Resveratrol directly targets DDX5 resulting in suppression of the mTORC1 pathway in prostate cancer.

Resveratrol has various attractive bioactivities, such as prevention of cancer, neurodegenerative disorders, and obesity-related diseases. Therefore, identifying its direct binding proteins is expected to discover druggable targets. Sirtuin 1 and phosphodiesterases have so far been found as the direct molecular targets of resveratrol. We herein identified 11 novel resveratrol-binding proteins, including the DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5, also known as p68), using resveratrol-immobilized beads. Treatment with resveratrol induced degradation of DDX5 in prostate cancer cells. Depletion of DDX5 caused apoptosis by inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling. Moreover, knockdown of DDX5 attenuated the inhibitory activities of resveratrol against mTORC1 signaling and cancer cell growth. These data show that resveratrol directly targets DDX5 and induces cancer cell death by inhibiting the mTORC1 pathway.

Rice stripe virus 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells.

The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9 K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9 K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9 K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9 K protein antiserum. Moreover, electron-dense masses labeled with 23.9 K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9 K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.

Induction of anesthesia with ketamine reduces the magnitude of redistribution hypothermia.

UNLABELLED: Hypothermia after induction of general anesthesia results largely from core-to-peripheral redistribution of body heat. Both central inhibition of tonic thermoregulatory vasoconstriction in arteriovenous shunts and anesthetic-induced arteriolar and venous dilation contribute to this redistribution. Ketamine, unique among anesthetics, increases peripheral arteriolar resistance; in contrast, propofol causes profound venodilation that other anesthetics do not. We therefore tested the hypothesis that induction of anesthesia with ketamine causes less core hypothermia than induction with propofol. Twenty patients undergoing elective surgery were randomly assigned to anesthetic induction with either 1.5 mg/kg ketamine (n = 10) or 2.5 mg/kg propofol (n = 10). Anesthesia in both groups was subsequently maintained with sevoflurane and 60% nitrous oxide in oxygen. Forearm minus finger, skin-temperature gradients <0 degrees C were considered indicative of significant arteriovenous shunt vasodilation. Ketamine did not cause vasodilation just after induction, whereas propofol rapidly induced vasodilation. Core temperatures in the patients given ketamine remained significantly greater than those in the patients induced with propofol. These data suggest that maintaining vasoconstriction during induction of anesthesia reduces the magnitude of redistribution hypothermia. IMPLICATIONS: Core hypothermia during the first hour of anesthesia was less after induction of anesthesia with ketamine than propofol. Maintaining arteriovenous shunt vasoconstriction during induction of anesthesia reduces the magnitude of redistribution hypothermia.

Cytokine production and helper T cell subsets in Vogt-Koyanagi-Harada's disease.

PURPOSE: To investigate helper T (Th) cell subsets and cytokine production in patients with Vogt-Koyanagi-Harada's (VKH) disease. METHODS: Nine patients in the acute stage of VKH disease and 9 healthy controls were studied. Interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and IL-4 mRNA from peripheral blood mononuclear cells (PBMC) was detected by reverse transcriptase-polymerase chain reaction. Cytokine levels in plasma, cerebrospinal fluid (CSF) and PBMC culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The proportions of each cytokine-producing CD4+ or CD8+ cells in PBMC cultured with or without anti-CD3 antibody stimulation were analyzed by flow cytometry. RESULTS: We observed a significant increase of cytokine mRNA positive cases of VKH patients in comparison with controls only in IFN-gamma mRNA detection from PBMC. ELISA detected IFN-gamma in CSF from only 1 patient and no cytokine in plasma. The levels of IFN-gamma and IL-2 were significantly higher in the stimulated cell culture supernatant of the patients than those of controls. The proportions of IFN-gamma- or IL-2-producing CD4+ cells were significantly higher in the patients than in controls in both stimulated and unstimulated conditions. However, no significant difference was found in IL-4-producing CD4+ cells. In CD8+ cells, significant difference was found only in IL-2-producing cells in the stimulated condition. CONCLUSIONS: Th cells from VKH patients produced predominantly Th1 cytokines, especially after stimulation in vitro. It is therefore suggested that activated Th cells produce predominantly Th1 cytokines, which then produce pathologic changes.

[Hydrothorax during diagnostic laparoscopy].

