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Original Articles, 定型精神病は、満田が提唱した疾患概念であり、急性に発症し、挿間性ないし周期性の経過をとる予後が良好なものである。こういった特徴は、統合失調症の経過や病態とは異なっており、また満田は臨床遺伝学的にも両疾患が異なったものであるとしている。今回、非定型精神病患者に HTPP テストを行う機会を得た。従来、統合失調症患者の HTPP テストの特徴として、「歪んだ印象」の描画が顕著に目立つこと、また、形式分析及び内容分析の中で統合失調症サインと考えられる特徴の約半数が、正常者と比較して有意に多く出現すること等が報告されている。今回得られた結果を統合失調症の HTPP テストの特徴と比較したところ、「歪んだ印象」はなく、奇妙なところもなかった。また、統合失調症サインの出現頻度は非常に少ないことがわかった。以上の結果から、非定型精神病と統合失調症は心理学的見地からも異なった特性を示す疾患である可能性が示唆された。Atypical psychosis, a disease concept originally proposed by Mitsuda in the 1940s, has a good prognosis that develops acutely and takes an intermittent or periodic course. These characteristics differ from the course and pathology of schizophrenia, and Mitsuda argued that the two diseases have different clinical genetics. In this study, we performed an HTPP test on a patient with atypical psychosis. Conventionally, the HTPP test of patients with schizophrenia is characterized by conspicuous drawings of a “distorted impression” and about half of the features considered to be schizophrenia signs appear significantly more frequently in formal analysis and content analysis than in normal subjects. However, when the HTPP test results from the patient with atypical psychosis were compared with those reported as characteristic of schizophrenia, there was no such “distorted impression” or anything else considered to be notably strange. The frequency of occurrence of signs of schizophrenia according to HTPP test was found to be very low. From a psychological point of view, these results suggest that atypical psychosis and schizophrenia may be diseases with different characteristics.

This study aimed to clarify the treatment experience of patients undergoing negative pressure wound therapy (NPWT).This study used a qualitative design.Seventeen inpatients were semi-structured interviewed about their experiences of treatment with negative pressure wound therapy.Inpatients' answers were categorized into seven themes: pain and discomfort associated with treatment, physical limitations owing to attached device, mental burden owing to the odour and noises of the attached device, social limitations owing to the attached device, advances in medical care and science, device personification and mixed feelings towards medical staff. The patients were able to tolerate the aforementioned limitations while feeling attachment and gratitude towards the device created through advances in medical care and science, and towards medical staff who helped them heal. In the future, we plan to develop an NPWT care guide.

本稿は、保育者養成において、学生がより良い「読み語り」の実践ができるための授業のあり方について検討し、課題を探ることを目的とする。より良い「読み語り」とは、聞き手に様々な空想体験を提供し、読み手との「心地よい交流」を通して、乳幼児の人格形成に関わる豊かな感性を育む土壌づくりになるものである。第一筆者は、保育者が行う「読み語り」が保育者と子ども、子どもと子どもの心を繋ぎ、良好な関係を築くことができるという保育体験から、学生が絵本の魅力や価値について認識を深め、「読み語り」への意識やスキルを高める取り組みが大切と考えた。そこで、2018年度前期に、担当する授業の中に絵本に関する教育プログラムを組み込み、実践を試みた。取り組みの中で、学生が多様なジャンルの絵本と出会い、絵本への興味・関心を高めるとともに「読み語り」のスキルを向上させたいという意欲の高まりが明らかになった。本稿では、絵本に関わる教育プログラムの取り組みについて報告する。

