Search

We applied microsatellite analysis to prenatal diagnosis of Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive severe muscular dystrophy associated with brain malformations. Recent identification of the FCMD gene locus at 9q31-q33 provided the basis for prenatal diagnosis and carrier detection. We recently developed new microsatellite markers which are closer to the FCMD gene and improved the phenotype probability. Nine fetuses in eight unrelated FCMD families, including a twin pregnancy, were analysed using the newly developed markers. Four fetuses showed over 99% probability of being healthy either as normal homozygote (n = 1) or heterozygote carrier (n = 3) and were born without signs of FCMD. The other five fetuses were diagnosed with a probability of FCMD of 99% or greater; all of the latter parents decided to terminate the pregnancies. Brain malformations characteristic of FCMD in one of the aborted fetuses confirmed the diagnosis of FCMD at 19 weeks of gestation. [ABSTRACT FROM PUBLISHER]

A complete hydatidiform mole coexisting with a fetus is a rare condition. The diagnosis is often difficult because of the morphological similarity to a partial mole, but is crucial to management in the postmolar course. We present a case of molar pregnancy coexisting with a fetus in which DNA polymorphism analysis revealed a different genetic origin for the fetal and molar parts. This is the only known case of a complete mole in a twin pregnancy complicated by pre-eclampsia followed by maternal pulmonary oedema. During follow-up, the patient developed a clinically invasive mole which was successfully treated with chemotherapy. In this case, genetic analysis unequivocally diagnosed a twin pregnancy consisting of a complete hydatidiform mole and a fetus. [ABSTRACT FROM AUTHOR]

Japan, with over 75% forest cover, is one of the most heavily forested countries in the world. Various types of climax forest are distributed according to latitude and altitude. At the same time, human intervention in Japan has historically been intensive, and many forest habitats show the influence of various levels of disturbance. Furthermore, Japanese landscapes are changing rapidly, and a system of efficient monitoring is needed. The aim of this research was to identify major historical trends in Japanese landscape change and to develop a system for identifying and monitoring patterns of landscape change at the national level. To provide a base for comparison, Warmth Index (WI) climatic data was digitalized and utilized to map potential climax vegetation for all of Japan. Extant Land Use Information System (LUIS) data were then modified and digitalized to generate national level Land Use/Land Cover (LU/LC) distribution maps for 1900, 1950 and 1985. In addition, MODIS data for 2001 acquired by the Tokyo University of Information Sciences were utilized for remote LU/LC classification using an unsupervised method on multi-temporal composite data. Eight classification categories were established using the ISODATA (cluster analyses) method; alpine plant communities, evergreen coniferous forest, evergreen broad-leaved forest, deciduous broad-leaved forest, mixed forest, arable land (irrigated rice paddy, non-irrigated, grassland), urban area, river and marsh. The results of the LUIS analyses and MODIS classifications were interpreted in terms of a Landscape Transformation Sere model assuming that under increasing levels of human disturbance the landscape will change through a series of stages. The results showed that overall forest cover in Japan has actually increased over the century covered by the data; from 72.1% in 1900 to 76.9% in 2001. Comparison of the actual vegetation and the potential vegetation as predicted by WI, however, indicated that in many areas the climax vegetation has been replaced by secondary forests such as conifer timber plantations. This trend was especially strong in the warm and mid temperate zones of western Japan. This research also demonstrated that classification of moderate resolution remote sensing data, interpreted within a LTS framework, can be an effective tool for efficient and repeat monitoring of landscape changes at the national level. In the future, the authors plan to continue utilizing this approach to track rapidly occurring changes in Japanese landscapes at the national level.

Pays tribute to Norio Kato, professor emeritus of Nagoya University in Japan, who died on April 5, 2002. Educational background; Contributions of Kato to the study of x-ray diffraction topography; Affiliations of Kato.

