Biomedical subjects
Publications and source records attributed to J Inoue.
A 100-member combinatorial library of alpha-ketoamides, which was designed to search potent cysteine protease inhibitors, was generated by a parallel solution-phase synthesis. A two-step one-pot synthesis, in which the Ugi reaction followed by pyridinium dichromate (PDC) oxidation was employed in the same reaction vessel, easily afforded the alpha-ketoamides in a short time.
We calculate the electronic states of the Mn-doped semiconductors and show that resonant states are formed at the top of the down spin valence band due to magnetic impurities and that they give rise to a strong and long-ranged ferromagnetic coupling between Mn moments. We propose that the coupling of the resonant states, in addition to the intra-atomic exchange interaction between the resonant and nonbonding states, is the origin of the ferromagnetism of (Ga-Mn)As. The mechanism is thus called "double resonance." The resonant states bring about the spin-dependent resistivity to produce magnetoresistive properties in (Ga-Mn)As and their junctions.
BCMA (B cell maturation) is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B lymphocytes. Previously, we reported in a human malignant myeloma cell line that BCMA is not primarily present on the cell surface but lies in a perinuclear structure that partially overlaps the Golgi apparatus. We now show that in transiently or stably transfected cells, BCMA is located on the cell surface, as well as in a perinulear Golgi-like structure. We also show that overexpression of BCMA in 293 cells activates NF-kappa B, Elk-1, the c-Jun N-terminal kinase, and the p38 mitogen-activated protein kinase. Coimmunoprecipitation experiments performed in transfected cells showed that BCMA associates with TNFR-associated factor (TRAF) 1, TRAF2, and TRAF3 adaptor proteins. Analysis of deletion mutants of the intracytoplasmic tail of BCMA showed that the 25-aa protein segment, from position 119 to 143, conserved between mouse and human BCMA, is essential for its association with the TRAFs and the activation of NF-kappa B, Elk-1, and c-Jun N-terminal kinase. BCMA belongs structurally to the TNFR family. Its unique TNFR motif corresponds to a variant motif present in the fourth repeat of the TNFRI molecule. This study confirms that BCMA is a functional member of the TNFR superfamily. Furthermore, as BCMA is lacking a "death domain" and its overexpression activates NF-kappa B and c-Jun N-terminal kinase, we can reasonably hypothesize that upon binding of its corresponding ligand BCMA transduces signals for cell survival and proliferation.
Down syndrome (DS) leads to cardiac defects which are common and significant in babies with DS. We recently generated chimeric mice carrying a human chromosome (hChr) 21. The contribution ratio of embryonic stem (ES) cells containing a hChr 21 was specifically low in the heart, compared to other organs, and cardiovascular malformations were observed, suggesting that an additional copy of hChr 21 also disrupts the normal development of heart in mice. Here we describe that the presence of hChr 21 in ES cells delays the appearance of beating cardiomyocyte during differentiation, whereas differentiation into other cell types is not disrupted. Furthermore, the defect in cardiogenesis was restored following the deletion of a specific region of hChr 21. Therefore, we conclude that the imbalance of specific gene(s) on hChr 21 may lead to the disturbance of cardiogenesis and that this may be a useful system to model and investigate the cardiac defects of human DS.
In order to develop new inhibitors of NF-kappaB activation, we designed and synthesized dehydroxymethyl derivatives of epoxyquinomicin C, namely, DHM2EQ and its regioisomer DHM3EQ. These derivatives were synthesized from 2,5-dimethoxyaniline in 5 steps. Since DHM2EQ was more active and less toxic than DHM3EQ, its stereochemical configuration was determined by X-ray crystallographic analysis. Each enantiomer of the protected DHM2EQ was separated by a chiral column and deprotected. DHM2EQ inhibited TNF-alpha-induced activation of NF-kappaB in human T cell leukemia cells, and also inhibited collagen-induced arthritis in a rheumatoid model in mice.