Tendon is a dense connective tissue that transmits high mechanical forces from skeletal muscle to bone. The transcription factor scleraxis (Scx) is a highly specific marker of both precursor and mature tendon cells (tenocytes). Mice lacking scx exhibit a specific and virtually complete loss of tendons during development. However, the functional contribution of Scx to wound healing in adult tendon has not yet been fully characterized. Here, using ScxGFP-tracking and loss-of-function systems, we show in an adult mouse model of Achilles tendon injury that paratenon cells, representing a stem cell antigen-1 (Sca-1)–positive and Scx-negative progenitor subpopulation, display Scx induction, migrate to the wound site, and produce extracellular matrix (ECM) to bridge the defect, whereas resident tenocytes exhibit a delayed response. Scx induction in the progenitors is initiated by transforming growth factor β (TGF-β) signaling. scx-deficient mice had migration of Sca-1–positive progenitor cell to the lesion site but impaired ECM assembly to bridge the defect. Mechanistically, scx-null progenitors displayed higher chondrogenic potential with up-regulation of SRY-box 9 (Sox9) coactivator PPAR-γ coactivator-1α (PGC-1α) in vitro, and knock-in analysis revealed that forced expression of full-length scx significantly inhibited Sox9 expression. Accordingly, scx-null wounds formed cartilage-like tissues that developed ectopic ossification. Our findings indicate a critical role of Scx in a progenitor-cell lineage in wound healing of adult mouse tendon. These progenitor cells could represent targets in strategies to facilitate tendon repair. We propose that this lineage-regulatory mechanism in tissue progenitors could apply to a broader set of tissues or biological systems in the body.

Early detection of extravasation is important, but conventional methods of detection lack objectivity and reliability. This study evaluated the predictive validity of thermography for identifying extravasation during intravenous antineoplastic therapy. Of 257 patients who received chemotherapy through peripheral veins, extravasation was identified in 26. Thermography was performed every 15 to 30 minutes during the infusions. Sensitivity, specificity, positive predictive value, and negative predictive value using thermography were 84.6%, 94.8%, 64.7%, and 98.2%, respectively. This study showed that thermography offers an accurate prediction of extravasation.

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal–regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin–containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp–mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.

HIV-1-specific cytotoxic T-lymphocytes (CTLs) with strong abilities to suppress HIV-1 replication and recognize most circulating HIV-1 strains are candidates for effector T cells for cure treatment and prophylactic AIDS vaccine. Previous studies demonstrated that the existence of CTLs specific for 11 epitopes was significantly associated with good clinical outcomes in Japan, although CTLs specific for one of these epitopes select for escape mutations. However, it remains unknown whether the CTLs specific for the remaining 10 epitopes suppress HIV-1 replication in vitro and recognize circulating HIV-1. Here, we investigated the abilities of these CTLs to suppress HIV-1 replication and to recognize variants in circulating HIV-1. CTL clones specific for 10 epitopes had strong abilities to suppress HIV-1 replication in vitro. The ex vivo and in vitro analyses of T-cell responses to variant epitope peptides showed that the T cells specific for 10 epitopes recognized mutant peptides which are detected in 84.1% to 98.8% of the circulating HIV-1 strains found in HIV-1-infected Japanese individuals. In addition, the T cells specific for 5 epitopes well recognized target cells infected with 7 mutant viruses that had been detected in >5% of tested individuals. Taken together, these results suggest that CTLs specific for the 10 epitopes effectively suppress HIV-1 replication and broadly recognize the circulating HIV-1 strains in the HIV-1-infected individuals. This study suggests the use of these T cells in clinical trials. IMPORTANCE In recent T-cell AIDS vaccine trials, the vaccines did not prevent HIV-1 infection, although HIV-1-specific T cells were induced in the vaccinated individuals, suggesting that the T cells have a weak ability to suppress HIV-1 replication and fail to recognize circulating HIV-1. We previously demonstrated that the T-cell responses to 10 epitopes were significantly associated with good clinical outcome. However, there is no direct evidence that these T cells have strong abilities to suppress HIV-1 replication and recognize circulating HIV-1. Here, we demonstrated that the T cells specific for the 10 epitopes had strong abilities to suppress HIV-1 replication in vitro. Moreover, the T cells cross-recognized most of the circulating HIV-1 in HIV-1-infected individuals. This study suggests the use of T cells specific for these 10 epitopes in clinical trials of T-cell vaccines as a cure treatment.