An 83-yr-old, 44-kg woman with a 2-month history of abdominal distension received diagnostic laparoscopy. Except for chronic treated hypertension, she was healthy. The preoperative chest X-ray demonstrated small pleural effusion occupying the lower left hemithorax, but she did not present with dyspnea or chest pain. After premedication with intravenous ranitidine 50 mg, anesthesia was induced with thiopental 150 mg, vecuronium 7 mg and maintained by 1-2% sevoflurane in 50% N2O/O2. SpO2 decreased after insufflation of CO2, but breath sound was audible on both lungs. At completion of operation, chest X-ray revealed the left hemilateral hydrothorax and 650 ml of pleural fluid was suctioned. Blood gas improved and the tracheal tube was removed. The diagnosis of tuberculous peritonitis was established by the demonstration of granulomas of the peritoneum. We speculated on four reasons for the increased pleural effusion on the left thorax: 1) Increase of systemic and capillary pressure caused by CO2 insufflation. 2) Increase of capillary permeability by tuberculous pleuritis. 3) Decrease of colloid osmotic pressure by hypoalbuminemia. 4) Decreased pleural fluid removal because of venous compression caused by increased intrathoracic pressure. Peritoneal insufflation of CO2 to create the pneumoperitoneum may induce hydrothorax in patients with tuberculous pleuritis.

Effects of Leukotriene B(4) Receptor Antagonist on Experimental Autoimmune Uveoretinitis in Rats.

Purpose: We evaluated the potential of leukotriene B(4) receptor antagonist (LTB(4) RA) as an anti-inflammatory agent on experimental autoimmune uveoretinitis (EAU).Materials and Methods: LTB(4) RA was administered to Lewis rats twice a day on days 0-10, 0-5, and 6-10 after immunization. Rats treated on days 0-10 after immunization were subdivided into three groups according to the dosage of LTB(4) RA. The eyes were examined histopathologically, and the expression of CD 45 RC in CD(+)T cells was analyzed.Results: The inflammatory changes in the eyes of EAU were decreased in all groups treated with LTB(4) RA in a dose-dependent manner. The treatment with LTB(4) RA on days 0-10 after immunization achieved much higher uveitis suppression. The infiltration into eye tissues by neutrophils and lymphocytes was decreased by treatment with LTB(4) RA. In treated groups, the CD 45 RC(high) subset decreased in the induction phase of EAU as compared with the untreated group.Conclusion: The suppressive mechanisms of LT-B(4) RA on EAU may be dependent on suppression of the activation of neutrophils and CD 4(+)T cells.

Complete nucleotide sequence of Northern cereal mosaic virus and its genome organization.

We determined the complete nucleotide sequence, 13, 222 nucleotides (nts) of the Northern cereal mosaic virus (NCMV). The genome had 273 nt 5' trailer sequence and 90nts 3' leader sequence. It formed a terminal complementarity in 25 nts of both terminal sequences. A characteristic intergenic sequence (consensus) separating genes, 3'-AUUCUUUUUGACUCUAGU-5' was presented. The genome had nine open reading frames (ORFs) on the viral complementary sequence. Five putative proteins of NCMV were postulated by its molecular weight or comparison of the similarities to other rhabdovirus proteins: nucleocapsid (N), non-structural protein or phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L). A series of four small ORFs (genes 3 to 6) were also presented between P and M gene sequences. The proposed NCMV genome organization was 3'leader-N-P-3-4-5-6-M-G-L-5'trailer. N and L proteins of NCMV had low but distinct similarities to those of lettuce necrotic yellows virus and Sonchus yellow net virus, respectively.

[Effects of leukotriene B4 receptor antagonist on experimental autoimmune uveoretinitis in rats].

PURPOSE: We evaluated the potential of leukotriene B4 receptor antagonist (LTB4 RA) as an anti-inflammatory agent on experimental autoimmune uveoretinitis (EAU). MATERIALS AND METHODS: LTB4 RA was administered to Lewis rats twice a day on days 0-10, 0-5, and 6-10 after immunization. Rats treated on days 0-10 after immunization were subdivided into three groups according to the dosage of LTB4 RA. The eyes were examined histopathologically, and the expression of CD 45 RC in CD 4+T cells was analyzed. RESULTS: The inflammatory changes in the eyes of EAU were decreased in all groups treated with LTB4 RA in a dose-dependent manner. The treatment with LTB4 RA on days 0-10 after immunization achieved much higher uveitis suppression. The infiltration into eye tissues by neutrophils and lymphocytes was decreased by treatment with LTB4 RA. In treated groups, the CD 45 RChigh subset decreased in the induction phase of EAU as compared with the untreated group. CONCLUSION: The suppressive mechanisms of LTB4 RA on EAU may be dependent on suppression of the activation of neutrophils and CD 4+T cells.