In an attempt to identify unknown target genes for SREBP-1, total RNA from a stable Chinese hamster ovary cell line (CHO-487) expressing a mature form of human SREBP-1a (amino acids 1-487) with a LacSwitch Inducible Mammalian Expression System was subjected to a polymerase chain reaction subtraction method. One of the fragments was found to have 90 and 86% homology with rat and human ATP citrate-lyase (ACL) cDNA, respectively. When Hep G2 cells are cultured under either sterol-loaded or -depleted conditions, expression of the gene is induced approximately 2-3-fold by sterol depletion. To investigate the direct effect of SREBP-1a on transcription, luciferase assays using the promoter of the human ACL gene were performed. These deletion studies indicated that a minimum 160-base pair segment contains the information required for the transcriptional regulation brought about by enforced expression of SREBP-1a. Luciferase assays using mutant reporter genes revealed that SREBP-dependent transcriptional regulation is mediated by two nearby motifs, the SREBP-binding site (a TCAGGCTAG sequence) and the NF-Y-binding site (a CCAAT box). It was confirmed by gel mobility shift assays that recombinant SREBP-1a binds to the sequence. Data from studies with transgenic mice and reporter assays show that the ACL gene promoter is activated by SREBP-1a more strongly than SREBP-2 in contrast to the HMG CoA synthase and LDL receptor gene promoters, which exhibit the same preference for the two factors. Therefore, SREBPs transcriptionally regulates ACL enzyme activity, which generates the cytosolic acetyl CoA required for both cholesterol and fatty acid synthesis.
We conduct a theoretical study of the temperature dependence of the spin polarization ( P) and the magnetoresistance (MR) ratio using the double exchange (DE) model for ferromagnetic tunnel junctions with half-metallic systems. It is shown that the strong exchange coupling in the DE model plays an important role in the temperature dependence of both P and the MR ratio; their values can be less than the maximum values expected for half-metallic systems at low temperatures, and the MR ratio decreases more rapidly than P with increasing temperature. The calculated results, however, indicate that the MR ratio may still be large at high temperatures near the Curie temperature.
The metal-interaction of aurine tricarboxylic acid (ATA) and its inhibitory effect on the DNA binding of NFkappaB were studied. Chemical speciation and spectroscopic studies have shown the strong interaction of ATA with metal ions present in the biological systems. EPR, FTIR and electronic spectral studies indicated the square planar structure of the metal-binding carboxylic and hydroxyl groups of ATA indicating the ground state 2B1g. Electrophoretic mobility shift assay using NFkappaB and 32P labeled DNA has shown that ATA was inhibitory against the DNA-NFkappaB binding at 30 microM. This activity was the strongest among the metal-chelating inhibitors of NFkappaB-DNA binding reported so far.
A novel series of 14 N-nitromethylsulfonanilide derivatives were synthesized and evaluated for their ability to inhibit recombinant aldose reductase. Computational docking simulations provided a good explanation for the observed structure-activity relationships. Kinetic analysis of (2-fluoro-5-methyl-N-methyl)-N-nitromethylsulfonanilide, 11, one of the most potent compounds in this series with an IC50 = 0.35 M, showed uncompetitive inhibition. Subsequent in vitro culture studies of rat lenses with 11 indicated that this series of aldose reductase inhibitors are effective in either preventing or retarding sugar cataract formation associated with diabetes.
Primary non-specific sarcoma of the ovary is extremely rare, and only 22 reported cases of pure leiomyosarcoma (LMS) are known to the authors. We present an autopsy case of a primary ovarian leiomyosarcoma in a 73-year-old woman. She had noticed an abdominal mass after difficulty in defecating for several months. The excision of tumor with bilateral salpingo-oophorectomy and hysterectomy was carried out. A diagnosis of pure leiomyosarcoma of the left ovary was made on pathological examination with immunohistochemistry. Adjuvant radio-chemotherapy was not given. At 18 months' follow up, abdomino-pelvic sonography revealed an abdominal tumor and hepatic metastasis. The patient died 3.5 years after the initial surgery. The post-mortem examination revealed a peritoneal recurrent tumor and extensive distant metastases of the liver, lungs, pancreas, gastric mucosa, muscle and skin. The prognosis of the ovarian LMS is poor from the pertinent literature. Several prognostic indicators on histology including mitotic activity, proliferative activity and p53 status of the tumor are discussed.
Bakuchiol, a meroterpene isolated from Psoralea corylifolia, prevented mitochondrial lipid peroxidation. Inhibition of oxygen consumption originating in lipid peroxidation was time-dependent. Bakuchiol protected mitochondrial respiratory enzyme activities against both NADPH-dependent and dihydroxyfumarate-induced peroxidation injury. Bakuchiol was shown to be effective to protect mitochondrial functions against oxidative stress.