HIV-1-specific cytotoxic T cells (CTLs) play an important role in the control of HIV-1 subtype B or C infection. However, the role of CTLs in HIV-1 subtype A/E infection still remains unclear. Here we investigated the association of HLA class I alleles with clinical outcomes in treatment-naive Vietnamese infected with subtype A/E virus. We found that HLA-C*12:02 was significantly associated with lower plasma viral loads (pVL) and higher CD4 counts and that the HLA-A*29:01-B*07:05-C*15:05 haplotype was significantly associated with higher pVL and lower CD4 counts than those for individuals without these respective genotypes. Nine Pol and three Nef mutations were associated with at least one HLA allele in the HLA-A*29:01-B*07:05-C*15:05 haplotype, with a strong negative correlation between the number of HLA-associated Pol mutations and CD4 count as well as a positive correlation with pVL for individuals with these HLA alleles. The results suggest that the accumulation of mutations selected by CTLs restricted by these HLA alleles affects HIV control. IMPORTANCE Most previous studies on HLA association with disease progression after HIV-1 infection have been performed on cohorts infected with HIV-1 subtypes B and C, whereas few such population-based studies have been reported for cohorts infected with the Asian subtype A/E virus. In this study, we analyzed the association of HLA class I alleles with clinical outcomes for 536 HIV-1 subtype A/E-infected Vietnamese individuals. We found that HLA-C*12:02 is protective, while the HLA haplotype HLA-A*29:01-B*07:05-C*15:05 is deleterious. The individuals with HIV-1 mutations associated with at least one of the HLA alleles in the deleterious HLA haplotype had higher plasma viral loads and lower CD4 counts than those of individuals without the mutations, suggesting that viral adaptation and escape from HLA-mediated immune control occurred. The present study identifies a protective allele and a deleterious haplotype for HIV-1 subtype A/E infection which are different from those identified for cohorts infected with HIV-1 subtypes B and C.

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to noncancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver matrix stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-β activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-β1 through canonical and noncanonical Smad such as PI3 kinase-mediated pathways. TGF-β1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared with wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrhosis in negatively regulating TGF-β bioavailability, which in turn modulates ECM remodeling and stiffening and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in a broader variety of chronic fibrotic diseases.

Based on the recently developed Gitaigo personality scale (Komatsu, Sakai, Nishioka, & Mukoyama, 2012), we investigated the relationship between perceived personality and leading/following roles in close friend dyads. Primary participants rated their own and one of their close friend's personality with Gitaigo personality scale. They also described who takes the role of leader in the relationship with the friend they rated. When one in the pair is reported as leader, the other is considered as follower. Subsidiary participants who were cited as close friends rated their own personality. Our analysis of the 215 pairs showed that the participants taking the role of follower were rated higher in the traits of Cowardliness and Mildness by the primary participants. Regarding Mildness, this tendency was also clear in subsidiary participants' self-ratings. Primary participants rated the Preciseness and Candidness of their friends lower if their friend was considered a follower. Gitaigo personality scale describes the perceived personality well, at least for several traits.

