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Background: While benefit-risk (B-R) assessment in the real-world setting is an important challenge for pharmacovigilance, few studies have explored this approach. To investigate the utility and limitations of B-R assessment using a health care database by applying the Benefit Risk Action Team (BRAT) framework, we have conducted a case study with erythropoietin agents., Methods: Postmarketing data from the Medical Data Vision health care database were used in a B-R comparison between methoxy polyethylene glycol-epoetin beta (continuous erythropoietin receptor activator; C.E.R.A.) and other erythropoiesis-stimulating agents (ESAs). Data were from patients with chronic kidney disease (CKD) treated with C.E.R.A. (n = 131: nondialysis, 109; hemodialysis, 22) or other ESAs (n = 542: nondialysis, 327; hemodialysis, 215) between July 2011 and March 2014., Results: The B-R profile for C.E.R.A. appeared to be similar to that for other ESAs in both nondialysis and hemodialysis patients with CKD, when benefits and risks were mainly assessed in terms of odds ratios. Despite various point estimates and confidence intervals for each outcome, the results of subgroup analyses showed no notable differences from the overall analysis in B-R assessment., Conclusions: B-R assessment can be performed using the BRAT framework with a health care database, but limitations exist when using a single data source. Care should be taken when selecting data for extraction and defining outcomes of interest. Further research is necessary to facilitate practical application of this approach.

This paper investigates dynamics of a management system for controlling a pair of energy storages. The system involves the following two characteristics: each storage behaves in a manner that reduces the number of charge noncharge cycles and begins to be charged when the price of power is lower than a particular price threshold. The price is proportional to the past total power flow from a power grid to all storages. A peak of the total power flow occurs when these storages are charged simultaneously. From the viewpoint of nonlinear dynamics, the energy storages can be considered as relaxation oscillators coupled by a delay connection. Our analytical results suggest that the peak can be reduced by inducing an antiphase synchronization in coupled oscillators. We confirm these analytical results through numerical simulations. In addition, we numerically investigate the dynamical behavior in 10 storages and find that time delay in the connection is important in reducing the peak.

Metastasis to the skeletal muscle from gastric cancer is relatively rare. We report cases of 3 patients undergoing chemotherapy for gastric cancer with metastasis to the skeletal muscle. Case 1: A man in his 70s was diagnosed with advanced gastric cancer (cT4N3M1P0, stage IV), with metastasis to the lung, brain, lymph node, and iliopsoas muscle. Case 2: A man in his 60s was diagnosed with advanced gastric cancer (cT3N3M1P0, stage IV), with metastasis to the brain, lung, lymph node, and iliopsoas muscle. Case 3: A man in his 50s was diagnosed with advanced gastric cancer (cT4N3M1P0, stage IV), with metastasis to the urinary duct, lymph node, back muscle, and iliopsoas muscle. All 3 patients died within 7-8 months after the diagnosis due to progressive disease despite chemotherapy. The prognosis of these 3 patients was significantly poorer than that of patients in our hospital with metastasis not involving the skeletal muscle (p<0.01). Accordingly, metastasis to the skeletal muscle may be an adverse prognostic factor in gastric cancer.

We present a procedure to systematically design the connection parameters that will induce amplitude death in oscillator networks with time-varying delay connections. The parameters designed by the procedure are valid in oscillator networks with any network topology and with any connection delay. The validity of the design procedure is confirmed by numerical simulation. We also consider a partial time-varying delay connection, which has both time-invariant and time-varying delays. The effectiveness of the partial connection is shown theoretically and numerically.