Epithelial-mesenchymal transition (EMT) in cancer promotes metastasis and chemotherapy resistance. A subset of triple-negative breast cancer (TNBC) exhibits a mesenchymal gene signature that is associated with poor patient outcomes. We previously identified PTK6 tyrosine kinase as an oncogenic driver of EMT in a subset of TNBC. PTK6 induces EMT by stabilizing SNAIL, a key EMT-initiating transcriptional factor. Inhibition of PTK6 activity reverses mesenchymal features of TNBC cells and suppresses their metastases by promoting SNAIL degradation via a novel mechanism. In the current study, we identify membrane-associated RING-CH2 (MARCH2) as a novel PTK6-regulated E3 ligase that promotes the ubiquitination and degradation of SNAIL protein. The MARCH2 RING domain is critical for SNAIL ubiquitination and subsequent degradation. PTK6 inhibition promotes the interaction of MARCH2 with SNAIL. Overexpression of MARCH2 exhibits tumor suppressive properties and phenocopies the effects of SNAIL downregulation and PTK6 inhibition in TNBC cells, such as inhibition of migration, anoikis resistance, and metastasis. Consistent with this, higher levels of MARCH2 expression in breast and other cancers are associated with better prognosis. We have identified MARCH2 as a novel SNAIL E3 ligase that regulates EMT and metastases of mesenchymal TNBC., Significance: EMT is a process directly linked to drug resistance and metastasis of cancer cells. We identified MARCH2 as a novel regulator of SNAIL, a key EMT driver, that promotes SNAIL ubiquitination and degradation in TNBC cells. MARCH2 is oncogene regulated and inhibits growth and metastasis of TNBC. These insights could contribute to novel strategies to therapeutically target TNBC., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Chromosomal translocation generates the MLL-AF4 fusion gene, which causes acute leukemia of multiple lineages. MLL-AF4 is a strong oncogenic driver that induces leukemia without additional mutations and is the most common cause of pediatric leukemia. However, establishment of a murine disease model via retroviral transduction has been difficult owning to a lack of understanding of its regulatory mechanisms. Here, we show that MLL-AF4 protein is post-transcriptionally regulated by RNA-binding proteins, including those of KHDRBS and IGF2BP families. MLL-AF4 translation is inhibited by ribosomal stalling, which occurs at regulatory sites containing AU-rich sequences recognized by KHDRBSs. Synonymous mutations disrupting the association of KHDRBSs result in proper translation of MLL-AF4 and leukemic transformation. Consequently, the synonymous MLL-AF4 mutant induces leukemia in vivo. Our results reveal that post-transcriptional regulation critically controls the oncogenic activity of MLL-AF4; these findings might be valuable in developing novel therapies via modulation of the activity of RNA-binding proteins., (© 2022. The Author(s).)

Direct cardiac reprogramming, in which fibroblasts are converted into induced cardiomyocytes (iCMs) with cardiogenic transcription factors, may be a promising approach for myocardial regeneration. Here, we present a protocol for cardiac reprogramming using a handmade hydrogel culture system. This system can recapitulate substrate stiffness comparable to that of the native myocardium. This protocol features improved efficiency of cardiac reprogramming by generating threefold more beating iCMs on the Matrigel-based hydrogel culture system compared to that on conventional polystyrene dishes. For complete details on the use and execution of this protocol, please refer to Kurotsu et al. (2020)., Competing Interests: S.K. is an employee of Otsuka Pharmaceutical Co., Ltd. I.H. is an employee of Canon Inc. M.I. holds a patent related to this work: U.S. Patent 9,517,250 entitled “Methods for Generating Cardiomyocytes,” issued on October 19, 2012. Inventors: Deepak Srivastava and Masaki Ieda., (© 2022 The Author(s).)

