Biomedical subjects
Publications and source records attributed to Y Kobayashi.
[structure: see text] Three additional NMR databases, 1-3, in a chiral solvent are presented. The C.21-C.38 portion of oasomycin A is used to demonstrate the scope and limitation of the universal NMR database approach in a chiral solvent for assignment of relative and absolute stereochemistry without degradation and/or derivatization.
[structure: see text] Examples of (13)C NMR desymmetrization of meso compounds are presented. On analysis of the NMR profiles of 1,3-diols, the additivity is recognized to predict the NMR profiles of 1,3,5-tirols.
It is generally believed that apoptosis is not associated with inflammation. However, we have found that phagocytosis of apoptotic cells by PMA-treated THP-1 cells and human monocyte-derived macrophages led to the production of proinflammatory cytokines, notably IL-8. These macrophages were obtained either by PMA treatment or by M-CSF treatment, possibly affecting the cytokine production after phagocytosis of apoptotic cells. In order to exclude the possibility, we employed resident tissue macrophages such as Kupffer cells and alveolar macrophages in this study and examined the production of cytokines after phagocytosis of apoptotic cells. Kupffer cells produced proinflammatory cytokines MIP-2 and TNF-alpha at the mRNA level. The MIP-2 protein was also detected by means of ELISA. Alveolar macrophages also produced the MIP-2 protein after phagocytosis of apoptotic cells. Furthermore, apoptotic thymocytes induced a similar response by these macrophages. These findings do support the notion that macrophages are apt to produce proinflammatory cytokines after phagocytosis of apoptotic cells.
[reaction: see text] BF(3)-promoted 1,4-addition of bulky aryl groups to alpha-iodo enones, prepared from the parent enones, afforded beta-aryl-alpha-iodo ketones. Subsequent reaction with EtMgBr furnished the magnesium enolates, which upon reactions with ClP(O)(OEt)(2) and aldehydes gave enol phosphates and aldols, respectively. This method was applied successfully to a synthesis of Delta(1)-trans-tetrahydrocannabinol.
Genes associated with regulation of membrane-type matrix metalloproteinase-1 (MT1-MMP)-mediated pro-MMP-2 processing were screened in 293T cells by a newly developed expression cloning method. One of the gene products, which promoted processing of pro-MMP-2 by MT1-MMP was claudin-5, a major component of endothelial tight junctions. Expression of claudin-5 not only replaced TIMP-2 in pro-MMP-2 activation by MT1-MMP but also promoted activation of pro-MMP-2 mediated by all MT-MMPs and MT1-MMP mutants lacking the transmembrane domain (DeltaMT1-MMP). A carboxyl-terminal deletion mutant of pro-MMP-2 (proDeltaMMP-2) was processed to an intermediate form by MT1-MMP in 293T cells and was further converted to an activated form by introduction of claudin-5. In contrast to the stimulatory effect of TIMP-2 on pro-MMP-2 activation by MT1-MMP, activation of pro-MMP-2 by DeltaMT1-MMP in the presence of claudin-5 and proDeltaMMP-2 processing by MT1-MMP were both inversely repressed by expression of exogenous TIMP-2. These results suggest that TIMP-2 is not involved in cluadin-5-induced pro-MMP-2 activation by MT-MMPs. Stimulation of MT-MMP-mediated pro-MMP-2 activation was also observed with other claudin family members, claudin-1, claudin-2, and claudin-3. Amino acid substitutions or deletions in ectodomain of claudin-1 abolished stimulatory effect. Direct interaction of claudin-1 with MT1-MMP and MMP-2 was demonstrated by immunoprecipitation analysis. MT1-MMP was co-localized with claudin-1 not only at cell-cell borders, but also at other parts of the cells. TIMP-2 enhanced cell surface localization of MMP-2 mediated by MT1-MMP, and claudin-1 also stimulated it. These results suggest that claudin recruits all MT-MMPs and pro-MMP-2 on the cell surface to achieve elevated focal concentrations and, consequently, enhances activation of pro-MMP-2.