Biological processes require close cooperation of multiple transcription factors that integrate different signals. Thyroid hormone receptors (TRs) induce Krüppel-like factor 9 (KLF9) to regulate neurogenesis. Here, we show that triiodothyronine (T3) also works through TR to induce KLF9 in HepG2 liver cells, mouse liver, and mouse and human primary hepatocytes and sought to understand TR/KLF9 network function in the hepatocyte lineage and stem cells. Knockdown experiments reveal that KLF9 regulates hundreds of HepG2 target genes and modulates T3 response. Together, T3 and KLF9 target genes influence pathways implicated in stem cell self-renewal and differentiation, including Notch signaling, and we verify that T3 and KLF9 cooperate to regulate key Notch pathway genes and work independently to regulate others. T3 also induces KLF9 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) and this effect persists during differentiation to definitive endoderm and hiPSC-derived hepatocytes. Microarray analysis reveals that T3 regulates hundreds of hESC and hiPSC target genes that cluster into many of the same pathways implicated in TR and KLF9 regulation in HepG2 cells. KLF9 knockdown confirms that TR and KLF9 cooperate to regulate Notch pathway genes in hESC and hiPSC, albeit in a partly cell-specific manner. Broader analysis of T3 responsive hESC/hiPSC genes suggests that TRs regulate multiple early steps in ESC differentiation. We propose that TRs cooperate with KLF9 to regulate hepatocyte proliferation and differentiation and early stages of organogenesis and that TRs exert widespread and important influences on ESC biology. Stem Cells 2015;33:416–428

HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. IMPORTANCE Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro . This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals.

Prion diseases are fatal neurodegenerative disorders characterized by accumulation of PrP Sc , vacuolation of neurons and neuropil, astrocytosis, and microglial activation. Upregulation of gene expressions of innate immunity-related factors, including complement factors and CD14, is observed in the brains of mice infected with prions even in the early stage of infections. When CD14 knockout (CD14 −/− ) mice were infected intracerebrally with the Chandler and Obihiro prion strains, the mice survived longer than wild-type (WT) mice, suggesting that CD14 influences the progression of the prion disease. Immunofluorescence staining that can distinguish normal prion protein from the disease-specific form of prion protein (PrP Sc ) revealed that deposition of PrP Sc was delayed in CD14 −/− mice compared with WT mice by the middle stage of the infection. Immunohistochemical staining with Iba1, a marker for activated microglia, showed an increased microglial activation in prion-infected CD14 −/− mice compared to WT mice. Interestingly, accompanied by the increased microglial activation, anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor β (TGF-β) appeared to be expressed earlier in prion-infected CD14 −/− mice. In contrast, IL-1β expression appeared to be reduced in the CD14 −/− mice in the early stage of infection. Double immunofluorescence staining demonstrated that CD11b- and Iba1-positive microglia mainly produced the anti-inflammatory cytokines, suggesting anti-inflammatory status of microglia in the CD14 −/− mice in the early stage of infection. These results imply that CD14 plays a role in the disease progression by suppressing anti-inflammatory responses in the brain in the early stage of infection.

Based on the recently developed Gitaigo personality scale (Komatsu, Sakai, Nishioka,Mukoyama, 2012), we investigated the relationship between perceived personality and leading/following roles in close friend dyads. Primary participants rated their own and one of their close friend's personality with Gitaigo personality scale. They also described who takes the role of leader in the relationship with the friend they rated. When one in the pair is reported as leader, the other is considered as follower. Subsidiary participants who were cited as close friends rated their own personality. Our analysis of the 215 pairs showed that the participants taking the role of follower were rated higher in the traits of Cowardliness and Mildness by the primary participants. Regarding Mildness, this tendency was also clear in subsidiary participants' self-ratings. Primary participants rated the Preciseness and Candidness of their friends lower if their friend was considered a follower. Gitaigo personality scale describes the perceived personality well, at least for several traits.

Acute liver injury causes massive hepatocyte apoptosis and/or fatal liver damage. Fibronectin, an extracellular matrix glycoprotein, is prominently expressed during adult tissue repair. However, the extent of fibronectin dependence on hepatocyte response to acute liver damage remains to be defined. Because identification of hepatic survival factors is critical for successful therapeutic intervention in liver failure, this relationship has been investigated using a fibronectin-deficient mouse model of acute liver injury. Here, we show that lack of fibronectin induces significantly increased hepatocyte apoptosis, which is accompanied by significant down-regulation of the antiapoptotic protein, B-cell lymphoma—extra large (Bcl-xL). Furthermore, fibronectin deficiency leads to a significantly elevated production of hepatocyte growth factor in hepatic stellate cells postinjury, which, in turn, results in an earlier onset and acceleration of hepatocyte regeneration. Primary hepatocytes on fibronectin are protected from reactive oxygen species–induced cellular damage, retaining the expression of Bcl-xL, whereas those on type I collagen are not. This retained expression of Bcl-xL is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Conclusion: We provide evidence that fibronectin-mediated matrix survival signals for hepatocytes are transduced through the PI3K/Bcl-xL-signaling axis in response to injury. This work defines fibronectin as a novel antiapoptotic factor for hepatocytes after acute liver injury, but demonstrates that fibronectin is not essential for subsequent hepatocyte proliferation. (HEPATOLOGY 2012;56:311–321)