Determining the nucleotide sequence and capsid-coding region of himetobi P virus: a member of a novel group of RNA viruses that infect insects.

We determined the complete genome sequence of Himetobi P virus (HiPV), an insect picorna-like virus, which was isolated from the small brown planthopper, Laodelphax striatellus. The genome of HiPV consists of 9,275 nucleotides excluding the poly (A) tail, and contains two large open reading frames (ORFs), which were separated by a 176-nucleotide noncoding region. The deduced amino acid sequence of the first ORF contains core motifs of picornaviral helicase, protease, and RNA-dependent RNA polymerase. The capsid protein-coding region was mapped onto the second ORF by determining the N-terminal amino acid sequences of the capsid proteins. Subgenomic RNA for the capsid protein gene was not detected in the infected tissue. The capsid protein precursor gene of HiPV lacks an AUG initiation codon at the expected position and the upstream sequence of the gene is predicted to form several stem-loop structures, suggesting that the precursor is produced by internal ribosome entry site (IRES) mediated-translation, as occurs in Plutia stali intestine virus (PSIV). These characteristics of the HiPV genome are similar to those of a new group of RNA viruses consisting of Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), and PSIV.

The complete nucleotide sequence of the rice grassy stunt virus genome and genomic comparisons with viruses of the genus Tenuivirus.

Rice grassy stunt virus (RGSV, IRRI isolate) has six genomic RNA segments. The nucleotide (nt) sequences of RNAs 1-4 were determined. The cumulative length of the RGSV genome, including RNAs 5 and 6, was 25142 nt. All six RNA segments had an ambisense coding strategy and almost identical terminal sequences over 17 nt. The virus complementary (vc) sequence of the largest segment, RNA1, had an open reading frame encoding a protein of Mr 339133 (the 339.1K protein), while the virus sense (v) sequence encoded a protein of Mr 18910 in the 5'-proximal region. The predicted 339.1K protein contained the highly conserved motifs of the RNA-dependent RNA polymerase and a short but distinct Arg/Gly-rich stretch at the C terminus. The putative RNA polymerase showed strong similarity with that of rice stripe tenuivirus (RSV); they shared 37.9% amino acid identity over 2140 residues. The predicted proteins of Mr 23280 on vRNA2 and 93 879 on vcRNA2 were only slightly similar in sequence to the proteins encoded by vRNA2 and vcRNA2 of other tenuiviruses. The predicted proteins encoded by RNA3 and RNA4 did not show significant similarity to any database proteins. Only the putative RNA polymerase encoded on RNA1 was well-conserved between RGSV and RSV. The low sequence similarities in proteins encoded by RNAs 2, 5 and 6, together with the unique RNA segments 3 and 4, indicate that RGSV may be distinct from other tenuiviruses.

The proteins encoded by rice grassy stunt virus RNA5 and RNA6 are only distantly related to the corresponding proteins of other members of the genus Tenuivirus.

The genome of rice grassy stunt virus (RGSV) consists of six RNA segments. The nucleotide (nt) sequences of the two smallest segments, RNAs 5 and 6, were determined and found to comprise 2704 and 2584 nt, respectively. The 5'- and 3'-terminal sequences of both RNAs were identical over a length of 21 nt and could potentially form a panhandle-like structure due to intramolecular complementarity. Each RNA segment contained a virus (v) sense open reading frame (ORF) in the 5'-proximate region, and a virus complementary (vc) ORF in the 3'-proximate region, indicating an ambisense coding strategy. The protein encoded by the ORF on the vc strand of RNA5 was identified as the viral nucleocapsid protein (M(r) 35927). The ORF on the v strand of RNA6 encoded a protein of M(r) 20581 which represented the major nonstructural protein, previously shown to be produced in RGSV-infected rice tissues. The predicted proteins encoded by RGSV RNAs 5 and 6 were only distantly similar in sequence to the four proteins encoded by RNAs 3 and 4 of other viruses belonging to the genus Tenuivirus. These low sequence similarities, together with the apparently distinct number of genome segments, set RGSV apart from the other tenuiviruses and indicate that it should be placed in a taxonomically separate genus.

[Cell biology in endogenous uveitis].