Hearing loss is the most widespread sensory disorder, with an incidence of congenital genetic deafness of 1 in 1600 children. For many ethnic populations, the most prevalent form of genetic deafness is caused by recessive mutations in the gene gap junction protein, beta 2, 26 kDa (GJB2), which is also known as connexin 26 (Cx26). Despite this knowledge, existing treatment strategies do not completely recover speech perception. Here we used a gene delivery system to rescue hearing in a mouse model of Gjb2 deletion. Mice lacking Cx26 are characterized by profound deafness from birth and improper development of cochlear cells. Cochlear delivery of Gjb2 using an adeno-associated virus significantly improved the auditory responses and development of the cochlear structure. Using gene replacement to restore hearing in a new mouse model of Gjb2-related deafness may lead to the development of therapies for human hereditary deafness., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The present paper deals with the dynamics of bus networks, which consist of several identical dc bus systems connected by resistors. It is analytically guaranteed that the stability of a stand-alone dc bus system is equivalent to that of the networks, independent of the number of bus systems and the network topology. In addition, we show that a decentralized delayed-feedback control can stabilize an unstable operating point embedded within the networks. Moreover, this stabilization does not depend on the number of bus systems or the network topology. A systematic procedure for designing the controller is presented. Finally, the validity of the analytical results is confirmed through numerical examples.

The present paper studies time-delayed-connection induced amplitude death in high-dimensional oscillator networks. We provide two procedures for design of a coupling strength and a transmission delay: these procedures do not depend on the topology of oscillator networks (i.e., network structure and number of oscillators). A graphical procedure based on the Nyquist criterion is proposed and then is numerically confirmed for the case of five-dimensional oscillators, called generalized Rössler oscillators, which have two pairs of complex conjugate unstable roots. In addition, for the case of high-dimensional oscillators having two unstable roots, the procedure can be systematically carried out using only a simple algebraic calculation. This systematic procedure is numerically confirmed for the case of three-dimensional oscillators, called Moore-Spiegel oscillators, which have two positive real unstable roots.

The benefits and challenges of incorporating biomarkers into the development of anticancer agents have been increasingly discussed. In many cases, a sensitive subpopulation of patients is determined based on preclinical data and/or by retrospectively analyzing clinical trial data. Prospective exploration of sensitive subpopulations of patients may enable us to efficiently develop definitively effective treatments, resulting in accelerated drug development and a reduction in development costs. We consider the development of a new molecular-targeted treatment in cancer patients. Given preliminary but promising efficacy data observed in a phase I study, it may be worth designing a phase II clinical trial that aims to identify a sensitive subpopulation. In order to achieve this goal, we propose a Bayesian randomized phase II clinical trial design incorporating a biomarker that is measured on a graded scale. We compare two Bayesian methods, one based on subgroup analysis and the other on a regression model, to analyze a time-to-event endpoint such as progression-free survival (PFS) time. The two methods basically estimate Bayesian posterior probabilities of PFS hazard ratios in biomarker subgroups. Extensive simulation studies evaluate these methods' operating characteristics, including the correct identification probabilities of the desired subpopulation under a wide range of clinical scenarios. We also examine the impact of subgroup population proportions on the methods' operating characteristics. Although both methods' performance depends on the distribution of treatment effect and the population proportions across patient subgroups, the regression-based method shows more favorable operating characteristics., (Copyright © 2014 John Wiley & Sons, Ltd.)

Brn4, which encodes a POU transcription factor, is the gene responsible for DFN3, an X chromosome-linked, non-syndromic type of hearing loss. Brn4-deficient mice have a low endocochlear potential (EP), hearing loss, and ultrastructural alterations in spiral ligament fibrocytes, however the molecular pathology through which Brn4 deficiency causes low EP is still unclear. Mutations in the Gjb2 and Gjb6 genes encoding the gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) genes, respectively, which encode gap junction proteins and are expressed in cochlear fibrocytes and non-sensory epithelial cells (i.e., cochlear supporting cells) to maintain the proper EP, are responsible for hereditary sensorineural deafness. It has been hypothesized that the gap junction in the cochlea provides an intercellular passage by which K+ is transported to maintain the EP at the high level necessary for sensory hair cell excitation. Here we analyzed the formation of gap junction plaques in cochlear supporting cells of Brn4-deficient mice at different stages by confocal microscopy and three-dimensional graphic reconstructions. Gap junctions from control mice, which are composed mainly of Cx26 and Cx30, formed linear plaques along the cell-cell junction sites with adjacent cells. These plaques formed pentagonal or hexagonal outlines of the normal inner sulcus cells and border cells. Gap junction plaques in Brn4-deficient mice did not, however, show the normal linear structure but instead formed small spots around the cell-cell junction sites. Gap junction lengths were significantly shorter, and the level of Cx26 and Cx30 was significantly reduced in Brn4-deficient mice compared with littermate controls. Thus the Brn4 mutation affected the assembly and localization of gap junction proteins at the cell borders of cochlear supporting cells, suggesting that Brn4 substantially contributes to cochlear gap junction properties to maintain the proper EP in cochleae, similar to connexin-related deafness.

Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.

This paper tackles a destabilizing problem of a direct-current (dc) bus system with constant power loads, which can be considered a fundamental problem of dc power grid networks. The present paper clarifies scenarios of the destabilization and applies the well-known delayed-feedback control to the stabilization of the destabilized bus system on the basis of nonlinear science. Further, we propose a systematic procedure for designing the delayed feedback controller. This controller can converge the bus voltage exactly on an unstable operating point without accurate information and can track it using tiny control energy even when a system parameter, such as the power consumption of the load, is slowly varied. These features demonstrate that delayed feedback control can be considered a strong candidate for solving the destabilizing problem.

Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyper-stabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs.

Nephron morphogenesis is a complex process that generates blood-filtration units (glomeruli) connected to extremely long and patterned tubular structures. Hepatocyte nuclear factor 1β (HNF1β) is a divergent homeobox transcription factor that is expressed in kidney from the first steps of nephrogenesis. Mutations in HNF1B (OMIM #137920) are frequently found in patients with developmental renal pathologies, the mechanisms of which have not been completely elucidated. Here we show that inactivation of Hnf1b in the murine metanephric mesenchyme leads to a drastic tubular defect characterized by the absence of proximal, distal and Henle's loop segments. Nephrons were eventually characterized by glomeruli, with a dilated urinary space, directly connected to collecting ducts via a primitive and short tubule. In the absence of HNF1β early nephron precursors gave rise to deformed S-shaped bodies characterized by the absence of the typical bulge of epithelial cells at the bend between the mid and lower segments. The lack of this bulge eventually led to the absence of proximal tubules and Henle's loops. The expression of several genes, including Irx1, Osr2 and Pou3f3, was downregulated in the S-shaped bodies. We also observed decreased expression of Dll1 and the consequent defective activation of Notch in the prospective tubular compartment of comma- and S-shaped bodies. Our results reveal a novel hierarchical relationship between HNF1β and key genes involved in renal development. In addition, these studies define a novel structural and functional component of S-shaped bodies at the origin of tubule formation.

The bound state of water in PEG (polyethylene glycol)-H(2)O systems was studied by using HFS and DSC methods. Samples with the mixing ratios EO:H(2)O = 1:1 (a) and 1:10 (b), where EO indicates the ethylene oxide units in PEG chain, were heated from frozen state to melt state. Sample (a) gave an HFS peak corresponding to the weakly bound water, while sample (b) gave the peaks of both the bound and free water. DSC measurements gave quite similar patterns to those of HFS measurements, but with additional peaks corresponding to cold crystallization. The difference between the two measurements was reasonably interpreted by considering the origin of the cold crystallization.

In the mammalian cochlea, both the sensory cells-called hair cells (HCs)-and nonsensory cells such as supporting cells (SCs) and mesenchymal cells participate in proper auditory function through the expression of various functional molecules. During development, expression of certain genes is repressed through genomic methylation, one of the major epigenetic regulatory mechanisms. We explored the genomic regions that were differentially methylated in rat auditory epithelium at postnatal day 1 (P1) and P14 using amplification of intermethylated sites (AIMS). An AIMS fragment was mapped to the 3'-flanking region of Pou3f3/Brn-1. Bisulfite-converted PCR and quantitative methylation-specific PCR showed that the methylation frequency of the AIMS region and the adjacent CpG island was increased at P14, when the expression of Pou3f3 and the noncoding RNAs nearby decreased. Expression of de novo DNA methyltransferases 3a and 3b also suggests a role of epigenetic regulation during postnatal inner ear development. Immunohistochemical analysis showed that Pou3f3 was expressed specifically in the SCs and mesenchymal cells in the cochlea and established that Pou3f3 is a new cell-type marker for studying inner ear development. Mice deficient in Pou3f3 or Pou3f2 plus Pou3f3 did not exhibit any abnormality in the embryonic cochlea. Absence of Pou3f3 affected neither the proliferation nor the differentiation activities of HC progenitor cells. Pou3f3 may, however, be important for the maintenance or functional development of the postnatal cochlea. This is the first report to study involvement of an epigenetic regulatory mechanism in the developing mammalian auditory epithelium.