Gitaigo is a subtype of mimetic words (onomatopoeia) in the Japanese language, which can be regarded as words that imitate actions or states. This study develops a personality scale, with six subscales, using 60 gitaigo words as items for rating the personality of the self and others. We asked 1 054 participants to rate their own personality and 905 participants to rate a close friend's personality, using 158 gitaigo words as items to describe personality. We found that a six-factor model, found in our previous study, was also applicable to the present study of ratings of participants' own personality. We also found six groups of words in the ratings of close friends' personality, although the factor structure is slightly different from the self-rating factors. We selected ten words that exhibited high loadings for each of the six factors to develop a personality scale with six subscales showing high reliability. We named those factors: cowardliness, slowness, preciseness, irritableness, candidness, and frivolousness. The average scores for self-ratings were significantly lower for two subscales (preciseness and candidness) and higher for other four subscales compared to the rating of others.

Summary Mechanical forces influence homeostasis in virtually every tissue [1, 2]. Tendon, constantly exposed to variable mechanical force, is an excellent model in which to study the conversion of mechanical stimuli into a biochemical response [3–5]. Here we show in a mouse model of acute tendon injury and in vitro that physical forces regulate the release of active transforming growth factor (TGF)-β from the extracellular matrix (ECM). The quantity of active TGF-β detected in tissue exposed to various levels of tensile loading correlates directly with the extent of physical forces. At physiological levels, mechanical forces maintain, through TGF-β/Smad2/3-mediated signaling, the expression of Scleraxis (Scx), a transcription factor specific for tenocytes and their progenitors. The gradual and temporary loss of tensile loading causes reversible loss of Scx expression, whereas sudden interruption, such as in transection tendon injury, destabilizes the structural organization of the ECM and leads to excessive release of active TGF-β and massive tenocyte death, which can be prevented by the TGF-β type I receptor inhibitor SD208. Our findings demonstrate a critical role for mechanical force in adult tendon homeostasis. Furthermore, this mechanism could translate physical force into biochemical signals in a much broader variety of tissues or systems in the body.

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG1 Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253–262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263–270). Similarly, anti-Lex phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Lex)-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.

The purpose of this study was to investigate the role of the mutant CUGn RNA in the induction of stress in type 1 myotonic dystrophy (DM1) cells and in the stress-mediated inhibition of protein translation in DM1. To achieve our goals, we performed HPLC-based purification of stress granules (SGs), immunoanalysis of SGs with stress markers TIA-1, CUGBP1, and ph-eIF2, site-specific mutagenesis, and examinations of RNA-protein and protein-protein interactions in myoblasts from control and DM1 patients. The cause-and-effect relationships were addressed in stable cells expressing mutant CUG repeats. We found that the mutant CUGn RNA induces formation of SGs through the increase of the double-stranded RNA-dependent protein kinase (PKR) and following inactivation of eIF2α, one of the substrates of PKR. We show that SGs trap mRNA coding for the DNA repair and remodeling factor MRG15 (MORF4L1), translation of which is regulated by CUGBP1. As the result of the trapping, the levels of MRG15 are reduced in DM1 cells and in CUG-expressing cells. These data show that CUG repeats cause stress in DM1 through the PKR-ph-eIF2α pathway inhibiting translation of certain mRNAs, such as MRG15 mRNA. The repression of protein translation by stress might contribute to the progressive muscle loss in DM1.—Huichalaf, C., Sakai, K., Jin, B., Jones, K., Wang, G.-L., Schoser, B., Schneider-Gold, C., Sarkar, P., Pereira-Smith, O. M., Timchenko, N., Lubov, T. Expansion of CUG RNA repeats causes stress and inhibition of translation in myotonic dystrophy 1 (DM1) cells.

