in 2 groups: I, control and II, conjugated equine estrogens (CCE). Total RNA was isolated from uterus, and differential expression was analyzed by array technology and RT-PCR. RESULTS: A total of 32 candidate genes were shown to be upregulated or downregulated in groups I or II. Among them, differential expression was already confirmed by RT-PCR for IGFBP5, S12, c-kit, and VEGF, genes whose expression was up regulated during CCE therapy, and casein kinase II and serine kinase expression was the same level in both groups. CONCLUSION: We have demonstrated that cDNA array represents a powerful approach to identify key molecules in the estrogens therapy. A number of the candidates reported here should provide new markers that may contribute to the detection of target estrogen receptor. This information may also aid the development of new approaches to therapeutic intervention.     

Thyroid hormone regulation of alpha-myosin heavy chain promoter activity assessed by in vivo DNA transfer in rat heart

We have studied the thyroid-hormone responsiveness of the alpha-myosin heavy chain (MHC) gene in vivo by directly injecting an expression vector containing the alpha-MHC 5' regulatory sequences (-613 to +421 base pairs) into the rat heart. In the expression vector pAM1Luc the alpha-MHC promoter elements direct the synthesis of firefly luciferase. Although thyroxine administration of both euthyroid and thyroidectomized rats for 5 days increased alpha-MHC promoter activity, the pAM1Luc gene construct did not mimic expression of the endogenous gene. These studies of direct gene transfer into mammalian myocardium suggest that additional cis-acting elements necessary for the in vivo response to thyroid hormone reside outside the -613 to +421 region.     

Identification and characterization of novel and unknown mouse epididymis-specific genes by complementary DNA microarray technology

To examine epididymal function, we attempted to identify highly expressed genes in mouse epididymis using a cDNA microarray containing PCR products amplified from a mouse epididymal cDNA library. We isolated one novel and four known genes-lymphocyte cytosolic protein 1 (Lcp1), complement subcomponents C1r/C1s, Uegf protein, and bone morphogenetic protein and zona pellucida-like domains 1 (Cuzd1), transmembrane epididymal protein 1 (Teddm1), and whey acidic protein 4-disulfide core domain 16 (Wfdc16)-with unknown functions in the epididymis. The novel gene, designated Serpina1f (serine peptidase inhibitor [SERPIN], clade A, member 1f), harbors an open reading frame of 1 233 bp encoding a putative protein of 411 amino acids, including a SERPIN domain. These five genes were predominantly expressed in the epididymis as compared to other organs. In situ hybridization analysis revealed their epididymal region-specific expression patterns. Real-time RT-PCR analysis revealed a significant increase in mRNA expression of these genes around puberty. Castration decreased their expression, except forLcp1. Testosterone (T) restored these reduced expressions, except forTeddm1; however, this restoration was not observed with 17 beta-estradiol (E2). Administration of T and E2 combination recovered the Serpina1f mRNA concentration; this recovery was also observed with T alone. However, the recovery of Cuzd1and Wfdc16mRNA concentrations was inadequate. Neonatal diethylstilbestrol treatment suppressed the Cuzd1, Wfdc16, and Serpina1f mRNA expression in the epididymis of 8-week-old mice; this was not observed with E2. These results suggest that our microarray system can provide a novel insight into the epididymal function on a molecular basis, and the five genes might play important roles in the epididymis.     

Thyroid hormone regulation of flavocoenzyme biosynthesis

The means by which thyroid hormone regulates flavocoenzyme biosynthesis was studied in hyper-, eu-, and hypothyroid rats by determining the activities of flavocoenzyme-forming enzymes, viz., flavokinase and FAD synthetase, as well as those of flavocoenzyme-degrading enzymes, viz., FMN phosphatase and FAD pyrophosphatase. Flavokinase activity was increased in hyperthyroid animal and decreased in hypothyroid animals. Correspondence of flavokinase activity with the amount of a high-affinity flavin-binding protein quantitated immunologically in hypo-, eu-, and hyperthyroid rats indicated that the thyroid response is caused by an increased amount of enzyme; moreover, the concomitant decrease in a low-affinity flavin-binding protein suggests an inactive precursor form of flavokinase. FAD synthetase activity showed a similar but less pronounced trend than flavokinase. Activities of FMN phosphatase and FAD pyrophosphatase were not influenced by thyroid hormone. Overall results indicate that the mechanism of thyroid hormone regulation of flavocoenzyme level is in the steps of biosynthesis, especially at flavokinase, rather than in degradation steps.     