To identify cytoplasmic male sterility (CMS)-associated genes in tomato, we determined the genome sequences of mitochondria and chloroplasts in three CMS tomato lines derived from independent asymmetric cell fusions, their nuclear and cytoplasmic donors, and male fertile weedy cultivated tomato and wild relatives. The structures of the CMS mitochondrial genomes were highly divergent from those of the nuclear and cytoplasmic donors, and genes of the donors were mixed up in these genomes. On the other hand, the structures of CMS chloroplast genomes were moderately conserved across the donors, but CMS chloroplast genes were unexpectedly likely derived from the nuclear donors. Comparative analysis of the structures and contents of organelle genes and transcriptome analysis identified three genes that were uniquely present in the CMS lines, but not in the donor or fertile lines. RNA-sequencing analysis indicated that these three genes transcriptionally expressed in anther, and identified different RNA editing levels in one gene, orf265, that was partially similar to ATP synthase subunit 8, between fertile and sterile lines. The orf265 was a highly potential candidate for CMS-associated gene. This study suggests that organelle reorganization mechanisms after cell fusion events differ between mitochondria and chloroplasts, and provides insight into the development of new F1 hybrid breeding programs employing the CMS system in tomato., (© 2021. The Author(s).)

Chronic myeloid leukemia (CML) is a form of myeloproliferative neoplasm caused by the oncogenic tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitors have dramatically improved the prognosis of patients with CML, several problems such as resistance and recurrence still exist. Immunological control may contribute to solving these problems, and it is important to understand why CML patients fail to spontaneously develop anti-tumor immunity. Here, we show that differentiation of conventional dendritic cells (cDCs), which are vital for anti-tumor immunity, is restricted from an early stage of hematopoiesis in CML. In addition, we found that monocytes and basophils, which are increased in CML patients, express high levels of PD-L1, an immune checkpoint molecule that inhibits T cell responses. Moreover, RNA-sequencing analysis revealed that basophils express genes related to poor prognosis in CML. Our data suggest that BCR-ABL not only disrupts the "accelerator" (i.e., cDCs) but also applies the "brake" (i.e., monocytes and basophils) of anti-tumor immunity, compromising the defense against CML cells., (© 2021. The Author(s).)

Mechanical stimuli including loading after birth promote bone growth. However, little is known about how mechanical force triggers biochemical signals to regulate bone growth. Here, we identified a periosteal-osteoblast-derived secretory peptide, Osteocrin (OSTN), as a mechanotransducer involved in load-induced long bone growth. OSTN produced by periosteal osteoblasts regulates growth plate growth by enhancing C-type natriuretic peptide (CNP)-dependent proliferation and maturation of chondrocytes, leading to elongation of long bones. Additionally, OSTN cooperates with CNP to regulate bone formation. CNP stimulates osteogenic differentiation of periosteal osteoprogenitors to induce bone formation. OSTN binds to natriuretic peptide receptor 3 (NPR3) in periosteal osteoprogenitors, thereby preventing NPR3-mediated clearance of CNP and consequently facilitating CNP-signal-mediated bone growth. Importantly, physiological loading induces Ostn expression in periosteal osteoblasts by suppressing Forkhead box protein O1 (FoxO1) transcription factor. Thus, this study reveals a crucial role of OSTN as a mechanotransducer converting mechanical loading to CNP-dependent bone formation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background/aim: Patients with attention-deficit hyperactivity disorder (ADHD) manifest symptoms of hyperactivity, impulsivity, and/or inattention. ADHD medications available in Japan are limited compared with those in Western countries. Prescribing status has not been sufficiently evaluated in clinical settings in Japan. This study investigated the current use of ADHD medications and characteristics of patients who received multiple ADHD medications in a clinical setting in Japan., Methods: Study participants were those who visited the Department of Child and Adolescent Psychiatry, Kohnodai Hospital between April 2015 and March 2020. We investigated patients who received osmotic-controlled release oral delivery system methylphenidate, atomoxetine, or guanfacine. A retrospective case-control design was used to evaluate the characteristics of patients who received multiple ADHD medications. Patients who were given three ADHD medications were defined as the case group. Randomly sampled sex- and age-matched patients diagnosed with ADHD were defined as the control group. We compared data for child-to-parent violence, antisocial behavior, suicide attempt or self-harm, abuse history, refusal to attend school, and two psychological rating scales (the ADHD-Rating Scale and Tokyo Autistic Behavior Scale)., Results: Among the 878 patients who were prescribed any ADHD medications, 43 (4.9%) received three ADHD medications. Logistic regression revealed that children with severe ADHD symptoms, autistic characteristics, or tendency of child-to-parent violence were more likely to have been prescribed three medications during their treatment., Conclusions: Our findings suggest the approach to prevent the use of multiple ADHD medications. A prospective study to investigate the causality between prescribing status and clinical characteristics is warranted., Competing Interests: The authors have declared that no competing interests exist.

