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本論文的目的是從潮汕的角度出發，探討客家與潮汕之間邊界的不穩定性。目前，對客家和潮州族群的研究成果已經不小。然而，他們的關係往往被先驗地視為不同性質的族群，關於客家和潮汕的邊界是如何在政治、經濟和社會上被建構的，至今尚未有充分的解釋。本論文將針對這一點，指出客家邊界其實含有相當程度的模糊性。本論文以台灣客家為中心，並將其文化與大陸潮汕地區的文化進行比較，基於客家的“名”與文化之間的鬆散關聯可能已轉化為強烈關聯的假設進行討論。討論的事例是韓愈和三山國王信仰、在潮州地區和台灣北部的客家、以及在移民來源和目的地有不同族群性的劉氏家族。從這些事例的分析中可以看出，“名”和文化元素在時間或空間上是無法固定的。換言之，客家人或潮州人的文化元素可能會隨著時間的推移而改變，即使是同名的族群也會因居住地區的不同而改變其文化元素。如果這伴隨著該地區社會群體的社會經驗，就更有可能成為現實，否則就不容易被確立為該地區或群體的“文化”。可是，需要注意的是，有一個潛在的考慮，即客家文化可能是另一個族群的文化。, The purpose of this article is to discuss the unstable boundary between Hakka and Chaozhou ethnicity from the perspective of Chaozhou culture. There has been considerable research on the ethnicity of the Hakka and Chaozhou people. However, the two groups have often been treated a priori as different ethnic groups, and how the boundaries between Hakka and Chaozhou have been constructed politically, economically, and socially has not been sufficiently analyzed. In this article, I would like to address this issue and point out that the boundary between Hakka and Chaozhou is quite ambiguous.In this article, I will focus mainly on the Hakka in Taiwan and compare them with the Hakka in the Chaoshan region of mainland China based on the hypothesis that their culture has been transformed from a loose relationship with the Hakka name into a strong one. The cases I will discuss are Han Yu beliefs, Sanshan GuoWang beliefs, and the Liu lineage, which has different ethnicities in the source and destination areas such as the Chaozhou people in the Chaoshan district and the Hakka in northern Taiwan. What we can see from the analysis of these cases is that “name” and cultural content cannot be fixed in time or space. In other words, the cultural content of even Hakka and Chaozhou can change over time, and ethnic groups with the same name can have different cultural content depending on the region in which they live. If these cultural contexts are accompanied by social experiences for the social group in the region, they are more likely to contain a sense of reality; if not, they will not be easily established as “culture” for the region or ethnic group. What is important here, however, is that there is an underlying sense that what is considered to be the Hakka culture may be the culture of another ethnic group., 本論文の目的は，客家エスニシティの境界を潮州からの視点で眺めることによって両者の間にある境界の不安定さについて考察を深めることにある。これまで客家と潮州のエスニシティについて少なからぬ研究が蓄積されてきた。しかし，両者の関係性をアプリオリにもともと異なるエスニック集団として扱うことが多く，どのように客家または潮州という境界が政治・経済・社会的に構築されてきたのかについては十分に分析されてこなかった。本論文はこの点を改め，客家の境界が実際には相当程度あいまいさを含んでいることを指摘したい。 本論文では，主として台湾の客家に焦点を当て，大陸の潮汕地域と比較することで彼らの文化が客家という「名」とゆるやかな結びつきを保っていたものが，強固なものへと変換されていったのではないかという仮説に基づき議論を進める。取り上げる事例は韓愈信仰や三山国王信仰，それに潮汕地域では潮州人であり台湾北部では客家と移住元・移住先それぞれで異なったエスニシティを持つ劉氏一族である。これらの事例の分析から分かるのは，「名」と文化要素を時間的にも空間的にも固定することはできないということである。つまり，客家や潮州といってもその文化要素は時間の経過とともに変わる可能性があり，同じ名前を持つエスニック集団であっても居住する地域によって文化要素は変わりうる。これが当該地域の社会集団にとっての社会経験をともなうものであればリアリティを持ちやすく，そうでないならばその地域や集団にとっての「文化」として容易に定着することはないだろう。だがここで重要なのは，その客家文化は他のエスニック集団の文化かもしれないという配慮が根底に存在することである。

The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1 mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors.

As a reflective film material, Ag has the highest reflectivity in the visual light range (400-800nm) and is used in many cases. However, Ag is a thermally active material and we have concerns about the decrease in reflectivity by aggregation. In this study we investigate an ultra high heat proof protective layer for a thin Ag reflective film. Conventionally, SiO2 thin film is used as a protective layer for Ag reflective film. But it is difficult to obtain heat proofing > 723K. In this research we consider a SnO2 protective layer which combines high heat proofing and high crystallinity. We formed a SnO2 protective layer on an Ag thin film under various conditions, and investigated its heat proofing characteristics. We succeeded in obtaining ultra-high heat proofing exceeding 723K using 5 nm protective layer. Furthermore, in trying to understand the mechanism of this outstanding high heat proofing reflective film system, it became clear that the crystallinity in high temperature diffusion plays a major role.

It is known that some kinase inhibitors are sensitive to the phosphorylation state of the kinase, and therefore those compounds can discriminate between a phosphorylated and unphosphorylated protein. In this study, we prepared two colony stimulating factor-1 receptor (CSF-1R) tyrosine kinase proteins: one highly phosphorylated by autophosphorylation and the other dephosphorylated by phosphatase treatment. These kinases were subjected to an activity-based assay to investigate the effect of their phosphorylation state on the potency of several kinase inhibitors. Dasatinib, sorafenib, PD173074 and staurosporine showed similar inhibition against different phosphorylation states of CSF-1R, but pazopanib, sunitinib, GW2580 and imatinib showed more potent inhibition against dephosphorylated CSF-1R. Binding analysis of the inhibitors to the two different phosphorylation forms of CSF-1R, using surface plasmon resonance spectrometry, revealed that staurosporine bound to both forms with similar affinity, but sunitinib bound to the dephosphorylated form with higher affinity. Thus, these observations suggest that sunitinib binds preferentially to the inactive form, preventing the activation of CSF-1R. Screening against different activation states of kinases should be an important approach for prioritizing compounds and should facilitate inhibitor design.