Gene expression in the normal adult human kidney assessed by complementary DNA microarray

The kidney is a highly specialized organ with a complex, stereotyped architecture and a great diversity of functions and cell types. Because the microscopic organization of the nephron, the functional unit of the kidney, has a consistent relationship to the macroscopic anatomy of the kidney, knowledge of the characteristic patterns of gene expression in different compartments of the kidney could provide insight into the functions and functional organization of the normal nephron. We studied gene expression in dissected renal lobes of five adult human kidneys using cDNA microarrays representing approximately 30,000 different human genes. Total RNA was isolated from sections of the inner and outer cortex, inner and outer medulla, papillary tips, and renal pelvis and from glomeruli isolated by sieving. The results revealed unique and highly distinctive patterns of gene expression for glomeruli, cortex, medulla, papillary tips, and pelvic samples. Immunohistochemical staining using selected antisera confirmed differential expression of several cognate proteins and provided histological localization of expression within the nephron. The distinctive patterns of gene expression in discrete portions of the kidney may serve as a resource for further understanding of renal physiology and the molecular and cellular organization of the nephron.     

Thyroid hormone regulation by stress and behavioral differences in adult male rats

Thyroid hormones are essential regulators of growth, development and normal bodily function and their release is coordinated by the hypothalamic-pituitary-thyroid (HPT) axis. While the HPT axis has been established as an acutely stress-responsive neuroendocrine system, relatively little is known about the mechanisms of its stress regulation. The present study examined acute stress-induced changes in peripheral hormone levels [triiodothyronine (T3); thyroxine (T4), thyroid-stimulating hormone (TSH), reverse triiodothyronine (rT3)] and central mRNA levels of regulators of the HPT axis [thyrotropin-releasing hormone (TRH), somatostatin (SST), type II deiodinase (D2)] in response to an inescapable tail-shock, a rodent model of stress. Additionally, we examined whether individual differences in spontaneous exploratory behavior in an open field test predicted basal levels of TH or differential susceptibility to the effects of stress. The stress condition was associated with decreases in peripheral T3, T4 and TSH, but not rT3, when compared with controls. No changes were observed in TRH or SST mRNA levels, but there was a trend suggesting stress-related increases in D2 mRNA. We also found that an animal's exploratory behavior in an unfamiliar open field arena was positively related to peripheral thyroid hormone levels and predicted the magnitude of stress-induced changes.In conclusion, we found suggestive evidence for stress-induced decrease in central drive HPT axis, but the central mechanisms of its stress regulation remain to be elucidated. Additionally, we found that individual differences in animals' exploratory behavior were correlated with peripheral TH levels.     

Thyroid hormone regulation of beta-adrenergic receptor number.

The effects of exogenous thyroid hormones (thyroxine and triiodothyronine) on beta-adrenergic receptors in the rat myocardium were investigated. The potent beta-adrenergic antagonist, (-)-[3H]dihydroalprenolol, was used to directly estimate the number and affinity of beta-adrenergic receptors in rat heart membranes from control and hyperthyroid rats. Cardiac membranes from hyperthyroid rats contained 196 +/- 7 fmol of (-)-[3H]dihydroalprenolol binding sites/mg of protein which was significantly (p less than 0.005) greater than the number of binding sites (89 +/- 5 fmol/mg of protein) present in control membranes. The equilibrium dissociation constant (KD) for the interaction of receptors with dihydroalprenolol was the same (2 to 15 nM) in membranes from control and hyperthyroid rats. Similarly, there was no significant difference between the control and hyperthyroid membranes in the affinity of the beta-adrenergic receptor binding sites for the beta-adrenergic agonist isoproterenol. The results of this study demonstrate that thyroid hormones can regulate the number of cardiac beta-adrenergic receptors. The increased numbers of receptors may be responsible, at least in part, for the enhanced catecholamine sensitivity of beta-adrenergic-coupled cardiac responses in the hyperthyroid state.     

Transcriptional regulation of nitrogen fixation genes by DNA supercoiling

DNA gyrase (ATP dependent topoisomerase type II, EC 5.99.1.3) was found to be essential for the expression of the Klebsiella pneumoniae nitrogen fixation gene cluster carried by plasmid pRD1 in Escherichia coli. In the absence of DNA gyrase activity, nitrogen fixation activity could be restored by providing a constitutively expressed nifA function in trans. Our results suggest that nif gene regulation by oxygen may be mediated through the alteration of the superhelical status of the promoter of the nifLA regulatory operon, in addition to the action of the nifL gene product.Communicated by J. Schell     

Cloning and sequencing of bullfrog growth hormone complementary DNA

Total mRNA was isolated from the pituitary glands of bullfrog (Rana catesbeiana), purified by affinity chromatography with oligo(dT)-cellulose columns. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The cDNA library was screened by hybridization with 32P-labeled duck growth hormone (GH) cDNA. A positive clone was selected and sequenced. The full-length bullfrog GH cDNA contains 950 nucleotide pairs with an open reading frame coding for the precursor GH of 215 amino-acid residues. The partial amino-acid sequence from the protein confirms that derived from the cDNA, with Phe as the first residue in the mature bullfrog GH preceded by a 25-residue hydrophobic signal peptide. The bullfrog GH shares sequence homology with those of other vertebrate species in the following order: duck (61% protein sequence homology; 67% cDNA homology), rat (56%; 61%), human (47%; 57%) and salmon (42%; 50%).     

DNA microarray analysis of genes differentially expressed in adipocyte differentiation

In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a ≥10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RT-PCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPARγ2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.