Direct cardiac reprogramming holds great potential for regenerative medicine. However, it remains inefficient, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo, suggesting that undefined extrinsic factors may regulate cardiac reprogramming. Previous in vitro studies mainly used hard polystyrene dishes, yet the effect of substrate rigidity on cardiac reprogramming remains unclear. Thus, we developed a Matrigel-based hydrogel culture system to determine the roles of matrix stiffness and mechanotransduction in cardiac reprogramming. We found that soft matrix comparable with native myocardium promoted the efficiency and quality of cardiac reprogramming. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and suppression of fibroblast programs, which were activated on rigid substrates. Soft substrate further enhanced cardiac reprogramming with Sendai virus vectors via YAP/TAZ suppression, increasing the reprogramming efficiency up to ∼15%. Thus, mechanotransduction could provide new targets for improving cardiac reprogramming., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Mental health has emerged as an important public health concern in recent years. With a high proportion of children and adolescents affected by mental disorders, it is important to ensure that they are provided with proper care and treatment. With the goal of sharing the activities and good practices on child and adolescent mental health promotion, care, and treatment in Japan and the Philippines, the National Center for Global Health and Medicine conducted a training program on the promotion of mental health focused on treatment and care in Japan and the Philippines in September and November 2019., Key Highlights: The training program comprised of a series of lectures, site visits, and round table discussions in Japan and the Philippines. The lectures and site visits focused on the current situation of child and adolescent psychiatry, diagnosis of childhood mental disorders, abuse, health financing for mental disorders, pharmacotherapy, psychotherapy, and disaster child psychiatry in both countries. Round table discussions provided an opportunity to explore the similarities and differences between the two countries in terms of the themes discussed during the lectures.The training program identified the need to collaborate with other professionals to improve the diagnosis of mental disorders in children and adolescents and to increase the workforce capable of addressing mental health issues among children and adolescents. It also emphasized the importance of cooperation between government efforts during and after disasters to ensure that affected children and their families are provided with the care and support that they need., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

The physical properties of the extracellular matrix (ECM), such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin. Cell growth inhibition by doxorubicin was increased in response to ECM rigidity in a p53-dependent manner. The expression of Rho-associated coiled coil-containing protein kinase (ROCK) 2, which induces the activation of myosin II, was significantly higher when cells were cultured on stiffer ECM substrates. Knockdown of ROCK2 expression or pharmacological inhibition of ROCK decreased doxorubicin-induced p53 activation. Our results suggest that a soft ECM causes downregulation of ROCK2 expression, which drives resistance to chemotherapy by repressing p53 activation., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

MEKK1 (also known as MAP3K1), which plays a major role in MAPK signaling, has been implicated in mechanical processes in cells, such as migration. Here, we identify the actin-binding protein calponin-3 as a new MEKK1 substrate in the signaling that regulates actomyosin-based cellular contractility. MEKK1 colocalizes with calponin-3 at the actin cytoskeleton and phosphorylates it, leading to an increase in the cell-generated traction stress. MEKK1-mediated calponin-3 phosphorylation is attenuated by the inhibition of myosin II activity, the disruption of actin cytoskeletal integrity and adhesion to soft extracellular substrates, whereas it is enhanced upon cell stretching. Our results reveal the importance of the MEKK1-calponin-3 signaling pathway to cell contractility., (© 2016. Published by The Company of Biologists Ltd.)