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state. While the activation loop of the npMKK4/AMP complex was disordered, in the npMKK4/AMP/p38 complex it configured a long α-helix, which prevented substrate access to the active site and αC-helix movement to the active configuration of MKK4.

To evaluate the anti-angiogenic efficacy of CB-12181 [an azasugar derivative that has inhibitory actions against matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE)], we investigated the suppressing ability on in vitro (tube formation by endothelial cells) and in vivo (retinal neovascularization on murine ischemia-induced proliferative retinopathy) models of angiogenesis. For in vitro analysis, a capillary-like tube formation model using human umbilical vein endothelial cells (HUVECs) and fibroblasts co-culture assay was employed. Tube formation of HUVECs was stimulated by vascular endothelial growth factor (VEGF) and incubated with different concentrations of CB-12181 (0.1-100 microM) for 11 days. For in vivo analysis, mice were exposed to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Then, the mice were removed from the oxygen treatment and treated with CB-12181 (1, 15, or 50 mg/kg) by daily subcutaneous injection from the time of reintroduction to room air at P12 until P16. At P17, pathological and physiological angiogenesis was quantified using retinal flat-mounts visualized by fluorescent angiography. In the in vitro angiogenesis model, CB-12181 significantly suppressed VEGF-induced HUVEC tube formation. Furthermore, in the in vivo angiogenesis model, administration of CB-12181 significantly suppressed retinal neovascularization without any apparent side effects on physiological revascularization to the oxygen-induced obliteration area. These results suggest that CB-12181 might be useful in the treatment of various diseases that depend on pathologic angiogenesis, and especially valuable for the treatment of diabetic retinopathy and retinopathy of prematurity.

Extracellular signal-regulated kinase (ERK) is a member of the MAP kinase family, and can regulate several cellular responses. The isoforms ERK1 and ERK2 have markedly similar amino acid sequences, but exhibit distinctive physiological functions. As well as ERK2, ERK1 was auto- and mono-phosphorylated at Tyr204 in the activation loop during Escherichia coli production, resulting in basal level activity, approximately 500-fold less compared with fully-active ERK1 dual-phosphorylated at Thr202 and Tyr204. Crystal structure demonstrated that the mono-phosphorylated ERK1 kinase possessed a novel conformation distinguishable from the un-phosphorylated (inactive) and the dual-phosphorylated (full-active) forms. The characteristic structural features in both the C-helix and the activation loop likely contribute to the basal activity of the mono-phosphorylated ERK1. The structural dissection of ERK1 compared to ERK2 suggests that the structural differences in the D-motif binding site and in the backside binding site are putative targets for development of selective ERK1/ERK2 inhibitors.

We have developed a new multilayered reflection film structure containing Ag films for achieving slab waveguide based optical transmission interconnections between large scale integrated chips for next generation high-speed computers. We applied Cr to both adhere the Ag to the SiO2 optical layer and to inhibit diffusion of Ag into SiO2. Although we found that Cr tends to decrease the reflectance, we found that it is possible to satisfy both adhesiveness and reflectance requirements by adjusting the film thickness

Summary Background The basic function of epithelia is to provide a boundary between tissue and its external environment, and is achieved by a wide variety of components including extracellular molecules. Multiple monoclonal antibodies raised against epithelial antigens have helped identify a range of distinct, novel protein epitopes. Object In this study, we raised a monoclonal antibody to detect a novel epithelial molecular component. Methods We have produced a mouse monoclonal antibody using normal human amniotic tissue as an immunogen. The monoclonal antibody was subsequently immunohistochemically screened, and the target antigen was cloned using an immunoscreening method. Result In the course of the screening, we identified unique antibody staining patterns within the cytoplasm of a subset of amniotic cells at intervals within the normal placental epithelia. By immunoscreening, we identified this candidate gene as laminin receptor (LR). By dot blot analysis, this antibody reacted with recombinant LR. The same localization of the antigen and LR was proved by a double staining immunofluorescence test in the placenta. This monoclonal antibody unexpectedly demonstrated linear staining within the dermal–epidermal junction of normal human skin but failed to react within the keratinocyte cytoplasm. Conclusion We have produced and characterized a novel monoclonal antibody 29A that recognizes an LR-related molecule, which demonstrated a unique staining pattern. This monoclonal antibody might be a useful tool for further investigations into the epithelial tissues and the cutaneous basement membrane (BM).

Members of the epidermal growth factor (EGF) family are the most important growth factors involved in epithelialization during cutaneous wound healing. Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is thought to play an important role in skin wound healing. To investigate the in vivo function of HB-EGF in skin wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter. Studies of wound healing revealed that wound closure was markedly impaired in keratinocyte-specific HB-EGF-deficient mice. HB-EGF mRNA was upregulated at the migrating epidermal edge, although cell growth was not altered. Of the members of the EGF family, HB-EGF mRNA expression was induced the most rapidly and dramatically as a result of scraping in vitro. Combined, these findings clearly demonstrate, for the first time, that HB-EGF is the predominant growth factor involved in epithelialization in skin wound healing in vivo and that it functions by accelerating keratinocyte migration, rather than proliferation.