Objective: a part of patients with primary biliary cirrhosis (PBC) has anti-human carbonic anhydrase II (CA II) autoantibodies, although several contradictional reports followed. Since immunization of mice with CA II results in cholangitis in a susceptible strain, PBC with anti-CA II antibody may have distinct clinical features. Thus, we tested the sera of patients with PBC for anti-CA II antibodies and compared clinical characteristics of PBC patients with and without anti-CA II antibodies in Japanese patients. Methods: anti-CA II antibodies were detected in nine of 50 (18%) PBC patients by immunoblotting. The evaluation of these patients included various clinical parameters, autoantibodies, and immunological backgrounds. Results: the levels of serum liver tests and the prevalence of serum anti-mitochondrial antibody (77.8 vs. 92.7%) were not different between the patients with and without anti-CA II antibody. However, the prevalence of anti-nuclear antibody (ANA) was significantly higher in the patients with anti-CA II antibody than that in the patients without anti-CA II antibody (66.7 vs. 25.6%, P=0.044), although their mean titers were not statistically different. Association of Sjøgren's syndrome tended to be more frequent in the patients with anti-CA II antibody than those without it (33.3 vs. 14.6%, P=0.327). Studies of HLA class I allotype revealed that three of five (60.0%) patients with anti-CA II antibodies and one patients from 34 (3.0%) patients without anti-CA II antibodies had HLA B51 allotype; the difference in the prevalence of this allotype was significant (P=0.004, Pc=0.01), and the prevalence of other HLA class I and HLA DR allotypes was similar between the patients with and those without anti-CA II antibody. Administration of ursodeoxycholic acid (600 mg per day) was accompanied by change in liver tests in a similar way between the two patient groups. Conclusions: These results suggest that, although clinical features are not distinctive, PBC patients with anti-CA II antibody may have a genetic background, which may contribute to a susceptibility to immune-mediated cholangitis.
We generated transgenic mice that expressed a highly expanded 239 polyglutamine (polyQ) repeat under the control of the human androgen receptor promoter. These transgenic mice developed progressive neurological phenotypes of muscular weakness and ataxia, small body size and short life-span. PolyQ nuclear inclusions (NIs) were remarkable and widespread but found in selective regions of the central nervous system (CNS) such as the spinal cord, cerebrum and cerebellum as well as in selective peripheral visceral organs. This distribution pattern resembled that of spinal and bulbar muscular atrophy somewhat, but was more widespread. In neuronal tissues, NIs were present in astrocytes as well as neurons. Cytoplasmic and axonal inclusions were not observed. In the CNS regions with abundant NIs, neuronal populations were well-preserved, and neither neuronal cell death, reactive astrogliosis nor microglial invasions were detected. These findings suggest that polyQ alone can induce the neuronal dysfunction that precedes gross neuronal degeneration and provides a clue for investigating molecular mechanisms that underly the pathway to neuronal dysfunction from polyQ expansion.
Inherited deficiency of adenosine deaminase (ADA) results in one of the autosomal recessive forms of severe combined immunodeficiency. This report discusses 2 patients with ADA deficiency from different families, in whom a possible reverse mutation had occurred. The novel mutations were identified in the ADA gene from the patients, and both their parents were revealed to be carriers. Unexpectedly, established patient T-cell lines, not B-cell lines, showed half-normal levels of ADA enzyme activity. Reevaluation of the mutations in these T-cell lines indicated that one of the inherited ADA gene mutations was reverted in both patients. At least one of the patients seemed to possess the revertant cells in vivo; however, the mutant cells might have overcome the revertant after receiving ADA enzyme replacement therapy. These findings may have significant implications regarding the prospects for stem cell gene therapy for ADA deficiency.
TAMA300, an interferometric gravitational-wave detector with 300-m baseline length, has been developed and operated with sufficient sensitivity to detect gravitational-wave events within our galaxy and sufficient stability for observations; the interferometer was operated for over 10 hours stably and continuously. With a strain-equivalent noise level of h approximately 5x10(-21)/sqrt[Hz], a signal-to-noise ratio of 30 is expected for gravitational waves generated by a coalescence of 1.4M-1.4M binary neutron stars at 10 kpc distance. We evaluated the stability of the detector sensitivity with a 2-week data-taking run, collecting 160 hours of data to be analyzed in the search for gravitational waves.