Background: Antipsychotic drug treatment can potentially lead to adverse events such as leukopenia and neutropenia. Although these events are rare, they represent serious and life-threatening hematological side effects., Case Presentation: We present a case study of a patient with schizoaffective disorder in a 50-year-old woman. We report a case of paliperidone extended-release (ER)-induced leukopenia and neutropenia in a female patient with schizoaffective disorder. Initiating lithium carbonate treatment and decreasing the dose of valproic acid improved the observed leukopenia and neutropenia. This treatment did not influence psychotic symptoms., Conclusion: The combination of paliperidone ER and valproic acid induces increased paliperidone ER plasma levels. Lithium carbonate was successfully used to treat paliperidone ER-induced leukopenia and neutropenia.

Although extracellular matrix (ECM) stiffness is an important aspect of the extracellular microenvironment and is known to direct the lineage specification of stem cells and affect cancer progression, the molecular mechanisms that sense ECM stiffness have not yet been elucidated. In this study, we show that the proline-rich linker (PRL) region of vinculin and the PRL-region-binding protein vinexin are involved in sensing the stiffness of ECM substrates. A rigid substrate increases the level of cytoskeleton-associated vinculin, and the fraction of vinculin stably localizing at focal adhesions (FAs) is larger on rigid ECM than on soft ECM. Mutations in the PRL region or the depletion of vinexin expression impair these responses to ECM stiffness. Furthermore, vinexin depletion impairs the stiffness-dependent regulation of cell migration. These results suggest that the interaction of the PRL region of vinculin with vinexin α plays a crucial role in sensing ECM stiffness and in mechanotransduction.

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

Oncogenic Ras induces cell transformation and promotes an invasive phenotype. The tumor suppressor p53 has a suppressive role in Ras-driven invasion. However, its mechanism remains poorly understood. Here we show that p53 induces activation of the mitochondrial protease high-temperature requirement A2 (HtrA2; also known as Omi) and prevents Ras-driven invasion by modulating the actin cytoskeleton. Oncogenic Ras increases accumulation of p53 in the cytoplasm, which promotes the translocation of p38 mitogen-activated protein kinase (MAPK) into mitochondria and induces phosphorylation of HtrA2/Omi. Concurrently, oncogenic Ras also induces mitochondrial fragmentation, irrespective of p53 expression, causing the release of HtrA2/Omi from mitochondria into the cytosol. Phosphorylated HtrA2/Omi therefore cleaves β-actin and decreases the amount of filamentous actin (F-actin) in the cytosol. This ultimately down-regulates p130 Crk-associated substrate (p130Cas)-mediated lamellipodia formation, countering the invasive phenotype initiated by oncogenic Ras. Our novel findings provide insights into the mechanism by which p53 prevents the malignant progression of transformed cells.

We describe the design and validation of an equibiaxial stretching device in which cells are confined to regions of homogeneous strain. Using this device, we seek to overcome a significant limitation of existing equibiaxial stretching devices, in which strains are not homogeneous over the entire region of cell culture. We cast PDMS in a mold to produce a membrane with a cylindrical wall incorporated in the center, which was used to confine cell monolayers to the central membrane region subjected to homogeneous equibiaxial strain. We demonstrated that the presence of the wall to hold the culture medium did not affect strain homogeneity over the majority of the culture surface and also showed that cells adhered well onto the PDMS membranes. We used our device in cyclic strain experiments and demonstrated strain-dependent changes in extracellular signal-regulated kinase (ERK) and tyrosine phosphorylation upon cell stretching. Furthermore, we examined cell responses to very small magnitudes of strain ranging from 1% to 6% and were able to observe a graduated increase in ERK phosphorylation in response to these strains. Collectively, we were able to study cellular biochemical response with a high degree of accuracy and sensitivity to fine changes in substrate strain. Because we have designed our device along the lines of existing equibiaxial stretching technologies, we believe that our innovations can be incorporated into existing systems. This device would provide a useful addition to the set of tools applied for in vitro studies of cell mechanobiology.