Psoriasis is a common and chronic skin disease characterised by skin inflammation and hyperproliferation of epidermal keratinocytes, but the pathogenesis remains to be elucidated. Recently, anticytokine therapy, for example, the blockade of tumour necrosis factor (TNF)-α, was approved as treatment for psoriasis, indicating that TNF-α is a main mediator of chronic skin inflammation in psoriatic lesions. TNF-α is produced in diverse immune cells and keratinocytes and released after ectodomain shedding. TNF-α converting enzyme (TACE/ADAM17) is the primary enzyme that cleaves membrane-bound TNF-α at the ectodomain site and is classified within the disintegrin and metalloproteinase (ADAM) family. Hyperproliferation of keratinocytes is associated with activation of epidermal growth factor receptor (EGFR). The EGFR ligands are also generated in a membrane-bound form and released after ectodomain shedding by ADAMs and matrix metallo-proteinases (MMPs). Herein, the pathological and physiological implication of MMP...

次世代光・電子システムにむけたアド・ドロップグレーティング方式の光伝送路において, 課題であった光伝送系の反射膜として, 酸化物系光導波路に適したAg系反射膜を開発した。そしてわれわれは, この熱的・化学的に不安定なAg系薄膜が安定な反射膜を保っメカニズムを明らかにした。そのメカニズムは, Cr薄膜によってAg表面拡散が抑制されAgの結晶粒成長が抑制される, というものである。またこのとき, Agの酸化物系SiO2およびガラス基板に対する接着力と反射率の安定性には2nm近傍の極薄膜Crが有効であることを見出した。一方, この拡散支配型の結晶粒成長理論は, 薄膜が均一な膜を維持している範囲でのみ成立する。そして, これらの知見はその他の薄膜の熱安定性に関する材料・プロセス設計への展開可能性を示している。

Summary Background: Oculocutaneous albinism (OCA) is a heterogeneous congenital disorder. Tyrosinase is a key enzyme in melanin biosynthesis, and tyrosinase gene mutations cause the OCA1 subtype. Objective: This study was intended evaluate the frequency and details of tyrosinase gene mutations in Japanese OCA patients. Patients and methods: We examined nine non-consanguineous OCA families, sequenced the tyrosinase gene of the patients and also confirmed a splicing site mutation using exon trapping system. Results: Tyrosinase gene mutations were identified in five out of nine OCA families (55%). IVS2-10deltt-7t-a was present in 3 out of 18 alleles in three families (16%), P310insC was present in three alleles in three families (16%) and R278X was found in three alleles (16%), including those in one heterozygous and one compound homozygous patient. G97V (290 G-T) was found in 1 out of 18 alleles, and we could not find G97V in the mutation database. We have added this mutation as 9th mutation of Japanese OCA1 patients. In 8 of 18 alleles, four families, no tyrosinase mutations were identified. They were presumed not to be OCA1, but other subtypes of OCA. Exon trapping system demonstrated IVS2-10deltt-7t-a mutation generated the abnormal splicing site, and inserted the codon 4 bases in mRNA level resulting in premature termination codon downstream. Conclusion: This study provided new information about OCA1 mutations, and highlights the requirement of broader detailed search to make precise diagnosis of OCA.

Summary Background Darier disease (DD), an autosomal dominant genodermatosis characterized by warty papules and plaques over seborrhoeic areas, is caused by mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca2+ ATPase type 2 isoform (SERCA2). While markedly different clinical severity within DD-affected family members is known, the pathomechanism has not been elucidated. Objectives Based on the hypothesis that multiple ATP2A2 mutations might contribute to the pathomechanism, we have analysed two DD families in which the clinical severity differs markedly within a single pedigree, and, as controls, eight DD families without differing clinical severity. Methods All the exons and intron–exon borders of ATP2A2 were directly sequenced from the genomic DNA extracted from all the subjects. Results We identified the heterozygous mutations, G233R in pedigree 1 and C318R in pedigree 2, respectively, whereas no other ATP2A2 mutations in any of severely affected individuals were found. In eight DD pedigrees as control, we have found M1V, N39D, L180R, A838P and 2170 insertion G in each of five pedigrees, but no mutation was found in three DD pedigrees. Conclusions Our results together with previous data indicate that the distribution of mutations is scattered over the entire ATP2A2 without any, as yet, discernible ‘hotspots’. The mutations in pedigrees 1 and 2 with intrafamiliar clinical differences occurred around the Ca2+-binding sites on SERCA2, which might be associated with differences in clinical severity. These variations in ATP2A2 mutations alone cannot account for the clinical heterogeneity within DD pedigrees.

The in vivo effects of IL-18 on bone metabolism were investigated by histopathology in IL-18 transgenic mice. Deformed cortical bone and decreased turnover rate of lumbar trabecular bone are consistent with increased expression of IFN-gamma and IL-18 in the bone marrow. Interleukin (IL)-18 has been demonstrated to inhibit osteoclastogenesis in an in vitro co-culture system. We investigated the effects of IL-18 overexpression on bone metabolism by comparing bone characteristics in male IL-18 transgenic (TG) mice, which secrete mature murine IL-18 from their B- and T-cells, and their wildtype littermates (WT). Histopathological analysis revealed that the cortical bone of the femur was thinner and more deformed in IL-18 TG mice. Bone histomorphometry showed that the cortical bone area of the mid-diaphysis of the femur and the trabecular bone volume of the lumbar vertebrae were significantly reduced in IL-18 TG mice. IL-18 TG mice also exhibited significantly fewer osteoclasts and a reduced bone formation rate in the trabecular bones of their lumbar vertebrae. Real-time reverse transcriptase-polymerase chain reaction amplification of bone marrow cell mRNA revealed that interferon (IFN)-gamma mRNA expression was significantly increased, whereas IL-4 mRNA expression was significantly reduced, in IL-18 TG mice. However, the expression ratio of receptor activator of NFkappaB ligand and osteoprotegerin mRNA was not significantly altered. Thus, deformed cortical bone and a decreased turnover rate of lumbar trabecular bone are characteristic of IL-18 TG mice, and these features might be associated with the increased expression of IFN-gamma and IL-18 in the bone marrow.