We studied the immune system in 18 cases of Behçet's disease with ocular involvement. The proportion of CD69+ cells in CD4+ cells was significantly higher in patients with active uveoretinitis than in normal controls (p < 0.01). After OKT-3 stimulation of cultured cells, the proportion was significantly increased in controls (p < 0.01) but not in patients. Fas-ligand positive cells in CD8+ cells in patients did not increase after OKT-3 stimulation. Thus, the T cells in patients were in an activated state in vivo but were not further activated by OKT-3 stimulation. Cultured lymphocytes of patients after OPT-3 activation and anti-Fas antibody stimulation showed that the T cells in the active stage of the disease were resistant to apoptosis and unlikely to undergo regression by activation-induced cell death (AICD). The mean level of soluble Fas antigen was significantly elevated in sera of patients with active uveoretinitis as compared with normal controls (p < 0.05). TdT-mediated dUTP-biotin nick end labelling (TUNEL)-positive infiltrating cells were present in the inflamed retina and the posterior chamber in experimental autoimmune uveoretinitis (EAU) in rats, suggesting the involvement of apoptosis of infiltrated cells in the regression of inflammation. Serum concentration of tumor necrosis factor-alpha (TNF-alpha) was significantly elevated after 9 days of immunization in rats (p < 0.02). The inflammation score was suppressed by intravenous administration of anti-TNF-alpha antibody from days 7 to 14. It is concluded that intraocular inflammation in Behçet's disease is associated with activation of T cells and abnormality in apoptosis and AICD mechanisms. Systemic anti-TNF-alpha antibody promises to be of value in the treatment of the disease.

[A case of bilateral MALT (mucosa-associated lymphoid tissue) type conjunctival malignant lymphoma].

A case of bilateral MALT (mucosa-associated lymphoid tissue) type conjunctival malignant lymphoma is reported. The patient, a 32 year old female, was referred to our hospital with the diagnosis of allergic conjunctivitis. We performed histopathological, immunohistochemical, and molecular genetical examination. The histopathological findings showed proliferation of CCL (centrocytelike) cells, lymphoepithelial lesion, and follicular colonization. Monoclonality of the tumor cells was revealed by molecular genetical analysis. On the basis of these detailed examinations, the patient was diagnosed as having MALT type malignant lymphoma. MALT type malignant lymphoma was first described by Isaacson in 1983. Both histological and clinical features are characteristic and quite different from other lymphomas. Some patients are affected bilaterally. Immunohistochemical and molecular genetical examinations in MALT type malignant lymphomas were found to be useful.

Non-viral sequences at the 5' termini of mRNAs derived from virus-sense and virus-complementary sequences of the ambisense RNA segments of rice stripe tenuivirus.

The three small segments of the four RNAs of the rice stripe tenuivirus (RSV) genome have an ambisense coding strategy. The mRNA transcripts corresponding to open reading frames for the non-structural protein (NS4) and nucleocapsid protein (N), which are encoded on virus-sense (v) RNA 4 and virus-complementary sense (vc) RNA 3, respectively, were recovered from polysomes of RSV-infected wheat leaves, and their 5' termini were analysed. The mRNAs derived from both v and vc sequences contained from 10 to 23 non-viral bases at their 5' termini. Results of nucleotide sequence similarity analyses indicated that these non-viral heterogeneous sequences may be derived from host cellular mRNAs. Taken together, these results suggest that the viral mRNA transcription of either v or vc sequences of ambisense segments of RSV is primed by non-viral oligonucleotides in vivo.

Nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus, the prototype of the tenuiviruses.

The complete nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus (RSV), was determined using two sets of overlapping cDNA clones. RNA segment 1 comprises 8970 nucleotides and on the viral complementary sequence has a single long open reading frame coding for a protein of 2919 amino acids with an estimated M(r) of 336860. Amino acid sequence comparisons of the putative protein indicated strong homology (30% amino acid identity over about 1500 residues) with the L protein of the genus Phlebovirus of the Bunyaviridae, but no detectable similarity with other members of the Bunyaviridae. However, weak similarity was detected with the L protein of Tacaribe arenavirus. The highly homologous sequence domain includes the conserved motifs of the putative RNA-dependent RNA polymerase. The data presented here, along with previous work clearly show significant similarities in genome organization, structure and expression between RSV and members of the genus Phlebovirus of the Bunyaviridae. Taken together, we propose that tenuiviruses should be included in the Bunyaviridae under the genus Tenuivirus.

Pathogenesis of idiopathic scoliosis: SEPs in chicken with experimentally induced scoliosis and in patients with idiopathic scoliosis.

