Role of Notch-1 intracellular domain in activation of rheumatoid synoviocytes.

OBJECTIVE: Notch family proteins are transmembrane receptors that control cell fate and proliferation. Rheumatoid arthritis (RA) is characterized by activation and abnormal proliferation/differentiation of synoviocytes. We examined the expression of Notch-1 and its role in the activation of RA synoviocytes. METHODS: The expression of Notch-1 protein was detected by a specific antibody raised against the Notch-1 intracellular domain. Notch-1 messenger RNA (mRNA) expression in synoviocytes was analyzed by Northern blotting. Notch-1 protein expression was confirmed by Western blotting with anti-Notch-1 antibody. To analyze the role of Notch-1 in synoviocyte proliferation, we examined the effects of antisense Notch-1 oligonucleotides (ODNs) and MW167, a gamma-secretase inhibitor. RESULTS: Notch-1 protein and mRNA were detected in synovium from all study subjects. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 antibody, whereas there was predominantly cytoplasmic staining of normal and osteoarthritis (OA) synoviocytes. Western blotting showed a distinct approximately 63-kd protein detected by anti-Notch-1 antibody in nuclear extracts from RA synoviocytes, indicating that nuclear staining of RA synovium and synoviocytes is likely to be the result of nuclear localization of Notch-1 intracellular domain (NICD). Furthermore, tumor necrosis factor alpha (TNFalpha) increased NICD nuclear translocation in a dose-dependent manner. Antisense Notch-1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFalpha-induced proliferation in both OA and RA synoviocytes. In addition, gamma-secretase inhibitor, which blocks the production of NICD, also inhibited TNFalpha-induced proliferation of RA synoviocytes. CONCLUSION: Our results demonstrate the expression of Notch-1 in synoviocytes and the presence of Notch-1 fragment in the nuclei of RA synoviocytes and suggest the involvement of Notch-1 signaling in the TNFalpha-induced proliferation of RA synoviocytes.

Dual roles of RNA helicase A in CREB-dependent transcription.

RNA helicase A (RHA) is a member of an ATPase/DNA and RNA helicase family and is a homologue of Drosophila maleless protein (MLE), which regulates X-linked gene expression. RHA is also a component of holo-RNA polymerase II (Pol II) complexes and recruits Pol II to the CREB binding protein (CBP). The ATPase and/or helicase activity of RHA is required for CREB-dependent transcription. To further understand the role of RHA on gene expression, we have identified a 50-amino-acid transactivation domain that interacts with Pol II and termed it the minimal transactivation domain (MTAD). The protein sequence of this region contains six hydrophobic residues and is unique to RHA homologues and well conserved. A mutant with this region deleted from full-length RHA decreased transcriptional activity in CREB-dependent transcription. In addition, mutational analyses revealed that several tryptophan residues in MTAD are important for the interaction with Pol II and transactivation. These mutants had ATP binding and ATPase activities comparable to those of wild-type RHA. A mutant lacking ATP binding activity was still able to interact with Pol II. In CREB-dependent transcription, the transcriptional activity of each of these mutants was less than that of wild-type RHA. The activity of the double mutant lacking both functions was significantly lower than that of each mutant alone, and the double mutant had a dominant negative effect. These results suggest that RHA could independently regulate CREB-dependent transcription either through recruitment of Pol II or by ATP-dependent mechanisms.

A Role of RNA Helicase A in cis-Acting Transactivation Response Element-mediated Transcriptional Regulation of Human Immunodeficiency Virus Type 1.

RNA helicase A (RHA) has two double-stranded (ds) RNA-binding domains (dsRBD1 and dsRBD2). These domains are conserved with the cis-acting transactivation response element (TAR)-binding protein (TRBP) and dsRNA-activated protein kinase (PKR). TRBP and PKR are involved in the regulation of HIV-1 gene expression through their binding to TAR RNA. This study shows that RHA also plays an important role in TAR-mediated HIV-1 gene expression. Wild-type RHA preferably bound to TAR RNA in vitro and in vivo. Overexpression of wild type RHA strongly enhanced viral mRNA synthesis and virion production as well as HIV-1 long terminal repeat-directed reporter (luciferase) gene expression. Substitution of lysine for glutamate at residue 236 in dsRBD2 (RHA(K236E)) reduced its affinity for TAR RNA and impaired HIV-1 transcriptional activity. These results indicate that TAR RNA is a preferred target of RHA dsRBDs and that RHA enhances HIV-1 transcription in vivo in part through the TAR-binding of RHA.