The present study was conducted to critically determine the protective role of IL-18 in host response to Mycobacterium tuberculosis infection. IL-18-deficient (knockout (KO)) mice were slightly more prone to this infection than wild-type (WT) mice. Sensitivity of IL-12p40KO mice was lower than that of IL-12p40/IL-18 double KO mice. IFN-γ production caused by the infection was significantly attenuated in IL-18KO mice compared with WT mice, as indicated by reduction in the levels of this cytokine in sera, spleen, lung, and liver, and its synthesis by spleen cells restimulated with purified protein derivatives. Serum IL-12p40 level postinfection and its production by peritoneal exudate cells stimulated with live bacilli were also significantly lower in IL-18KO mice than WT mice, suggesting that attenuated production of IFN-γ was secondary to reduction of IL-12 synthesis. However, this was not likely the case, because administration of excess IL-12 did not restore the reduced IFN-γ production in IL-18KO mice. In further studies, IL-18 transgenic mice were more resistant to the infection than control littermate mice, and serum IFN-γ level and its production by restimulated spleen cells were increased in the former mice. Taken together, our results indicate that IL-18 plays an important role in Th1 response and host defense against M. tuberculosis infection although the contribution was not as profound as that of IL-12p40.

The expression of intradermally injected DNA by keratinocytes is found mainly in the upper and middle layers of the epidermis. To investigate the mechanism of this selective expression, we observed the sequential changes in the distribution of interleukin-6-expressing keratinocytes after the introduction of the interleukin-6 gene. Transgene expression first occurred in basal keratinocytes and subsequently expanded to all epidermal layers and then remained in the upper layers. Semiquantitative analysis indicated that keratinocytes in the lower layers incorporated and lost DNA earlier than those in the upper layers. In order to examine the effect of the DNA size on the transgene expression, we constructed a plasmid containing a full-length 9 kb cDNA of type VII collagen and introduced it into keratinocytes. The expression pattern of type VII collagen in the epidermis was the same as those for smaller genes. This suggests that plasmid size has little or no effect on the expression pattern of the transfected gene. To trace the introduced plasmid, we intradermally injected a green fluorescence protein expression plasmid coupled with a rhodamine flag. Almost all keratinocytes in the injected areas showed rhodamine fluorescence. Furthermore, some cells also expressed green fluorescence protein. A lack of rhodamine fluorescence in the nucleus suggested an impairment of plasmid DNA transport from the cytoplasm to the nucleus. Collectively, our results show that the majority of keratinocytes take up the intradermally injected DNA regardless of its size, but that the transfer of DNA from the cytoplasm to the nucleus is limiting the transgene expression.

Previously we identified the novel type II lectin receptor, dectin-1, that is expressed preferentially by murine antigen presenting dendritic cells (DC) and is involved in co-stimulation of T cells by DC. To identify the human homologue (DECTIN-1), we employed degenerative PCR amplification of mRNA isolated from DC and subsequent cDNA cloning. DECTIN-1 is a type II lectin receptor with high homology to type II lectin receptors expressed by natural killer (NK) cells. It contains an immunoreceptor tyrosine-based activation motif within the cytoplasmic domain. Human DECTIN-1 mRNA is expressed predominantly by peripheral blood leukocytes and preferentially by DC. The mRNA likely encodes a 33 kDa glycoprotein. In human epidermis, the protein is expressed selectively by Langerhans cells, which are an epidermal subset of DC. A truncated form of DECTIN-1 RNA (termed T beta) encodes for a polypeptide lacking almost the entire neck domain, which is required for accessibility of the carbohydrate recognition domain to ligands. Genome analysis showed the deleted amino acid sequence in T beta to be encoded by an exon, indicating that T beta RNA is produced by alternative splicing. DECTIN-1 gene maps to chromosome 12, between p13.2 and p12.3, close to the NK gene complex (12p13.1 to p13.2) which contains genes for NK lectin receptors. Our results indicate that human DECTIN-1 shares many features with mouse dectin-1, including the generation of neck domain-lacking isoforms, which may down-regulate the co-stimulatory function of dectin-1.

Conformationally constrained peptide libraries have been made by grafting randomized amino acid sequences onto a rigid scaffold derived from natural proteins. Here, as a library scaffold, we propose a de novo designed helix-loop-helix motif. We constructed a peptide library of the loop region and screened against Aurora-A, which is a member of the Aurora family of serine/threonine protein kinases, to successfully isolate the inhibitory peptides. A semi-rational strategy, which combines phage-displayed libraries and de novo designed peptides, would provide a new way to generate selective peptide inhibitors for the protein kinase family.

1. 1. The mechanism of action of a new antiplatelet agent, KBT-3022 (ethyl 2-[4,5-bis(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetate) and its active main metabolite, desethyl KBT-3022, was investigated. 2. 2. KBT-3022 and desethyl KBT-3022 inhibited cyclooxygenase from ovine seminal gland with IC 50 values of 0.69 and 0.43 μM, respectively. 3. 3. At concentrations higher than those required for cyclooxygenase inhibition, desethyl KBT-3022 inhibited cAMP-phosphodiesterase, specific binding of U46619, and release of phosphatidic acid from thrombin-stimulated platelets. 4. 4. Oral administration of KBT-3022 inhibited the production of thromboxane B 2 during blood coagulation more potently than the production of 6-keto-prostaglandin F 1α from aortic strips in guinea pigs. 5. 5. These findings suggest that KBT-3022 may inhibit platelet activation principally via the inhibition of cyclooxygenase by desethyl KBT-3022.

