Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase.

The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

Selective interaction of vitamin D receptor with transcriptional coactivators by a vitamin D analog.

The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor. A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way. We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms. Unlike other vitamin D analogs tested, OCT (22-oxa-1alpha,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators. Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters.

Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor alpha.

The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERalpha (hERalpha) is enhanced through phosphorylation of the Ser(118) residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERalpha. Phosphorylation of hERalpha Ser(118) potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERalpha in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERbeta, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERalpha AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERalpha A/B domain was essential for the full activity of hERalpha AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERalpha AF-1 and strongly suggest that the interaction between p68 and the hERalpha A/B domain is regulated by MAPK-induced phosphorylation of Ser(118).

Caspase-like activity in programmed nuclear death during conjugation of Tetrahymena thermophila.

Apoptosis, or programmed cell death, is common in a variety of eucaryotes, from unicellular protozoa to vertebrates. The ciliated protozoan Tetrahymena thermophila has a unique apoptosis-like nuclear death during conjugation, called programmed nuclear death. This death program involves nuclear condensation (pyknosis) and oligonucleosomal DNA fragmentation in the parental macronucleus. Subsequently, the condensed nucleus is entirely resorbed in the autophagosome. Here we demonstrate that caspase-8- and -9-like activity was detected, but no caspase-3-like activity, by in vitro assay during the nuclear resorption process, suggesting that caspase-like activity is associated with both programmed cell death and apoptosis-like nuclear death in Tetrahymena. The use of indicator dye to detect the loss of mitochondrial membrane potential suggested the uptake of mitochondria and the degenerating macronucleus by the autophagosome. An involvement of mitochondria in the programmed nuclear death is discussed.

Characterization of a tandemly repeated DNA sequence family originally derived by retroposition of tRNA(Glu) in the newt.

A previous report from this laboratory showed that in vitro transcription of total genomic DNA of the newt Cynopus pyrrhogaster resulted in a discrete sized 8 S RNA, which represented highly repetitive and transcribable sequences with a glutamic acid tRNA-like structure in the newt genome. We isolated four independent clones from a newt genomic library and determined the complete sequences of three 2000 to 2400 base-pair PstI fragments spanning the 8 S RNA gene. The glutamic acid tRNA-related segment in the 8 S RNA gene contains the CCA sequence expected as the 3' terminus of a tRNA molecule. Further, the 11 nucleotides located 13 nucleotides upstream from one of the two transcription initiation sites of the 8 S RNA were found to be repeated in the region upstream from the termination site, suggesting that the original unit, which is shorter than the 8 S RNA, was retrotransposed via cDNA intermediates from the PolIII transcript. In the upstream region of the 8 S RNA gene, a 360 nucleotide unit containing the glutamic acid tRNA-related segment was found to be duplicated (clones NE1 and NE10) or triplicated (clone NE3). Except for the difference in the number of the 360 nucleotide unit, the three sequences of the 2000 to 2400 base-pair PstI fragment were essentially the same with only a few mutations and minor deletions. Inverse polymerase chain reaction and sequence determination of the products, together with a Southern hybridization experiment, demonstrated that the family consists of a tandemly repeated unit of 3300, 3700 or 4100 base-pairs. Thus during evolution, this family in the newt was created by retroposition via cDNA intermediates, followed by duplication or triplication of the 360 nucleotide unit and multiplication of the 3300 to 4100 base-pair region at the DNA level.

Tetrahymena actin. Cloning and sequencing of the Tetrahymena actin gene and identification of its gene product.

Actin is ubiquitous in eukaryotes, nevertheless its existence has not yet been clearly proven in Tetrahymena. Here we report the cloning and sequencing of an actin gene from the genomic library of Tetrahymena pyriformis using a Dictyostelium actin gene as a probe. The Tetrahymena actin gene has no intron. The predicted actin is composed of 375 amino acids like other actins and its molecular weight is estimated as 41,906. Both T. pyriformis and T. thermophila possess a single species of actin genes which differ in their restriction patterns. Northern hybridization analysis revealed that the actin gene was actively transcribed in vivo. To detect the gene product, we synthesized an N-terminal peptide of the deduced sequence and prepared its antibody. Using an immunoblotting technique, we identified Tetrahymena actin on a two-dimensional gel electrophoretic plate. The actin spot migrated near an added spot of rabbit skeletal muscle actin, but clearly differed from the latter in its isoelectric point and apparent molecular weight. The primary structure of Tetrahymena actin shares about 75% homology equally with those of other representative actins. This value is extremely low as a homology rate between known actins. Tetrahymena actin diverges not only in relatively variable regions of other actins, but also in relatively constant regions. The hydrophilicity levels of two regions (residues 190 to 200 and residues 225 to 235) are also quite different between the Tetrahymena actin and skeletal muscle actin. Thus, we conclude that actin is present in Tetrahymena, but it is one of the most unique actins among the actins known hereto.

Effects of repeated maternal stress on FOS expression in the hypothalamic paraventricular nucleus of fetal rats.