A primary ovarian leiomyosarcoma is extremely rare. Moreover, there is no established treatment modality other than surgery, and the prognosis is extremely poor. We report a case of a primary ovarian leiomyosarcoma. The patient was a gravida 4, para 2, 73-year-old female. She first presented with chief complaints of constipation and a pelvic mass. A physical examination revealed a solid tumor in the pelvic cavity. Which was about the size of an infant's head and had an irregular shape. The patient was suspected of having a subserosal myoma of the uterus or an ovarian tumor, and she was subjected to a laparotomy. A solid tumor about the size of an infant's head was found in the left uterine adnexa, and an intraoperative rapid pathological diagnosis of an ovarian leiomyosarcoma was made. A total abdominal hysterectomy, a bilateral salpingo-oophorectomy, a pelvic lymphadenectomy, and an omentectomy were then performed. The final pathological diagnosis confirmed a left primary ovarian leiomyosarcoma. After a series of discussions with the patient's family about her therapeutic plan, we decided to let the patient be followed-up without adjuvant therapy. Forty-two months after the surgery, the patient accumulated massive amounts of pleural effusion and ascites, with extensive organ metastasis. She received terminal care and soon died.
We herein report that mRNA expression of microsomal triglyceride transfer protein (MTP) and its protein synthesis decline in response to sterol depletion in HepG2 cells, and we functionally characterized the MTP gene promoter in an effort to investigate the molecular mechanisms by which MTP gene transcription is regulated. Luciferase assays using truncated versions of the reporter gene revealed that the region at -124 to +33 base pairs of the human promoter contains the elements required for the suppression of transcription by sterol depletion. Enforced expression of an active form of sterol regulatory element-binding protein (SREBP)-1 (amino acids 1-487) or -2 (amino acids 1-481), both of which are activated under sterol-depleted conditions, is able to mimic sterol-mediated down-regulation. Either further truncation of the promoter region or mutation of the putative SREBP-binding sequence (5'-GCAGCCCAC-3', -124 to -116 base pairs) abolishes the sterol- and SREBP-dependent transcriptional regulation. Gel mobility shift assay showed that recombinant SREBP-2-(1-481) is able to bind the sequence. Enforced expression of a truncated form of SREBP-2 (amino acids 31-481), which acts as an inhibitor of transcription of the low density lipoprotein receptor gene because it lacks the transcriptional activation domain, also diminishes the luciferase activity, suggesting that direct binding to the promoter region might be sufficient and that the mechanism by which SREBPs inhibit MTP gene expression is distinct from that for the transcriptional stimulation of sterol-regulated genes. Although the SREBP-binding site overlaps a negative insulin-responsive element, insulin negatively regulates MTP gene expression even when the amount of the active form of SREBPs is quite low under the sterol-loaded conditions, indicating that SREBPs only slightly mediate, if at all, the insulin effects. Overall, we conclude that SREBPs are responsible for regulation of lipoprotein secretion via their control of MTP gene expression. Moreover, our results describe for the first time a novel mechanism by which SREBPs negatively regulate expression of the gene encoding the protein involved in lipid metabolism.
A novel monoclonal antibody (ASH1a/256C) that recognizes atherosclerotic lesions in human and Watanabe heritable hyperlipidemic (WHHL) rabbit aortae is described. When (123)I-labeled ASH1a/256C antibody is injected intravenously into WHHL rabbits, it associates specifically with fatty streaks on the aorta. The antigen recognized by the antibody is lipid, based on extraction with chloroform and methanol from WHHL rabbit tissues. The antigen, purified by high performance liquid chromatography, was shown to be phosphatidylcholine (PC), which contains unsaturated fatty acyl groups based on analyses utilizing (1)H and (13)C nuclear magnetic resonance, Fourier transfer-infrared spectrum, and mass spectrometry. The antibody did not react with other classes of phospholipids or neutral lipids when tested using an enzyme-linked immunosorbent assay. When PC was mixed with either cholesterol, cholesteryl ester, or triacylglycerol, however, the reactivity of the antibody to PC increased up to 8-fold. Homogenates of aorta tissue obtained from normal and WHHL rabbits were fractionated using sucrose density gradient ultracentrifugation in which neutral lipid droplets, cellular membranes, and proteins are separated. The phospholipid content in cellular membrane fractions from WHHL rabbits was twice as high as that of normal rabbits, and there was an enormous difference in the antigenic activity in these fractions. The content of cholesterol in the cellular membrane fraction of WHHL rabbits was approximately 50 times higher than that of normal rabbits. Addition of neutral lipids to the cellular membrane fraction of normal rabbit markedly increased the antigenic activity. Atheromatous lesions in thickened WHHL rabbit aortic intima that were rich in lipid droplets were stained positively with ASH1a/256C immunohistochemically. These results strongly suggest that PC-neutral lipid complex domains are formed in atherosclerotic lesions.