Directional cellular migrations as a chemotactic response to spatially inhomogeneous growth factor stimulation play an important role in establishing physiological mechanisms and pathological events in cells. We developed epidermal growth factor (EGF)-immobilized microbeads by photoreaction and evaluated its local stimulatory effects on the dynamic chemotactic motility of fibroblasts. The local stimulation resulted in global activation of ERK 1/2 and directionality of cellular migration. The cellular migration by stimulation using 3-μm diameter EGF-immobilized microbeads persisted for a longer time, were involved a wider field and their number were further increased with stimulation. This effective technique allows cellular migration and biochemical analyses that will help elucidate the mechanisms involved in signal transduction by spatially inhomogeneous stimulation of the growth factor., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cells sense the rigidity of their substrate; however, little is known about the physical variables that determine their response to this rigidity. Here, we report traction stress measurements carried out using fibroblasts on polyacrylamide gels with Young's moduli ranging from 6 to 110 kPa. We prepared the substrates by employing a modified method that involves N-acryloyl-6-aminocaproic acid (ACA). ACA allows for covalent binding between proteins and elastomers and thus introduces a more stable immobilization of collagen onto the substrate when compared to the conventional method of using sulfo-succinimidyl-6-(4-azido-2-nitrophenyl-amino) hexanoate (sulfo-SANPAH). Cells remove extracellular matrix proteins off the surface of gels coated using sulfo-SANPAH, which corresponds to lower values of traction stress and substrate deformation compared to gels coated using ACA. On soft ACA gels (Young's modulus <20 kPa), cell-exerted substrate deformation remains constant, independent of the substrate Young's modulus. In contrast, on stiff substrates (Young's modulus >20 kPa), traction stress plateaus at a limiting value and the substrate deformation decreases with increasing substrate rigidity. Sustained substrate strain on soft substrates and sustained traction stress on stiff substrates suggest these may be factors governing cellular responses to substrate rigidity., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Recent studies reveal that the mechanical environment influences the behavior and function of various types of cells, including stem cells. However, signaling pathways involved in the mechanical regulation of stem cell properties remain largely unknown. Using polyacrylamide gels with varying Young's moduli as substrates, we demonstrate that mouse embryonic stem cells (mESCs) are induced to differentiate on substrates with defined elasticity, involving the Src-ShcA-MAP kinase pathway. While the dual inhibition of mitogen-activated protein (MAP) kinase and glycogen synthase kinase 3 (GSK3), termed "2i," was reported to sustain the pluripotency of mESCs, we find it to be substrate elasticity dependent. In contrast, Src inhibition in addition to 2i allows mESCs to retain their pluripotency independent of substrate elasticity. The alternative dual inhibition of Src and GSK3 ("alternative 2i") retains the pluripotency and self-renewal of mESCs in vitro and is instrumental in efficiently deriving mESCs from preimplantation mouse embryos. In addition, the transplantation of mESCs, maintained under the alternative 2i condition, to immunodeficient mice leads to the formation of teratomas that include differentiation into three germ layers. Furthermore, mESCs established with alternative 2i contributed to chimeric mice production and transmitted to the germline. These results reveal a role for Src-ShcA-MAP kinase signaling in the mechanical regulation of mESC properties and indicate that alternative 2i is a versatile tool for the maintenance of mESCs in serum-free conditions as well as for the derivation of mESCs., (Copyright © 2012 AlphaMed Press.)