The Tn-antigen, GalNAcalpha-Ser/Thr, is a tumour-associated carbohydrate antigen that may provide a sensitive and specific marker for pre-clinical detection of carcinoma and a target for cancer therapies. We recently reported that MLS128 monoclonal antibody treatment significantly inhibited colon and breast cancer cell growth. On the basis of our observations, the present study aimed to produce human anti-Tn-antigen antibodies with specificity similar to that of MLS128 monoclonal antibody, which recognizes a structure of three consecutive Tn-antigens (Tn3). Six phage clones displaying human single-chain variable fragments (scFvs) were isolated from a newly constructed phage library by panning and screening with a synthetic Tn3-peptide. Deduced amino-acid sequences of six anti-Tn3 scFvs exhibited a high degree of homology. Of those, anti-Tn3 4E10 and 4G2 scFv proteins were successfully purified from phage-infected Escherichia coli to near homogeneity. Surface plasmon resonance analyses revealed a K(D) of purified scFv proteins for Tn3 on an order of 10(-7) M, which is high for carbohydrate-specific monovalent antibodies. Further analyses suggested that both scFv proteins also bind to Tn2 and cultured colon and breast cancer cells. These results demonstrated the potential for use of these scFvs in developing antibody therapeutics targeting colon and breast cancer.

We investigated the effects of different essential oils on adipogenesis in rat subcutaneous adipocytes. Subcutaneous preadipocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing insulin, transferrin, fibroblast growth factor, dexamethasone, indomethacin, fetal bovine serum, and an essential oil at 37 degrees C in 5% CO2 to induce their differentiation. After 7 days, the number of viable cells and the amount of triglycerides accumulated in the cells were determined. Differentiation markers such as the enzymatic activity of glycerol-3-phosphate dehydrogenase (GPDH) and the expression of GPDH and peroxisome proliferator-activated receptor gamma (PPAR gamma) genes were also measured, as well as the intracellular Ca2+ levels. We found that grapefruit oil inhibited the accumulation of triglycerides in a dose-dependent manner at concentrations of 50 to 400 microg/mL. Furthermore, it suppressed the expression of GPDH and caused a 70% decrease in the enzymatic activity of GPDH at a concentration of 50 microg/mL. Grapefruit oil also caused a nearly 2-fold increase in the intracellular concentration of Ca2+ and suppressed the expression of PPAR gamma genes. Our results demonstrate that grapefruit oil efficiently inhibits adipogenesis in cultured subcutaneous preadipocytes and adipocytes.

Very-low-density lipoprotein receptor (VLDLR) is a multi ligand apolipoprotein E (apoE) receptor and is involved in brain development through Reelin signaling. Different forms of VLDLR can be generated by alternative splicing. VLDLR-I contains all exons. VLDLR-II lacks an O-linked sugar domain encoded by exon 16, while VLDLR-III lacks the third complement-type repeat in the ligand binding domain encoded by exon 4. We quantitatively compared lipoprotein binding to human VLDLR variants and analyzed their mRNA expression in both human cerebellum and mouse brain. VLDLR-III exhibited the highest capacity in binding to apoE enriched β-VLDL in vitro and was more effective in removing apoE containing lipoproteins from the circulation than other variants in vivo. In human cerebellum, the major species was VLDLR-II, while the second most abundant species was a newly identified VLDLR-IV which lacks both exon 4 and 16. VLDLR-I was present at low levels. In adult mice, exon 4 skipping varied between 30 and 47% in different brain regions, while exon 16 skipping ranged by 51–76%. Significantly higher levels of VLDLR proteins were found in mouse cerebellum and cerebral cortex than other regions. The deletions of exon 4 and exon 16 frequently occurred in primary neurons, indicating that newly identified variant VLDLR-IV is abundant in neurons. In contrast, VLDLR mRNA lacking exon 4 was not detectable in primary astrocytes. Such cell type-specific splicing patterns were found in both mouse cerebellum and cerebral cortex. These results suggest that a VLDLR variant lacking the third complement-type repeat is generated by neuron-specific alternative splicing. Such differential splicing may result in different lipid uptake in neurons and astrocytes.