Metallothionein (MT), a low molecular weight, cysteine-rich metal binding protein, has been associated with cytoprotection from heavy metals and cellular oxidants. As MT has the ability to scavenge hydroxyl radicals, MT may control intracellular redox status. In the present study, we examined whether MT regulates the activity of nuclear factor-kappaB (NF-kappaB), which is one of the redox-regulated transcription factors, using the MT null embryonic cell lines established from MT null mice. We first found that tumor necrosis factor (TNF)-induced activation of the binding of NF-kappaB protein to DNA in wild type MT+/+ cells was lower than that in MT-/- cells. The NF-kappaB activation in MT-expressing cells established from MT-/- cells by the transfection of mouse MT-1 gene was also significantly lower than that in MT-/- cells. In addition, transfection of the MT gene inhibited TNF-induced IkappaB degradation and suppressed NF-kappaB-dependent gene expression induced by TNF. These results demonstrate that MT may function as a negative regulator of NF-kappaB activity.
Interleukin-1 (IL-1) is a proinflammatory cytokine that has several effects in the inflammation process. When it binds to its cell-surface receptor, IL-1 initiates a signalling cascade that leads to activation of the transcription factor NF-kappaB and is relayed through the protein TRAF6 and a succession of kinase enzymes, including NF-kappaB-inducing kinase (NIK) and I kappaB kinases (IKKs). However, the molecular mechanism by which NIK is activated is not understood. Here we show that the MAPKK kinase TAK1 acts upstream of NIK in the IL-1-activated signalling pathway and that TAK1 associates with TRAF6 during IL-1 signalling. Stimulation of TAK1 causes activation of NF-kappaB, which is blocked by dominant-negative mutants of NIK, and an inactive TAK1 mutant prevents activation of NF-kappaB that is mediated by IL-1 but not by NIK. Activated TAK1 phosphorylates NIK, which stimulates IKK-alpha activity. Our results indicate that TAK1 links TRAF6 to the NIK-IKK cascade in the IL-1 signalling pathway.
CD40 signaling modulates the immune response at least in part by activation of nuclear factor kappaB (NFkappaB). It has been shown that two distinct domains in the CD40 cytoplasmic tail (cyt), namely cyt-N and cyt-C, independently activate NFkappaB. Although four members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF3, TRAF5, and TRAF6, bind to the CD40 cyt, how each TRAF protein contributes to the NFkappaB activation by CD40 is not clear. Here we report that TRAF2, TRAF3, and TRAF5 bind cyt-C, whereas TRAF6 binds cyt-N. cyt-N is conserved poorly between human and mouse CD40, while cyt-C is highly conserved. However, single aa substitution of Glu-235 in cyt-N of human CD40 with Ala abolishes the binding of TRAF6 to cyt-N and NFkappaB activation by cyt-N. Conservation of this Glu between mouse and human CD40 strongly suggests that TRAF6 could link cyt-N to signals essential for CD40-mediated immune response. Furthermore, NFkappaB activation by cyt-C is inhibited by a kinase-negative form of NFkappaB-inducing kinase more efficiently than that by cyt-N, consistent with the result that NFkappaB activation by TRAF2 and TRAF5 is inhibited by a kinase-negative form of NFkappaB-inducing kinase more efficiently than that by TRAF6. These results indicate that NFkappaB activating signals emanating from cyt-N and cyt-C are mediated by the different members of the TRAF family and could be regulated in a distinct manner.
BACKGROUND: TRAF6, a member of the tumour necrosis factor receptor-associated factor family, was first identified as a transducer of CD40 and interleukin-1 receptor (IL-1R) signals based on the interaction of TRAF6 with the cytoplasmic tail of CD40 and with the IL-1R associated kinase in vitro. However, the functions of TRAF6 in vivo remain unidentified. RESULTS: We show that TRAF6-/- mice exhibit severe osteopetrosis and are defective in osteoclast formation. In vitro culture experiments revealed that osteoclast precursor cells derived from TRAF6-/- mice are unable to differentiate to functional osteoclasts in response to osteoclast differentiation factor (ODF). In bone marrow of TRAF6-/- mice, the number of sIgM+B220+ immature B cells is markedly reduced while the ratio of proB to preB cells is not affected. In contrast, development of thymocytes is not affected. Furthermore, TRAF6-/- mice are defective in lymph node organogenesis and IL-1 signalling in thymocytes. CONCLUSIONS: The results identify TRAF6 as an essential component of ODF signalling pathway, and also show that TRAF6 plays pivotal roles in immune and inflammatory systems in vivo.