We studied somatosensory evoked potentials (SEPs) in 20 chickens with experimentally induced scoliosis after pinealectomy and in 100 patients with idiopathic scoliosis. We also studied 20 chickens without scoliosis and 20 healthy youngsters. In the chickens, SEPs after leg stimulation was significantly delayed in the scoliosis group compared to the controls. In patients, the latency of cortical potential (N37) after stimulation of tibial nerve was longer in the scoliosis group than in the controls. Our findings in both experimental and clinical studies strongly support the hypothesis that idiopathic scoliosis results from dysfunction in the central nervous system. The type of SEPs abnormalities described in idiopathic scoliosis suggest a pathology from the midbrain to the cortex.

Ambisense coding strategy of the rice stripe virus genome: in vitro translation studies.

Rice stripe virus (RSV), the type species of the tenuivirus group, contains four RNA segments as its genome. Sequence analyses of the three smaller segments indicated that all of them have ambisense coding strategies. To examine the ambisense nature of the genomic RNAs, we synthesized in vitro the RNAs carrying the putative open reading frames (ORFs) by transcribing cDNA clones for RNA segments 2, 3 and 4 in both directions using T7 RNA polymerase and translated each RNA in vitro using two systems: reticulocyte lysates and wheat-germ extracts. We detected the proteins encoded by the ORFs present in the 5'-proximal regions of both viral RNAs (vRNAs) and their complementary RNAs (cRNAs). Translation in vitro of total vRNA generated proteins encoded by the ORFs present in the 5' regions of vRNAs. The overall results are consistent with the prediction that RSV RNAs, at least up to segment 2, are ambisense in their coding strategy.

Nucleotide sequence and possible ambisense coding strategy of rice stripe virus RNA segment 2.

The complete nucleotide sequence (3514 nucleotides) of RNA segment 2 of rice stripe virus (RSV), the prototype member of tenuivirus group, was determined. In the virus-sense RNA an open reading frame (ORF) is present which encodes a 199 amino acid protein of M(r) 22,762. Another long ORF encoding an 834 amino acid protein with M(r) 94,047 (94K) exists in the virus-complementary RNA. Between these two ORFs, there is a long non-coding intergenic region of 299 nucleotides. The sequence suggests that RNA 2 has an ambisense coding strategy as found for RSV RNAs 3 and 4. The putative 94K protein carries stretches with an amino acid sequence showing weak similarity to parts of the membrane glycoproteins of Punta Toro and Uukuniemi phleboviruses of the family Bunyaviridae, suggesting a possible distinct evolutionary relationship between the animal phleboviruses and the plant tenuiviruses.

Clinical values of intraoperative ultrasonography for spinal tumors.

Intraoperative ultrasonography was conducted in 52 cases of spinal tumor, at 7.5 MHz, mainly by means of linear scanning, to evaluate its clinical usefulness. The procedure was effectively applied in such clinical purposes as: 1) locating the tumor, 2) deciding the resectability of intramedullary tumors, 3) deciding the site for intraspinal biopsy or shunt tube insertion, 4) establishing the topical relationship between the spinal cord and the tumor, and 5) differentiating neurilemoma from meningoma. Of 10 patients with intramedullary tumors, 5 (50%) were removed, because extirpation was possible when the spinal cord and the tumor were well demarcated on the ultrasonogram. Intratumorous cysts were found to exist in 73% of neurilemoma and 14% of meningioma cases, enabling the differential diagnosis between the two tumors. Intraoperative ultrasonography is an uninvasive method to reveal intradural and extradural conditions and thus constitutes a valuable diagnostic means to ensure safe and precise spinal surgery.

Genetically engineered rice resistant to rice stripe virus, an insect-transmitted virus.

The coat protein (CP) gene of rice stripe virus was introduced into two japonica varieties of rice by electroporation of protoplasts. The resultant transgenic plants expressed the CP at high levels (up to 0.5% of total soluble protein) and exhibited a significant level of resistance to virus infection. Plants derived from selfed progeny of the primary transformants also expressed the CP and showed viral resistance, indicating stable transmission of the CP gene and the trait of resistance to the next generation. Moreover, the virally encoded strip disease-specific protein was not detected in transgenic plants expressing CP 8 weeks after inoculation, indicating protection before viral multiplication. These studies demonstrated that CP-mediated resistance to virus infection can be extended to cereals and to the viruses transmitted by an insect vector (planthopper).