A ubiquitously expressed chaperone, heat shock protein 90 (HSP90) is of considerable interest as an oncology target because tumor cells and oncogenic proteins are acutely dependent on its activity. AT13387 (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, l-lactic acid salt) a novel, high-affinity HSP90 inhibitor, which is currently being clinically tested, has shown activity against a wide array of tumor cell lines, including lung cancer cell lines. This inhibitor has induced the degradation of specific HSP90 client proteins for up to 7 days in tumor cell lines in vitro. The primary driver of cell growth (mutant epidermal growth factor receptors) was particularly sensitive to HSP90 inhibition. The long duration of client protein knockdown and suppression of phospho-signaling seen in vitro after treatment with AT13387 was also apparent in vivo, with client proteins and phospho-signaling suppressed for up to 72 h in xenograft tumors after treatment with a single dose of AT13387. Pharmacokinetic analyses indicated that while AT13387 was rapidly cleared from blood, its retention in tumor xenografts was markedly extended, and it was efficacious in a range of xenograft models. AT13387's long duration of action enabled, in particular, its efficacious once weekly administration in human lung carcinoma xenografts. The use of longer-acting HSP90 inhibitors, such as AT13387, on less frequent dosing regimens has the potential to maintain antitumor efficacy as well as minimize systemic exposure and unwanted effects on normal tissues., (© 2011 Japanese Cancer Association and Astex Pharmaceuticals Ltd.)

Collective motion of cell sheets plays a role not only in development and repair, but also in devastating diseases such as cancer. However, unlike single-cell motility, collective motion of cell sheets involves complex cell-cell communication during migration; therefore, its mechanism is largely unknown. To elucidate propagation of signaling transduced by cell-cell interaction, we designed a hydrogel substrate that can cause local mechanical stretching of cell sheets. Poly (N-isopropyl acrylamide) (PNIPAAm) hydrogel is a temperature-responsive polymer gel whose volume changes isotropically in response to temperature changes below 37 °C. We designed a combined hydrogel substrate consisting of collagen-immobilized PNIPAAm as the local stimulation side and polyacrylamide (PAAm) as the non-stimulation side to assess propagation of mechanical transduction. Mardin-Darby canine kidney (MDCK) cells adhered to the collagen-immobilized PNIPAAm gel increased it area and were flattened as the gel swelled with temperature decrease. E-cadherin in these cells became undetectable in some domains, and actin stress fibers were more clearly observed at the cell base. In contrast, E-cadherin in cells adhered to the collagen-immobilized PAAm side was equally stained as that in cells adhered to the collagen-immobilized PAAm side even after temperature decrease. ERK1/2 MAPK activation of cells on the non-stimulated substrate occurred after partial stretching of the cell sheet suggesting the propagation of signaling. These results indicate that a change in the balance of mechanical tension induced by partial stretching of cell sheets leads to activation and propagation of the cell signaling.

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (-)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A new culture substrate was developed for cells to be equibiaxially stretched using fibronectin (Fn)-immobilized temperature-responsive hydrogel. The cells cultured on the gel substrate were equibiaxially stretched with swelling of the gel, which was accompanied by slight changes of temperature. During gel swelling, changes of cell shape were clearly observed by optical microscopy because of high transparency of the gel. ERK was highly and transiently activated by mechanical stimulation whereas focal adhesion kinase (FAK) was not, indicating that mechanical signals were transduced into biochemical signals in cells. We found that cells formed filopodia-like structures in response to mechanical cues, suggesting that mechanical forces facilitated actin polymerization at the peripheral region. In the cytoplasm, paxillin-containing fibrous structures were formed along actin fibers. These results indicate that we can perform both analysis of intracellular signal transduction and observation of cell shapes at high magnification in our method.