The effect of KBT-3022 (ethyl 2-[4,5-bis(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetate), a new cyclooxygenase inhibitor, on experimental brain edema was studied. In vitro, KBT-3022 (100 microM) and its metabolite desethyl KBT-3022 (10 and 100 microM), but neither acetylsalicylic acid nor indomethacin, inhibited arachidonic acid-induced swelling of guinea pig cortical slices. KBT-3022 (3-100 microM) and desethyl KBT-3022 (3-30 microM), but neither acetylsalicylic acid nor indomethacin, inhibited lipid peroxidation in guinea pig brain homogenate. In vivo, oral administration of KBT-3022 (1, 3 and 10 mg/kg) and indomethacin (10 and 30 mg/kg), but not acetylsalicylic acid, prevented brain edema induced by bilateral carotid occlusion and recirculation in gerbils. Indomethacin then prevented postischemic hyperthermia, but not KBT-3022. KBT-3022 (10 mg/kg) and indomethacin (30 mg/kg) inhibited lactate accumulation in gerbil brain after ischemia and recirculation. These results suggest that KBT-3022 prevents development of both cytotoxic edema in vitro and vasogenic edema in vivo.

The effects of the new anti-platelet agent KBT-3022, ethyl 2-[4,5-bis(4-methoxyphenyl)-thiazol-2-yl]pyrrol-1-ylacetate, and its metabolite desethyl KBT-3022 on rabbit neutrophil function were investigated in comparison with the effects of acetylsalicylic acid (ASA), ticlopidine hydrochloride (TP), cilostazol (CIL) and indomethacin (IM). The adhesion and migration of neutrophils induced by formyl-methionyl-leucyl-phenylalanine (fMLP) were inhibited by all the compounds tested, their rank order of potency being KBT-3022 = desethyl KBT-3022 > TP = CIL = IM > ASA. KBT-3022, desethyl KBT-3022, CIL and IM all suppressed fMLP-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) in neutrophils, their potencies correlating with their inhibitory effects on fMLP-induced adhesion and migration. KBT-3022 (1 microM), desethyl KBT-3022 (1-10 microM) and CIL (10 microM) but not IM significantly inhibited both neutrophil migration and the increase in [Ca2+]i induced by leukotriene B4 (LTB4). KBT-3022 (1 microM) and desethyl KBT-3022 (1 microM) suppressed the increase in [Ca2+]i induced by complement C5a. Although KBT-3022 and desethyl KBT-3022 did not influence [3H]LTB4 and [125I]C5a specific binding, [3H]fMLP specific binding was inhibited by desethyl KBT-3022 (IC50: 1.9 microM). Neutrophil adhesion and superoxide anion production stimulated by phorbol 12-myristate 13-acetate were partially inhibited by KBT-3022 (1 microM) and desethyl KBT-3022 (1-10 microM). These results suggest that KBT-3022 and desethyl KBT-3022 have a wider spectrum of action and are more potent inhibitors of neutrophil activation than ASA, TP, CIL and IM.

Pyoderma gangrenosum (PG) is a neutrophilic dermatosis characterized by destructive, necrotizing and noninfective ulceration of the skin mostly on lower extremities. PG is well known as a complication of Takayasu's arteritis in Japan. However, this association is not commonly observed in North American and European patients. We describe a case of PG that was associated with Takayasu's arteritis who was successfully treated with systemic cyclosporin. We have reviewed 35 well-documented PG cases with Takayasu's arteritis in comparison to 106 PG cases without Takayasu's arteritis. The results demonstrate that this association occurs predominantly in young females and that these cases exhibit more widespread PG lesions.

The effects of KBT-3022 and its metabolite desethyl KBT-3022 on platelet aggregation were determined in rat, guinea-pig, rabbit and human platelets in-vitro and ex-vivo. KBT-3022 and desethyl KBT-3022 inhibited platelet aggregation induced by arachidonic acid and collagen in-vitro more potently than aggregation induced by adenosine diphosphate, platelet-activating factor or thrombin, as well as by acetylsalicylic acid, and their effects were approximately 100 times more potent than those of acetylsalicylic acid. Desethyl KBT-3022, but not KBT-3022 or acetylsalicylic acid, inhibited thrombin-induced aggregation and 5-hydroxytryptamine release from platelets more potently than ticlopidine hydrochloride at higher concentrations. Oral administration of KBT-3022 inhibited both arachidonic acid- and collagen-induced platelet aggregation and reduced platelet retention in a glass-bead column approx. 100 times more potently than acetylsalicylic acid. KBT-3022 showed little or no anti-inflammatory effect on either ultraviolet-induced erythema or arachidonic acid induced ear oedema, and had lower gastro-ulcerogenicity than acetylsalicylic acid. These results suggest that KBT-3022 is a potent inhibitor of platelet activation with weak side-effects.

SummaryUsing guinea pigs, a study was conducted on the effects of KBT-3022, a new anti-platelet agent, on hemorheological properties in various tests including blood filterability, blood viscosity, shear stress-induced red blood cell (RBC) deformability and contents of ATP and 2,3-diphosphoglycerate (2,3-DPG). Oral administration of KBT-3022 at 1 and 10 mg/kg significantly increased blood filterability, and significantly reduced blood viscosity at 10 mg/kg without changing the hematocrit, plasma fibrinogen concentration or plasma viscosity. KBT-3022 (10 mg/kg, p.o.) improved RBC deformability in response to shear stress, which was evoked by passing the blood through a thin tube. This dose of KBT-3022 also increased the contents of ATP and 2,3-DPG in RBC. These findings indicate that KBT-3022 may reduce blood viscosity as a sequel to improvement of RBC deformability through direct action on RBC. The increase in the intracellular levels of ATP and 2,3-DPG was considered to be involved in this improvement of hemorheological properties. These hemorheological effects of KBT-3022 appear to be promising for the treatment of patients with ischemic vascular disease.