Invasive fungal infection (IFI) caused by Lecythophora mutabilis occasionally occurs in patients with impaired host immunity; such patients had eosinophilia at onset, and surviving patients were treated with fungal cell-membrane-targeted drugs. An 18-year-old man with mitochondrial encephalomyopathy accompanied with refractory anaemia and chronic renal failure developed septic shock caused by L. mutabilis, which was detected from a blood culture, and was identified morphologically and genetically. During the course of the infection, he had eosinophilia, although beta-d-glucan levels were within the normal range. He was treated with micafungin, but deteriorated and died, despite his treatment being changed to liposomal amphotericin B. On the basis of this we suggest that IFI caused by L. mutabilis should be suspected when a compromised host develops infection and eosinophilia, and that antifungal drugs that target beta-d-glucan are not advisable.

The neocortex and the hippocampus comprise several specific layers containing distinct neurons that originate from progenitors at specific development times, under the control of an adequate cell-division patterning mechanism. Although many molecules are known to regulate this cell-division patterning process, its details are not well understood. Here, we show that, in the developing cerebral cortex, the RP58 transcription repressor protein was expressed both in postmitotic glutamatergic projection neurons and in their progenitor cells, but not in GABAergic interneurons. Targeted deletion of the RP58 gene led to dysplasia of the neocortex and of the hippocampus, reduction of the number of mature cortical neurons, and defects of laminar organization, which reflect abnormal neuronal migration within the cortical plate. We demonstrate an impairment of the cell-division patterning during the late embryonic stage and an enhancement of apoptosis of the postmitotic neurons in the RP58-deficient cortex. These results suggest that RP58 controls cell division of progenitor cells and regulates the survival of postmitotic cortical neurons.

Brain-type fatty acid-binding protein (B-FABP) belongs to a family of intracellular lipid-binding proteins. B-FABP exhibits a binding affinity to long-chain fatty acids (FAs) whose effects on brain functions including development, emotion, learning and memory have been proposed. B-FABP is localized in the ventricular germinal cells in embryonic brain and astrocytes in developing and mature brain of rodents. In the present study we generated the mouse harboring a null mutation in the B-FABP gene and studied its phenotype. B-FABP mutant mice exhibited the enhanced anxiety and increased fear memory as well as the decreased content of docosahexaenoic acid (DHA) in their brain during the neonatal period without detection of any histological changes in the brain. In the adult brain, B-FABP was localized more numerously to the astrocytes in the amygdala and septal area than to those in the hippocampal area. Analysis of FA content in the amygdala of adult brain revealed that arachidonic and palmitic acids increased significantly in the mutant mice compared with wild-type. Furthermore, the response of N-methyl-d-aspartate receptor-mediated current to DHA in isolated neurons from B-FABP mutant brain was significantly decreased compared with that of wild-type, while no significant differences were detected in behavioral responses related to the spatial learning/memory or in the hippocampal long-term potentiation. These data indicate that B-FABP is crucially involved in the fear memory and anxiety through its binding with FAs and/or its own direct effects on pertinent metabolism/signaling of FAs.

Epithelial cysts are one of the fundamental architectures for mammalian organogenesis. Although in vitro studies using cultured epithelial cells have revealed proteins required for cyst formation, the mechanisms that orchestrate the functions of these proteins in vivo remain to be clarified. We show that the targeted disruption of the mouse Par3 gene results in midgestational embryonic lethality with defective epicardial development. The epicardium is mainly derived from epicardial cysts and essential for cardiomyocyte proliferation during cardiac morphogenesis. PAR3-deficient epicardial progenitor (EPP) cells do not form cell cysts and show defects in the establishment of apical cortical domains, but not in basolateral domains. In PAR3-deficient EPP cells, the localizations of aPKC, PAR6beta and ezrin to the apical cortical domains are disturbed. By contrast, ZO1 and alpha4/beta1 integrins normally localize to cell-cell junctions and basal domains, respectively. Our observations indicate that EPP cell cyst formation requires PAR3 to interpret the polarity cues from cell-cell and cell-extracellular matrix interactions so that each EPP cell establishes apical cortical domains. These results also provide a clear example of the proper organization of epithelial tissues through the regulation of individual cell polarity.