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules with well-defined functions in development, but their expression patterns and functions in many adult tissues are still largely unknown. We have detected substantial levels of the EphB2 and EphB6 receptors and the ephrin-B1 ligand in the adult mouse kidney by RT-PCR amplification. Immunolocalization experiments revealed that EphB2 is localized in the tubules of the inner and outer medulla and EphB6 is in the tubules of the outer medulla and cortex. By contrast, ephrin-B1 was detected in tubules throughout the whole nephron. Consistent with the overlapping expression of the EphB2 receptor and the ephrin-B1 ligand in the medulla, EphB2 is tyrosine-phosphorylated, and therefore activated, in the kidney. In the outer medulla, however, EphB2 signaling may be attenuated by the co-expressed kinase-inactive EphB6 receptor. Interestingly, we found that EphB signaling induces RhoA activation and Rac1 inactivation as well as cell retraction, enlargement of focal adhesions and prominent stress fibers in primary cultures of medullary tubule cells. These results suggest that EphB receptor signaling through Rho family GTPases regulates the cytoarchitecture and spatial organization of the tubule cells in the adult kidney medulla and, therefore, may affect the reabsorption ability of the kidney.

Galactose moiety as the hepatocyte anchorage was covalently coupled with chitosan for the development of synthetic extracellular matrix. Hepatocytes adhesion to galactosylated chitosan (GC)-coated polystyrene (PS) dish became as high as 94.7% after 2 h incubation whereas the hepatocytes adhesion to chitosan-coated PS dish was 69.1%, indication of galactose-specific recognition between GC molecules and asialoglycoprotein receptors of hepatocytes. The DNA synthesis of the hepatocytes adhered to GC-coated dish was increased in the presence of epidermal growth factor (EGF) at low concentration of GC (0.05 microg/ml) whereas the DNA synthesis of the hepatocytes adhered to GC-coated dish was decreased in the presence of EGF at high concentration of GC (5 microg/ml). The spreading shapes of the hepatocytes adhered to the surface in the presence of EGF at low concentration of GC (0.05 microg/ml) were enhanced than in the absence of EGF. The hepatocytes adhered to the surface at high concentration of GC (5 microg/ml) showed round shapes and exhibited many spheroid formation after 24 h in the presence of EGF.

Fetal growth is thought to be independent of the concentration of GH, although circulating levels of GH are high in the human fetus. To elucidate the role of GH in fetal development, levels of GH-binding protein (GHBP) were measured in the serum of nonpregnant and pregnant women and neonates as well as in amniotic fluid obtained at various stages of gestation. Total GHBP (the sum of free GHBP and GHBP bound to GH) is measured by a ligand-mediated immunofunctional assay. GHBP concentrations in adult serum were not changed by pregnancy or the stage of gestation. A significant correlation was observed between the concentration of GHBP in the umbilical artery and vein. No correlations were observed between the GHBP concentration and such measures of fetal growth as fetal weight and fetal age. Although the neonatal concentrations of GHBP were significantly lower than those of pregnant women, no correlation was observed between them. GHBP was also present in the amniotic fluid from early to late gestation at concentrations higher than in the cord serum of the neonate. The amniotic GHBP concentration in late gestation was significantly higher than in early gestation. GHBP appears to be derived from GH receptors of fetal organs (most probably fetal liver). The low level of GHBP in fetal serum may be the result of a decrease in GH receptors caused by high levels of circulating GH. GHBP levels in amniotic fluids may be related to the development or maturation of the fetus.

Raman spectra have been measured of a nonapeptide which has an amino acid sequence identical to that of the C-terminal region of the major coat protein subunit of filamentous bacteriophage Pf3. The peptide shows a strong tendency to form a beta-sheet structure in aqueous solution. The beta-sheet formation is significantly promoted by complexation with single-stranded DNA but not with double-stranded DNA. It is suggested that the C-terminal region of the Pf3 coat protein binds to the single-stranded DNA genome in the virion with a beta-sheet conformation, in sharp contrast with the alpha-helical binding in other filamentous bacteriophages.