The protective effect of KBT-3022 (ethyl 2-[4,5-bis-(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetate), a new cyclooxygenase inhibitor, in cerebral hypoxia and ischemia was studied and compared with those of indomethacin and acetylsalicylic acid (ASA). Oral administration of KBT-3022 (3-100 mg/kg) and indomethacin (3 and 10 mg/kg) significantly prevented KCN-induced death in mice, while ASA (100mg/kg) had no effect. KBT-3022 (3 and 10 mg/kg, p.o.) and indomethacin (10 mg/kg, p.o.) significantly prolonged the survival time of mice subjected to normobaric hypoxia, while ASA (100 mg/kg, p.o.) had no effect. KBT-3022 (3-30 mg/kg, p.o.) and indomethacin (3 mg/kg, i.p.) significantly ameliorated delayed neuronal death in the gerbil hippocampal CA1 sector after occlusion of bilateral carotid arteries for 5 min, while ASA (300 mg/kg, p.o.) had no effect. KBT-3022 (10 mg/kg, p.o.) significantly inhibited ATP depletion in the gerbil hippocampus after a 1-min occlusion of bilateral carotid arteries, but had no effect on ATP depletion after a 5-min occlusion and the recovery during recirculation. These results show that KBT-3022 exerts protective effects against cerebral anoxia and hypoxia and ameliorates delayed neuronal death in the hippocampus. KBT-3022 may therefore be useful for prophylaxis of ischemic cerebrovascular disorders.

This study is concerned with the problem of dynamic response of angle-ply laminated cylindrical shells under axial compressive impact loads. A mathematical formulation of shell theory is based on the Kirchhoff-Love hypothesis. Cylindrical shells are analyzed using the Donnell-type equations under the boundary condition for simply supported edges. Subsequently, certain perturbations are superimposed on this motion, and their behavior in time is investigated. The symmetric state of motion of the shell is called stable if the perturbations remain bounded. The solutions for the prebuckling motion and the perturbed motion are obtained using the Galerkin method. The inevitability of dynamically unstable behavior is proved analytically and the effects of various factors, such as compressive load ratio, lamination angle, dynamic unstable mode and dimensions of the cylinder are clarified.

Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in the p38 MAP kinase signal cascade that regulates various stress-induced responses and is associated with pathological conditions. The crystal structure of human non-phosphorylated MAP2K6 (npMAP2K6) complexed with an ATP analogue was determined at 2.6 A resolution and represents an auto-inhibition state of MAP2K6. Three characteristics of short α-helices configured in the activation loop region, termed activation helices (AH1, AH2 and AH3), are important in controlling the auto-inhibition mechanism. AH1 displaces the αC-helix, a component essential for forming the active configuration, away from the active site. AH1 and AH2 were found to enclose the γ-phosphate, the leaving group of ATP. A comparison with the related enzymes, MAP2K1 and MAP2K4 reveals that MAP2K6 has the unique auto-inhibition mechanism mediated by the three activation helices.

The effects of the novel calcium channel antagonist KB-2796, other calcium channel antagonists, an N-methyl- D -aspartate (NMDA) antagonist, non-NMDA antagonists, Mg2+, a Ca2+-chelator and a calcium channel agonist on neurotoxicity induced by a 10-min application of 100 μΜ glutamate were studied in rat hippocampal primary cell cultures. KB-2796 (0.1 and 1 μΜ), flunarizine (1 μΜ), nimodipine (10 μΜ), MK-801 (0.01-1 μΜ), Mg2+ (10 mM) and EGTA (10 mM) significantly prevented the neurotoxicity, but 6-cyano-7-nitro-quinoxalinedione (CNQX) (10 μΜ) and 6,7-dinitro-quinoxalinedione (DNQX) (10 μΜ) did not. Bay K 8644 (10 and 100 nM) enhanced the neurotoxicity. These findings indicate that KB-2796 protects the neuronal cell from the glutamate-induced neurotoxicity, presumably by blocking the Ca2+ influx into brain neurons.

Fyn-related kinase (Frk) was first identified using human breast cancer cells. It shares 51% identity with c-Src. Like all members of the Src family, Frk is thought to cause several cancers via dysregulations in signal transduction from cell-surface receptors. The excess activity of Frk on beta-cells has a crucial role in type-I diabetes. A silent mutation at Ile229 conferred a bacterial expression system on the kinase domains of Frk, which allowed for the quick expression and purification of one unphosphorylated and two mono-phosphorylated kinase domains. The C-terminal catalytic segment of the human Frk kinase conjugating hexahistidine purification tag (His-tag) was expressed in Escherichia coli. After first-step purification utilizing the His-tag, an anion-exchange chromatogram yielded three major peaks that had distinguishable phosphorylation characteristics as judged by Western blot analysis and measurement of kinase activity. This result of active protein production should promote drug discovery studies, including highthrough-put screening and structure-based drug design.

The hemorheological effect of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4,-trimethoxybenzyl)piperazine dihydrochloride, was studied in guinea pigs and rabbits in comparison with those of flunarizine (FNZ) and pentoxifylline (PXF). KB-2796 and FNZ at 10-100 microM dose-dependently prevented crenation of rabbit erythrocytes induced by the Ca2+ ionophore A23187. After incubation of guinea pig whole blood at 37 degrees C, blood micropore-filterability decreased and blood viscosity increased with the progress of erythrocyte crenation. After a 4-hr incubation, KB-2796 inhibited erythrocyte crenation and decreased blood filterability at a concentration of 30 microM, and it increased blood viscosity at 10 microM. Treatment with FNZ (30 microM) and PXF (100 microM) also inhibited erythrocyte crenation and decreased blood filterability. Intravenous administration of KB-2796 at 3 mg/kg significantly inhibited the decrease of blood micropore-filterability after occlusion of the bilateral carotid arteries in rabbits. Although FNZ (3 mg/kg, i.v.) had no effect, PXF (3 mg/kg, i.v.) produced significant inhibition. These results suggest that KB-2796 prevents increase of blood viscosity and decrease of blood filterability by inhibiting the crenation of erythrocytes and suggest that this effect may be useful for the improvement of hemorheology in ischemic disease.