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.

This study identifies a role for the gene for the POU transcription factor Brn1 in distal tubule formation and function in the mammalian kidney. Normal development of Henle's loop (HL), the distal convoluted tubule and the macula densa was severely retarded in Brn1-deficient mice. In particular, elongation and differentiation of the developing HL was affected. In the adult kidney, Brn1 was detected only in the thick ascending limb (TAL) of HL. In addition, the expression of a number of TAL-specific genes was reduced in the Brn1+/- kidney, including Umod, Nkcc2/Slc12a1, Bsnd, Kcnj1 and Ptger3. These results suggest that Brn1 is essential for both the development and function of the nephron in the kidney.

High-frequency spectroscopy has been applied to monitoring the stability of oil-in-water emulsions. It was found that stable emulsions showed absorption peaks at around 550 MHz; further, they shifted from lower to higher frequencies with time. The rate of increase of the frequencies was dependent on the amount of emulsifier added. These results show that the stability of emulsions can be monitored by the amount of the frequency shift.

The acrosome is a unique organelle that plays an important role at the site of sperm-zona pellucida binding during the fertilization process, and is lost in globozoospermia, an inherited infertility syndrome in humans. Although the acrosome is known to be derived from the Golgi apparatus, molecular mechanisms underlying acrosome formation are largely unknown. Here we show that Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), a recently identified Golgi-associated protein, is predominantly localized at the trans-Golgi region in round spermatids, and male mice in which GOPC has been disrupted are infertile with globozoospermia. The primary defect was the fragmentation of acrosomes in early round spermatids, and abnormal vesicles that failed to fuse to developing acrosomes were apparent. In later stages, nuclear malformation and an abnormal arrangement of mitochondria, which are also characteristic features of human globozoospermia, were observed. Interestingly, intracytoplasmic sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes were activated. Thus, GOPC provides important clues to understanding the mechanisms underlying spermatogenesis, and the GOPC-deficient mouse may be a unique and valuable model for human globozoospermia.

Formation of highly organized neocortical structure depends on the production and correct placement of the appropriate number and types of neurons. POU homeodomain proteins Brn-1 and Brn-2 are coexpressed in the developing neocortex, both in the late precursor cells and in the migrating neurons. Here we show that double disruption of both Brn-1 and Brn-2 genes in mice leads to abnormal formation of the neocortex with dramatically reduced production of layer IV-II neurons and defective migration of neurons unable to express mDab1. These data indicate that Brn-1 and Brn-2 share roles in the production and positioning of neocortical neuron development.

Measurements of DSC, NMR and high-frequency spectroscopy have been done on PEO-H2O and PEO-H2O-HQ mixed systems. DSC measurements show that water molecules contained in PEO are divided roughly into two species: freezable water and non-freezable water. By 1H-NMR measurements, PEO and H2O protons show an absorption peak around 3.6 ppm and 4.5 ppm, respectively. All the PEO-H2O samples containing H2O show NMR absorption signals, suggesting the existence of movable H2O. Samples with H2O molar ratio over 0.5, show one absorption peak of H2O protons, and more than two peaks of PEO. The results from DSC and NMR measurements show that the bound state of H2O in PEO, as well as the mobility of PEO itself, varies according to the water content in PEO-H2O samples. On the other hand, DSC measurements give the result that PEO-H2O-HQ systems can be considered as homogeneous so long as the content of H2O and/or HQ is not so high. Furthermore, the water molecules in the system exist as bound water. Results of high-frequency spectroscopic measurements for the PEO-H2O and PEO-H2O-HQ systems show good agreement with those of DSC and NMR measurements.

The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1-deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1-deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4-positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (approximately 600 D) toward the skin surface, whereas in the claudin-1-deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.