The autoimmune regulator protein has transcriptional transactivating properties and interacts with the common coactivator CREB-binding protein.

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, caused by mutations in the autoimmune regulator (AIRE) gene, is an autosomal recessive autoimmune disease characterized by the breakdown of tolerance to organ-specific antigens. The 545 amino acid protein encoded by AIRE contains several structural motifs suggestive of a transcriptional regulator and bears similarity to cellular proteins involved in transcriptional control. We show here that AIRE fused to a heterologous DNA binding domain activates transcription from a reporter promoter, and the activation seen requires the full-length protein or more than one activation domain. At the structural level AIRE forms homodimers through the NH(2)-terminal domain, and molecular modeling for this domain suggests a four-helix bundle structure. In agreement, we show that the common transcriptional coactivator CREB-binding protein (CBP) interacts with AIRE in vitro and in yeast nuclei through the CH1 and CH3 conserved domains. We suggest that the transcriptional transactivation properties of AIRE together with its interaction with CBP might be important in its function as disease-causing mutations almost totally abolish the activation effect.

Hypernuclear acetylation in atherosclerotic lesions and activated vascular smooth muscle cells.

Recent studies have implicated acetylation of several nuclear proteins such as histones and p53 on their epsilon-portion of lysine residues in eukaryotic transcription. Here we raised a specific polyclonal antibody against epsilon-acetylated lysine. Using the antibody, we detected hypernuclear acetylation (HNA) in atherosclerotic vascular smooth muscle cells (VSMCs). Thrombin, a humoral factor known to cause activation and proliferation of VSMCs, strongly potentiated HNA in cultured VSMCs. MAP kinase pathway and a signal coactivator CREB binding protein (CBP) were involved in thrombin-induced HNA of VSMCs. Our results suggest that coactivators cooperating with signal-dependent transcription activators play an important role in atherosclerogenesis via HNA in VSMCs.

Functional association between CBP and HNF4 in trans-activation.

CBP functions as a key transcriptional coactivator for a variety of transcription factors. We show here that the hepatocyte nuclear factor 4 (HNF4), a transcription factor in the nuclear receptor superfamily with no defined ligand, is cloned by yeast two-hybrid system using CBP as a bait. GST-pull down assay with nuclear extracts or in vitro translation products revealed that CBP and HNF4 interact with each other at the middle portion (aa 119-375) of HNF4 and two distinct regions (aa 271-451 and 1626-2259) of CBP, respectively, in the ligand-independent manner. Co-transfection experiments indicated that CBP is capable of activating HNF4 site-mediated transcription. These results suggested a functional association between CBP and HNF4 in trans-activation.

[Clinical studies on the complaints of survivors living more than 3 months after total gastrectomy].

From 1973 to 1983, 802 patients with gastric cancer were operated on. Out of them, 292 (36.4%) received total gastrectomy. Reconstruction was performed mainly by the Billroth II procedure associated with the closure of the afferent loop according to Plenk's method. 90 patients living more than 3 months complained of the following: heartburn, 18 (20%); reflux, 12 (13.3%); retrosternal pain, 3 (3.3%); stenotic sensation, 23 (25.6%); diarrhea, 10 (11.1%); abdominal pain, 14 (15.6%); and dumping syndrome, 6 (6.7%). It seems to indicate that the quality of life after total gastrectomy is satisfactory.

[Histological classification and prognosis of mammary cancer-histological classification devised by the Japan Mammary Cancer Society and WHO classification].

Between 1966 and 1977, 427 patients with mammary cancer underwent surgery at the Shikoku Cancer Center Hospital. Using these subjects, the histological classification devised by the Japan Mammary Cancer Society was compared with the WHO classification. Since the WHO classification places 80.4% of all cases into the category of invasive duct carcinoma, the significance of the histological classification as a factor in predicting prognosis is reduced. Thus, some other subclassification is needed for practical application. We classified invasive duct carcinoma according to cellular atypism (CAT), structural atypism (SAT) and infiltration mode (INF), and examined their relationship to the 5-year survival rate; there was a positive correlation.

Effect of 2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate (MN-1695) on gastric ulcers and gastric secretion in experimental animals.