Human Lyn tyrosine kinase is expressed in hematopoietic tissues and plays crucial roles in the signal transduction of hematopoietic immune system. Its excess activity is involved in several tumors. The crystal structure has revealed that the potent inhibitor staurosporine binds to human Lyn kinase domain at the ATP-binding site. The remarkable structural features of the staurosporine-binding region will offer valuable structural insights for the structure-based design of novel Lyn-selective inhibitors.

To evaluate the anti-angiogenic efficacy of CB-12181 [an azasugar derivative that has inhibitory actions against matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE)], we investigated the suppressing ability on in vitro (tube formation by endothelial cells) and in vivo (retinal neovascularization on murine ischemia-induced proliferative retinopathy) models of angiogenesis. For in vitro analysis, a capillary-like tube formation model using human umbilical vein endothelial cells (HUVECs) and fibroblasts co-culture assay was employed. Tube formation of HUVECs was stimulated by vascular endothelial growth factor (VEGF) and incubated with different concentrations of CB-12181 (0.1-100 microM) for 11 days. For in vivo analysis, mice were exposed to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Then, the mice were removed from the oxygen treatment and treated with CB-12181 (1, 15, or 50 mg/kg) by daily subcutaneous injection from the time of reintroduction to room air at P12 until P16. At P17, pathological and physiological angiogenesis was quantified using retinal flat-mounts visualized by fluorescent angiography. In the in vitro angiogenesis model, CB-12181 significantly suppressed VEGF-induced HUVEC tube formation. Furthermore, in the in vivo angiogenesis model, administration of CB-12181 significantly suppressed retinal neovascularization without any apparent side effects on physiological revascularization to the oxygen-induced obliteration area. These results suggest that CB-12181 might be useful in the treatment of various diseases that depend on pathologic angiogenesis, and especially valuable for the treatment of diabetic retinopathy and retinopathy of prematurity.

Lyn is a member of the Src family of non-receptor protein kinase. As well as all members of the Src family, Lyn is thought to participate in signal transduction from cell surface receptors. The crystal structure of Lyn would have a better understanding of Lyn function in various cells. For the purpose of crystallization, C-terminal catalytic segment of human Lyn kinase conjugating hexahistidine purification tag (His-tag) was expressed in Sf21 insect cells. After first step purification utilizing His-tag, an anion-exchange chromatogram yielded four major peaks which had distinguishable phosphorylation manner as judged by Western blot analysis, Native-PAGE analysis and kinase activity measurements. The fractioned samples were separately examined for crystallization screening using a commercial available screening kit. The mono-phosphorylated protein was crystallized with a small rod-shaped and needle clusters. The higher phosphorylated samples corresponding to the other three fractions on the anion-exchange chromatogram were aggregated or precipitated under the above conditions. A crystal of the mono-phosphorylated sample was diffracted to 3.2A with synchrotron source at Photon Factory and a complete X-ray diffraction data set was collected. The coarse structure was solved by a molecular replacement method and further structural refinement is currently underway.

Summary We report a patient with epidermodysplasia verruciformis (EV) who had severe generalized verrucous skin lesions for 50 years without any immunological abnormality. Microscopic examination showed two histopathological features, including seborrhoeic keratosis and common warts. The detected human papilloma virus (HPV) types were found to be HPV 3, 50, 5, and 76, using a degenerate PCR method. EV and generalized verrucosis are distinguished by slight differences in clinical symptoms or HPV types, so there should be no apparent differential points common to both diseases. Therefore, we propose that an abnormal susceptibility specific to HPV, which is the most characteristic feature in EV, should be regarded as a differential point in these two diseases.

We report two atypical cases of membranous aplasia cutis surrounded by a rim of hairs, one case associated with dense dermal melanocytosis and the other with naevus flammeus, with characteristic clinical features. A rim of hypertrichosis, 'hair collar' sign, is proposed to have a close association with neuroectodermal defects. A failure of the normal closure of the cranial neural tube might have affected foetal skin development, including melanoblast migration and capillary network formation. The changes in the present cases, as well as the hair collar sign may suggest a complex hamartomatous nature of membranous aplasia cutis.

We report that clearance of Pseudomonas aeruginosa , accumulation of neutrophils, and synthesis of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the infected lung were not largely different in interleukin-18 (IL-18) knockout or transgenic mice compared with control mice. Our results suggest a limited role for IL-18 in the host defense against P. aeruginosa .

Several cases in which the disease course has undergone a transition from pemphigus vulgaris (PV) to pemphigus foliaceus (PF) or vice versa have been reported in the literature. The disease transition in these cases was determined by means of clinical and histopathological observations and immunoblot analysis. To our knowledge, the case described herein represents the first report to clearly demonstrate the serological transition from mucous PV to mucocutaneous PV to PF using a desmoglein (Dsg) enzyme-linked immunosorbent assay (ELISA) system.