The effects of repeated prenatal stress with different severity (restraint and immobilization) on Fos expression in the maternal and fetal hypothalamic paraventricular nucleus (PVN) were examined in rats. Acute stress treatment was performed for 30 min on gestational day 21, and repeated stress treatment for 30 min daily for 5 days from gestational days 17-21. In the parvocellular region of the maternal PVN, the stress-induced increases in the number of Fos-immunoreactive neurons were smaller in the repeated stress groups than the acute stress groups, indicating an adaptation of Fos expression to repeated stress. The attenuated Fos expression observed in the maternal PVN following repeated mild stress did not occur in the fetal PVN. In contrast, repeated immobilization stress caused a much smaller increase in Fos expression in the fetal PVN than did acute immobilization stress. The reduced Fos expression in the fetal PVN following repeated severe stress was thought to be due to cell death, since the fetal PVN in the chronic immobilization group revealed a reduction in the total number of cells and an increase in the number of apoptotic cells. In the female but not male fetuses, repeated restraint stress induced a significant increase in the number of apoptotic cells in the PVN. These findings suggest that the fetal PVN shows no adaptation of Fos expression to repeated maternal stress, but great vulnerability to cell death, including apoptosis. In addition, stress-induced apoptosis may more easily occur in the fetal PVN in females than males.

A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts.

The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor alpha (ERalpha) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.

Prospects for drug screening using the reverse two-hybrid system.

Rational drug-screening strategies have been limited by the number of available protein targets. The fields of genomics and functional genomics are now merging into 'chemical genomics' approaches, in which large numbers of potential target proteins can be used in standardized high-throughput drug-screening assays. Because protein-protein interactions are critical to most biological processes and can be tested in standardized assays, they may represent optimal targets in the chemical-genomics era. The reverse two-hybrid system appears to have several properties that would be critical for the success of this approach.

Transcription in vitro promoted by the Agrobacterium VirG protein.

Expression of the virulence genes (vir) on the hairy-root-inducing plasmid pRiA4 is induced by plant signals in Agrobacterium cells through a two-component regulatory system, the VirA-VirG system. We constructed an in vitro transcription system that consisted of the purified VirG protein and the Agrobacterium RNA polymerase holoenzyme. Both versions of VirG, the non-phosphorylated form and the VirA-phosphorylated form, were active but showed different patterns of the pH-dependency for transcriptional activation.

Characterization of the virA gene of the agropine-type plasmid pRiA4 of Agrobacterium rhizogenes.

We sequenced a 4.2-kb DNA region encompassing the vir A locus of the hairy-root-inducing plasmid pRiA4, and compared its sequence with the published vir A region sequences of four tumor-inducing plasmids. An open reading frame capable of coding for 829 amino acids was identified for vir A. Deletion mutants of vir A constructed by fusing to lacZ, but not the wild-type game itself, were efficiently expressed in Escherichia coli when they were put downstream front the lac promoter. These fused gene products became soluble or insoluble depending on the length of their lacZ moieties.

Exon-intron organization of the Arabidopsis thaliana protein kinase genes CDC2a and CDC2b.

We have previously shown by cDNA cloning that a higher plant, Arabidopsis thaliana, possesses at least two CDC2 genes (CDC2a and CDC2b) similar to the cell-cycle-controlling cdc2 gene of Schizosaccharomyces pombe. To understand the exon-intron organization of these genes, genomic clones were isolated and their nucleotide sequences determined. The coding and 5'-untranslated regions of CDC2a were interrupted by seven and one introns, respectively, whilst CDC2b contained three introns within the coding portion. These intron positions partly overlapped with each other and with those of the yeast cdc2 gene, nevertheless the lengths and sequences of the corresponding introns were diverse.

Antibody coating of liposomes with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide and the effect on target specificity.

Liposome surfaces were modified with normal rabbit IgG or rabbit anti-sheep erythrocyte IgG by adsorption or by coupling with a water soluble cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). Modification with normal IgG reduced the non-specific binding of the liposomes to erythrocytes. The immunological binding capacity of the immune IgG on liposomes was better for the chemically coupled preparation than for the adsorbed one. Complement dependent cytolytic activity of the immune IgG towards target erythrocytes was diminished as a consequence of liposome binding by either method, but to a lesser extent in the coupling method. Antibody coating by EDCI thus provides liposomes which can bind effectively to antigenic target cells.

Coating of liposomes with subunits of monoclonal IgM antibody and targeting of the liposomes.

SH-bearing subunits of IgM antibody were coupled through the Fc portion to sonicated liposomes consisting of N-(m-maleimidobenzoyl)dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine and cholesterol. Liposomes coated with subunits of monoclonal IgM antibody against a mouse mammary tumor-associated antigen selectively bound to the antigenic target cells. Assays of the liposomes for complement dependent cytotoxicity and spleen cell binding revealed inactivation of the liposome-coupled antibodies in both assays.

Agglutination microassay of hapten- or protein-modified liposomes using a multiple cell culture harvester.