Two-photon absorption (TPA) properties of a series of cationic, linear π-conjugated systems with donor(D)/acceptor(A) substitution have been examined systematically in terms of the type and length of the π-conjugation core and symmetric and asymmetric D/A-arrangement. The TPA spectra of the chromophores measured by the femtosecond open-aperture Z-scan method reveal that the double-bond core results in much more intense TPA than the triple-bond one, with a significant red shift of the TPA peak while the TPA becomes more intense as the chain is extended for the triple-bond core compounds with a little red shift. It is also found that the difference in the D/A-arrangement gives a small difference in the amplitude of the TPA spectrum although the spectral shapes differ considerably.

Differentiation of myocytes is impaired in patients with myotonic dystrophy type 1, DM1. CUG repeat binding protein, CUGBP1, is a key regulator of translation of proteins that are involved in muscle development and differentiation. In this paper, we present evidence that RNA-binding activity of CUGBP1 and its interactions with initiation translation complex eIF2 are differentially regulated during myogenesis by specific phosphorylation and that this regulation is altered in DM1. In normal myoblasts, Akt kinase phosphorylates CUGBP1 at Ser28 and increases interactions of CUGBP1 with cyclin D1 mRNA. During differentiation, CUGBP1 is phosphorylated by cyclinD3-cdk4/6 at Ser302, which increases CUGBP1 binding with p21 and C/EBPbeta mRNAs. While cyclin D3 and cdk4 are elevated in normal myotubes; DM1 differentiating cells do not increase these proteins. In normal myotubes, CUGBP1 interacts with cyclin D3/cdk4/6 and eIF2; however, interactions of CUGBP1 with eIF2 are reduced in DM1 differentiating cells and correlate with impaired muscle differentiation in DM1. Ectopic expression of cyclin D3 in DM1 cells increases the CUGBP1-eIF2 complex, corrects expression of differentiation markers, myogenin and desmin, and enhances fusion of DM1 myoblasts. Thus, normalization of cyclin D3 might be a therapeutic approach to correct differentiation of skeletal muscle in DM1 patients.

This study aimed to examine the goal consciousness scale as lifespan developmental index in young adult workers, and the relationship between work values and the goal consciousness. Survey data from 240 young adult workers were used, two subscales in work values scale and five subscales in goal consciousness scale were confirmed by factor analyses. Analysis of variance (ANOVAs) showed that goal-confidence and time-management were high with high expert-orientation or low work-as-devise-orientation. Thirst for goal was low with low expert-orientation and low work-as-devise-orientation. Anxiety about the future was high with high work-as-devise-orientation in the case of males who have high expert-orientation. Planning was high with low work-as-devise-orientation in the case of male. These results indicated that expert-orientation promotes goal-consciousness, work-as-devise-orientation disturbs it, especially, expert-as-devise-orientation raises anxiety for the future.