The effect of 2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate (MN-1695) on various experimental gastric ulcers and gastric secretion in experimental animals was compared with that of cimetidine and cetraxate. MN-1695 significantly inhibited the formation of Shay, stress-induced, indomethacin-induced, and histamine-induced ulcers and significantly accelerated the healing of acetic acid-induced gastric ulcers. MN-1695 was much more effective in suppressing stress- and acetic acid-induced ulcers than indomethacin-induced, histamine-induced or Shay ulcers. Cimetidine was effective in preventing stress- and acetic acid-induced ulcers but had no significant effect on Shay, indomethacin-induced and histamine-induced ulcers. Cetraxate was effective in preventing only stress-induced ulcers and had almost no effect on other experimental ulcers. MN-1695 inhibited secretion of gastric juice, acid and pepsin in Shay rats, but had no influence on basal and secretagogue-stimulated acid secretion in the perfused stomach of urethanized rats. These findings suggest that MN-1695 is a new type of anti-ulcer agent.

Effect of 2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate (MN-1695) on gastric mucosal blood flow in dogs.

The effect of 2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate (MN-1695) on gastric mucosal blood flow ( GMBF ) and on the changes of GMBF induced by catecholamines, tetragastrin, histamine and indomethacin were investigated in anesthetized dogs. MN-1695 at doses up to 1 mumol/kg i.v. showed only a slight but prolonged increase of GMBF . MN-1695 tended to inhibit the decreases of GMBF induced by norepinephrine and the increase of GMBF induced by tetragastrin. The later phase of decreased GMBF induced by histamine was suppressed by MN-1695. However, MN-1695 had almost no effect on the increase of GMBF induced by epinephrine. MN-1695 produced a marked increase of antral mucosal blood flow which was reduced by pretreatment with indomethacin. Systemic blood pressure, respiration and the pressor responses induced by catecholamines and gastric secretagogues were not affected by MN-1695. These results suggest that MN-1695 may possess selective effects on the gastric mucosal microcirculation and that suppressive effects of MN-1695 on the GMBF decrease induced by ulcerogenic stimuli may be responsible for its anti-ulcer activities in various types of experimental gastric ulcers.

Effect of 2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate (MN-1695) on gastric mucosal damage induced by various necrotizing agents in rats.

The effect of 2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate (MN-1695) on the gastric mucosal damage induced in rats by various necrotizing agents was compared with those of cimetidine, cetraxate and prostaglandin (PG) E2. MN-1695 at doses of 0.37 mg (1 mumol) to 3.72 mg (10 mumol)/kg, significantly decreased in a dose-dependent manner the damage indices after the application of ethanol, HCl, NaOH, 25% NaCl or boiling water. Cimetidine in doses of 12.6 mg (50 mumol) to 126 mg (500 mumol)/kg was effective only against HCl-induced damage. Cetraxate in doses of 34.2 mg (100 mumol) to 342 mg (1 mmol)/kg was effective against damage due to ethanol, HCl, 25% NaCl or boiling water. However, cetraxate increased the damage index of NaOH-induced injury. PGE2 in a dose of 25 micrograms (70 nmol)/kg was effective against ethanol and 25% NaCl. The histological changes of gastric mucosal cells produced by ethanol were significantly decreased by MN-1695 and PGE2. These results suggest that MN-1695 has a cytoprotective action like that of PGE2 and that this cytoprotection inhibits the development of various kinds of experimental gastric ulcers.

Separate involvement of the spinal noradrenergic and serotonergic systems in morphine analgesia: the differences in mechanical and thermal algesic tests.

Experiments using 3 analgesic tests, the tail-pinch, hot-plate and tail-flick methods, were done to evaluate the roles of the spinal noradrenergic and serotonergic systems in the production of morphine analgesia in rats. To deplete noradrenaline or serotonin in the spinal cord, 6-hydroxydopamine or 5,6-dihydroxytryptamine was given intrathecally. 6-Hydroxydopamine suppressed the antinociceptive effects of morphine injected systemically or intracerebrally (into the nuclei reticularis gigantocellularis and paragigantocellularis or into the periaqueductal gray matter) in the tail-pinch test, but not significantly in the hot-plate and tail-flick tests. Conversely, 5,6-dihydroxytryptamine suppressed the antinociceptive effects of systemically given morphine in the hot-plate test, but not significantly in the tail-pinch and the tail-flick tests. The results not only provide further evidence for the involvement of the descending inhibitory systems in morphine antinociception, but also show that the extent of participation of the spinal noradrenergic and serotonergic systems in the effects of morphine has to be carefully assessed as different analgesic tests (tail-pinch, tail-flick and hot-plate) yield different results.