A simple agglutination microassay is described for studying the presence of antigenic determinants on the surface of liposomal membranes. Antigen-sensitized radioactive liposomes, reacted with specific immune IgG in the wells of a microtest plate, were passed through a glass fiber filter in a multiple cell culture harvester. The amount of agglutinate trapped on the filter as determined by the radioactivity, was proportional to the amount of antigen in the liposomes and to the amount of specific immune IgG in the reaction mixture. The specificity of the agglutination was determined by inhibition with hapten and carrier antigens. Liposomes actively sensitized with hapten-conjugated lipid, and passively sensitized with protein, were used in the assay. A new method of preparing protein-bound liposomes is also described.

Evidence that cyclosporine causes both intracellular migration and inappropriate urinary excretion of magnesium in rats.

We determined the effects of cyclosporine on calcium, magnesium, and potassium metabolism in rats. Thirty Sprague-Dawley rats were randomized into three groups of ten animals each--control rats given olive oil, rats given cyclosporine at a dosage of 5 mg/kg daily, and rats given 15 mg/kg daily for four weeks. Urinary excretion of calcium, magnesium, and potassium was determined before and after 2 and 4 weeks of cyclosporine therapy. All rats were sacrificed after 4 weeks of therapy, and calcium, magnesium, and potassium concentrations in serum and tissues were determined. Serum magnesium levels were significantly lower in the cyclosporine-treated groups than in the control group, but there was no significant difference between the control and either of the cyclosporine-treated groups with regard to total urinary excretion of magnesium after four weeks of treatment. Magnesium content in the kidney, muscle, and liver was significantly higher in the 15 mg/kg group than in the control group. Calcium content in the kidney and liver was significantly higher as well. Potassium content in any type of tissue was similar in the three groups. We conclude that the intracellular migration of magnesium plays an important role--as does impaired renal conservation of magnesium--in the pathogenesis of cyclosporine-induced hypomagnesemia and that there is a discrepancy between magnesium and potassium metabolism in cyclosporine-treated rats.

Materno-fetal coordination of stress-induced Fos expression in the hypothalamic paraventricular nucleus during pregnancy.

This study investigates whether maternal stress during pregnancy induces maternal and fetal hypothalamic paraventricular nucleus (PVN) neuronal activation and the effects of maternal stress on fetal hypothalamic and PVN brain-derived neurotrophic factor (BDNF) expression. Pregnant rats were exposed to three types of maternal stress with varying severity (restraint, forced walking and immobilization) for 30 min on gestational day 21. Severity of stress was assessed by measurement of maternal plasma corticosterone 30 min following the stimulus. Maternal plasma corticosterone increased in each stress response group (immobilization>forced walking>restraint). Further, the expression of Fos protein, a marker of neuronal activation, increased in the fetal and maternal PVN in direct relation to the severity of stress treatments. Forced walking and immobilized stress, but not restraint stress, significantly increased BDNF expression in the fetal hypothalamus.These findings suggest that the fetal hypothalamic-pituitary-adrenal (HPA) response following maternal stress mirrors maternal HPA activation. In addition, BDNF may play a role in protecting fetal brain neurons from damage caused by severe stress.

The morphological changes of exocrine pancreas in chronic pancreatitis.

The following changes were found by either light or electron microscopic observation of the pancreas in spontaneously developed chronic pancreatitis models (WBN/Kob rats, spontaneously hypertensive rats, and rats with common bile-pancreatic duct stones) and in experimental models of chronic pancreatitis (alcoholic pancreatitis, ischemic pancreatitis, and obstructive pancreatitis): 1) the units of lobules, which were constituted by acinar cell deletion, ductular proliferation, and fibrosis; and 2) tortuous or helical ductal channels of pancreatic ducts with periductal fibrosis, which had many crater-like depressions and very long cilia in their inner surface. These are considered to be the results of obstructive pancreatitis, which are caused by the reactions of defensive factors against the increase of pancreatic duct pressure, including the apoptosis of acinar cells, the hyperplasia and hypertrophy of duct cells, a tighter junctional complex of duct cells, and periductal fibrosis.

Morphological changes in the rat exocrine pancreas after pancreatic duct ligation.

In the present study, morphological changes of the exocrine pancreas in rats after pancreatic duct ligation were examined with light microscopy (hematoxylin-eosin, TUNEL, and PCNA staining) and scanning electron microscopy in order to elucidate the effects of increased pancreatic duct pressure. On the fifth day after pancreatic duct ligation, ductular proliferation, periductal fibrosis, and disappearance of acini were observed. TUNEL and PCNA staining demonstrated many apoptotic acinar cells and proliferating ductal cells immediately after ligation, which reached a maximal number on the 2nd or 3rd day. Tortuous or helical interlobular pancreatic ducts with inner surfaces containing many crater-like depressions and long cilia were found after ligation. These changes were almost identical to those observed in the pancreatic tissue of model chronic pancreatitis rats, WBN/Kob rats, and stroke-prone spontaneously hypertensive (SHRSP) rats. In summary, the morphological changes observed after pancreatic duct ligation were similar to those of chronic pancreatitis, therefore, the characteristic changes of pancreatic ducts observed in chronic pancreatitis may be caused by increased pancreatic duct pressure.