The antibody response to coproantigens of Cryptosporidium parvum was examined in saliva and sera of calves experimentally infected with C. parvum. Coproantigens of C. parvum with approximate molecular masses of 17, 15 and less than 14kDa were found in the feces of infected calves on day 3 or later, and 60 and 23kDa coproantigens observed between days 4 and 9 post-infection, respectively. The antibody reactivity to the coproantigens was mainly attributable to IgA class antibodies in saliva and was detectable during the convalescent phase of infection. A 15kDa protein isolated from the feces of infected calves by immunoaffinity adsorption using a monoclonal anti C. parvum antibody was recognized by IgA antibodies present in the saliva during the convalescent phase of infection. These results suggest that this coproantigen may be released from C. parvum sporozoites and may induce IgA antibody production in the mucosal immune system of infected calves.
By using the convenient protocol for conversion of 2-substituted furans into 4-oxo-2-alkenoic acids ((i) NBS, (ii) NaClO(2)), macrosphelide B (2) was synthesized from furyl alcohol 5 (>98% ee) and acid 6 (99% ee). The protocol was first applied to the PMB ether of 5 to afford acid 13b. On the other hand, DCC condensation of acid 6 with 5 gave 16 after deprotection of the TBS group. Condensation was again carried out between 13b and 16 to furnish the key ketone 17, which upon reduction with Zn(BH(4))(2) afforded anti alcohol 18 stereoselectively (15:1). After protection/deprotection steps, the furan 18 was converted to seco acid 3 by using the furan oxidation protocol mentioned above, and lactonization of 3 with Cl(3)C(6)H(2)COCl, Et(3)N, and DMAP afforded 22 (MOM ether of 2), which upon deprotection with TFA produced 2. Transformation of 22 to macrosphelide A (1) was then investigated. Although the chelation-controlled reduction of 22 should afford the desired anti alcohol 24, Zn(BH(4))(2) at <-90 degrees C gave a 2 approximately 1:1 mixture of anti/syn alcohols. On the contrary, reduction with NaBH(4) in MeOH at -15 degrees C produced the syn isomer 23 with >10:1 diastereoselectivity. Mitsunobu inversion of the resulting C(14)-hydroxyl group and deprotection of the MOM group with TFA afforded 1. Similarly, reduction of 2 with NaBH(4) afforded the C(14)-epimer of 1 stereoselectively. The observed stereoselectivity in the reductions of 22 and 2 could be explained on the basis of computer-assisted calculation, which showed presence of the low-energy conformers responsible for the stereoselective reduction. In addition, conversion of 2 to 1 was established, for the first time.
One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor alpha (hER alpha) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hER alpha A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogen-bound hER alpha in MCF7 cells and in partially purified complexes associated with hER alpha from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hER alpha and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hER alpha. These findings indicate that p72/p68 acts as an ER subtype-selective coactivator through ER alpha AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.
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The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.
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In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.
We examined whether repetitive transcranial magnetic stimulation (rTMS) at a low rate could influence autonomic function, specially heart rate variability (HRV) by power spectrum analysis. We studied 16 healthy male volunteers as a stimulation group and 16 others as a sham group. The stimulation group received magnetic stimulations from a circular coil over Cz at a frequency of 0.2 Hz and an intensity presenting 90% of the motor threshold. Experiments in both groups included four daily sessions; at each, a train of 70 stimuli was delivered over 350 s. HRV of low-frequency power (LF) in a standing position and high-frequency power (HF) in a supine position were measured before and after each session. After stimulation, HF and LF powers were significantly increased. After sham stimulation, the power of HF but not that of LF significantly increased. Neither actual nor sham stimulation produced a long-term effect detectable on day 5. The finding of transiently increased LF power following actual but not sham stimulation suggests that rTMS may activate the sympathetic nervous system.
BACKGROUND: Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress. METHODS: ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided. RESULTS: After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium. CONCLUSION: The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.