Effects of temperature and ionic strength (S) on the local structure of tobacco mosaic virus RNA in phosphate buffer solution are studied by analyzing the small-angle X-ray scattering (SAXS) curves. The root-mean-square radius of a cross-section of RNA chain was kept at 0.845+/-0.005 nm over a wide range of S from 0.2 to 0.003 at 20 degrees C, whereas it gradually diminished from 0.85 to 0.61 nm when the temperature is raised from 20 to 50 degrees C at S = 0.2. Nevertheless, all of SAXS curves reflecting the backbone structures were equally mimicked by theoretical ones of freely hinged rod (FHR) models, i.e. several straight rods joined with freely hinged joints in the form of a combination of the letter Y, if the constituent rod lengths in the models are adjusted. From these facts, it is suggested that the local structure of the RNA chain in aqueous solution is characterized by an essential feature that unpaired bases in the partially double-stranded helix are constantly far isolated from each other along the helix and the rod-like structure of the helix is preserved over a range of helical contents. Such a characteristic local structure of the chain is entirely collapsed in the formamide solution at 50 degrees C.

Danaparoid sodium (danaparoid) is a low molecular weight heparinoid with anticoagulation properties, which mainly consists of heparan sulfate. Compared with heparin sodium (heparin), danaparoid has a much higher anti-Xa/anti-thrombin ratio. We compared the effect of danaparoid on endotoxin-induced experimental disseminated intravascular coagulation (DIC) in rats with heparin. A bolus injection of endotoxin (10 mg/kg) induced gradual decreases in the platelet count, and the plasma fibrinogen, antithrombin III (AT-III) and heparin cofactor II levels, as well as an increase in the fibrinogen/fibrin degradation products level from 1 to 6 hours after the injection, indicating that both coagulation and fibrinolysis were activated. The intravenous administration of danaparoid or heparin 3 hours after the endotoxin injection inhibited the endotoxin-induced decreases in the platelet count and plasma fibrinogen level and also inhibited the endotoxin-induced increase in glomerular fibrin deposition in the kidney. Differences between danaparoid and heparin were observed in their effects on the plasma AT-III level and clotting time. Danaparoid significantly inhibited both the decrease in the plasma AT-III level and the prolongation of the prothrombin time induced by endotoxin, where as heparin showed no effect on those responses. Moreover, danaparoid enhanced the prolongation of the activated partial thromboplast in time induced by endotoxin to a lesser degree than heparin. These findings suggest that the effects of danaparoid on the endotoxin-induced decrease of the plasma AT-III level and the prolongation of the clotting time are more advantageous than those of heparin. The results may have been due to a higher anti-Xa/anti-thrombin ratio of danaparoid than that of heparin, indicating that danaparoid may be useful in the treatment of DIC.

Recently, the cell biology field has come to appreciate the dissimilarity between two- (2D) and three-dimensional (3D) environments in which cells routinely operate in vivo. Efficacy of most anti-cancer drugs have been evaluated in 2D environment but it must be desirable to evaluate it in 3D environment. We developed a novel 3D cell culture system named Cell-ableTM Oncology utilizing photo-sensitive materials that change to hydro-gel after UV irradiation and the optimized molecular design not to allow the cells adhere to the hydro-gel. In multi-well plates, the Cell-ableTM Oncology has type I collagen-coated cell-attaching areas on the each well surface, which are circle in 100 μm diameter and arrayed in every direction at 100 μm interval. Most cancer cells are able to attach these circles and grow to form spheroids of uniform size on the Cell-ableTM Oncology. In this study, we investigated the stem-like cell phenotypes of cancer spheroids formed on the Cell-ableTM Oncology in comparison with that of the 2D monolayer cells. Various cancer cell lines and patient-derived primary cancer cells proliferated and formed spheroids on the Cell-ableTM Oncology in serum-containing or serum-free media. In contrast to the 2D monolayer cells, the inside of the spheroids formed on the Cell-ableTM Oncology was hypoxic condition like in vivo tumor tissues in patients. The spheroids grown on the Cell-ableTM Oncology showed chemo-resistance against conventional chemotherapy, gemcitabine and paclitaxel compared with cells grown on collagen-coated 2D plates. The chemo-resistance of the spheroid cells was associated with high population of stem-kike cells (CD44high/CD24low) and high expression of mRNA for a stem cell marker, NANOG and an ABC transporter ABCG2. The combination of the Cell-ableTM Oncology and serum-free media showed the highest population of stem-like cells. These results indicate that the spheroids characterized by 3D structure formed on the ECM-coated surface and the hypoxic condition confer enriched the cancer stem-like cells. Thus, the spheroids on the Cell-ableTM Oncology could be suitable for research and development of drugs targeting cancer stem cells. The pharmacodynamic and pharmacokinetic studies using the cancer stem cell-rich spheroids could provide meaningful information relevant to clinical conditions. Citation Format: Koichi Yokota, Tomoko Jomura, Emiko Ozeki, Takeshi Ikeya. Stem-like cell characteristics of cancer spheroids grown in a microfabricated cell array three-dimensional culture system Cell-ableTM Oncology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3838. doi:10.1158/1538-7445.AM2013-3838

In this study, we examined the relative amounts of mRNA expressed in normal versus psoriatic epidermis, using in situ hybridization with a biotinylated oligonucleotide poly d(T) probe. The hybridization image was analyzed by Laser Scanning Confocal Microscopy (LSCM). In normal human skin, hybridization signals were detected homogeneously throughout the epidermis, mostly in nucleus. These signals disappeared following RNase T2 or RNase A treatment, indicating that the target for this probe is RNA; by implication, mRNA. In psoriatic lesions, the overall signal intensity was significantly elevated, especially in the basal/suprabasal layers. Moreover, those signals were most prominent in the cytoplasm, not in the nucleus. In contrast, the signals of uninvolved epidermis adjacent to the psoriatic lesions were indistinguishable from those of normal skin in both signal intensity and hybridization profile. Our data are consistent with the notion that one of the characteristic features of psoriasis is an elevated (or uncontrolled) synthesis of mRNA and proteins.