To investigate relationships between particle (as a model of aggregates) size in a nanomaterial test suspension and its cytotoxicity, a series of eleven sizes of polystyrene (PS) particles were tested in the cytotoxicity test and the chromosome aberration test by using a Chinese hamster cell line CHL. The PS particles were spheres with defined diameters ranging from 0.1 to 9.2 µm. A series of eight sizes of particles with diameters ranging from 0.92 to 4.45 µm showed stronger cytotoxicity than the others. There was a marked difference in cytotoxicity between the 4.45- and 5.26-µm particles. The 0.92- to 4.45-µm particles did not induce structural chromosome aberrations but induced a high frequency of polyploidy in the chromosome aberration test. The 5.26-µm particles showed very weak induction of polyploidy. The incorporation of the 4.45-µm particles into CHL cells was observed by scanning electron microscopy (SEM). Some cells incorporated more than 10 particles. The semi-quantitative measurement of incorporation of particles into cells was performed by flow cytometry with a parameter of side scattered light (SSC) intensity. It showed that CHL cells preferably incorporated the 4.45-µm particles to the 5.26-µm particles. These findings suggest that CHL cells may have a kind of size-recognition ability and incorporate a particular size of particles. The particles may prevent a normal cytokinesis resulting in polyploidy induction. Nanomaterials also may show size-dependent toxicity. Data on particle (or aggregate) size distribution in the test suspension should be provided to evaluate properly the results of toxicity tests of nanomaterials.

Background Catheter care is considered to be important for prevention of catheter-associated bloodstream infections (CABSIs) although epidemiological evidence is sparse. Objectives To identify problems associated with catheter care and evaluate the effectiveness of nurse-initiated interventions to reduce CABSIs. Design An intervention study with before and after comparison. Settings CABSI surveillance was conducted in a 560-bed acute hospital located in a major urban area in Japan. Participants Patients were enrolled in this study from April 2000 to December 2002 based on the following criteria: (1) adult inpatients; and (2) those in whom central venous lines or Swan-Ganz catheters were inserted for 2 days or longer. Methods In the first year, risk factors for CABSI and problems associated with catheter care were identified by inspection of the infection control nurse (ICN) or four trained link nurses, and the laboratory results. In the subsequent 2 years, the following interventions based on the surveillance results were implemented: (1) enhanced skin preparation by scrubbing with regular bathing soap and tap water; (2) a new method for stabilisation of the catheter inserted into the internal jugular vein, where additional dressing was placed over the sterilised dressing; (3) educating the staff on maximal sterile precautions by teaching staff members at their section meetings and displaying posters; (4) use of a check list and observation of catheter insertion by link nurses to monitor compliance; and (5) selection of a disinfectant that requires shorter contact time and has longer residual effect. Results After these interventions were implemented, the overall bloodstream infection (BSI) rate declined from 4.0/1000 device-days to 1.1/1000 device-days ( p Conclusions We identified four problems—those related to skin preparation, dressing, sterile precautions and disinfectant. We implemented a series of interventions to reduce CABSIs; the overall CABSI rate decreased significantly.

Recombinant antibody consisting of the single-chain variable fragment (scFv) of 1H7 monoclonal antibody against insulin-like growth factor-I receptor (IGF-IR) and human IgG(1) Fc domain, scFv-Fc, has been found to exhibit inhibitory effects on breast cancer growth in vitro and in vivo [Li et al. (2000) Cancer Immunol. Immunother. 49, 243; Sachdev et al. (2003) Cancer Res. 63, 627]. Various types of scFvs from hybridomas producing 1H7 or 3B7 mAb were constructed using conventional phage display technology to further characterize the specificity and affinity of anti-IGF-IR mAbs. Binding studies performed using either phage antibodies or soluble scFv proteins to IGF-IR or insulin receptor (IR) and IGF-IR pre-incubated with mAbs suggested that (i) 1H7 and 3B7 bind to IGF-IR but do not bind to its structurally related IR, (ii) either the VL-VH or VH-VL sequence order does not apparently affect specificity for IGF-IR and (iii) 1H7 and 3B7 bind the independent epitopes, located in or near the N-terminal (440-514) and C-terminal (62-184) domains of the alpha subunit, respectively. This study not only revealed new information on binding regions for two anti-IGF-IR mAbs, but also provided the scFv genes as tools for further manipulation of the affinity or development of new IGF-IR-targeted cancer therapeutics.