We have generated mutant mice for epidermal-type fatty acid binding protein by the gene targeting technique and examined the phenotype in detail. Despite a lack in the expression of epidermal-type fatty acid binding protein mRNA and its protein in the skin and other tissues of the mutant mice, the animals appeared normal in gross and histologic examination. Northern blot analysis of other fatty acid binding proteins revealed a distinct elevated gene expression of heart-type fatty acid binding protein in the skin of the homozygous mice. In analyses of the skin, no differences were observed in contents of major fatty acids, electron microscopic appearance as well as inflammatory responses in ear skin between the mutant and wild-type mice. Basal transepidermal water loss of homozygous mice was lower than that of the wild mice. When acetone was applied to the skin for disruption of the water permeability barrier, recovery in transepidermal water loss was delayed, although maximum transepidermal water loss upon acetone treatment was similar between homozygous and wild-type mice in terms of size and time course. The molecular mechanism by which epidermal-type fatty acid binding protein contributes to the water barrier function of the skin remains to be elucidated.

Apc is a gene associated with familial adenomatous polyposis coli (FAP) and its inactivation is a critical step in colorectal tumor formation. The protein product, adenomatous polyposis coli (APC), acts to down-regulate intracellular levels of beta-catenin, a key signal transducer in the Wnt signaling. Conditional targeting of Apc in the neural crest of mice caused massive apoptosis of cephalic and cardiac neural crest cells at about 11.5 days post coitum, resulting in craniofacial and cardiac anomalies at birth. Notably, the apoptotic cells localized in the regions where beta-catenin had accumulated. In contrast to its role in colorectal epithelial cells, inactivation of APC leads to dysregulation of beta-catenin/Wnt signaling with resultant apoptosis in certain tissues including neural crest cells.

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1(+/-)) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5-11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1(+/-) mice was apparently slower than that in Tsc2(+/-) mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.

Background: NK homeobox genes have been shown to play important roles in cell-type specification and organogenesis. Murine Bapx1, a member of NK homeobox gene family, is expressed in all the cartilageous tissues that undergo endochondral bone formation, and in gut mesentery during embryogenesis, suggesting that Bapx1 may be a key transcription factor ragulating the development of these organs., Results: We generated Bapx1-deficient mice by gene targeting. Bapx1-/- mice exhibited lethal skeletal dysplasia, with abnormal development of the vertebral column and some craniofacial bones, accompanied with asplenia and gastroduodenal malformation. We showed that the proliferative activity of the sclerotome cells, forming the vertebral column, was significantly reduced in Bapx1-/- embryos. The sclerotome cells of the mutants appeared to migrate and condense normally, but subsequent differentiation into the mature vertebral bodies and intervertebral discs were affected. The sclerotome cells in the vertebral bodies failed to differentiate into hypertrophic chondrocytes, as revealed by the undetected expression of Col10a1 and Osteopontin, and the sclerotome cells in the intervertebral discs failed to express the typical extracellular matrix proteins Col2a1, Col9a2 and aggrecan. Furthermore, we investigated the effect of loss of Bapx1 on the expression of some transcription factors, identified to be expressed in the developing sclerotome and be required for normal development of the vertebral column. Among them, we found that the expression of MFH-1 (mesenchyme forkhead-1), which was reported to regulate the proliferation and differentiation of sclerotome cells, was significantly reduced in ventromedial sclerotome cells in Bapx1-/- mice., Conclusion: Our analysis provided evidence that Bapx1 was indispensable for normal development of ventromedial structure of vertebral column and some of craniofacial bones, splenogenesis and morphogenesis of gastroduodenal tract.

Twenty post-stroke depressive patients who obtained more than 11 points on Self-Rating Questionnaire for Depression, were treated with 0.075 mg/day lisuride maleate for 12 weeks. The drug effect on depression was evaluated quantitatively by the Hamilton Rating Scale for Depression. The relationships between brain CT or MRI and SRQ-D score were investigated in 24 subjects. More than 80% of post-stroke depressive patients improved after lisuride maleate treatment for 8 or 12 weeks. In particular, depressed mood, hypobulia, sleep disturbance, anxiety, etc. were significantly improved compared to the baseline condition. As for the relationships with CT and/or MRI findings, the group with moderate to severe brain atrophy had a significantly higher grade of depressive state than those without.

This study was conducted to examine the effect of the intramuscular injection of levallorphan tartrate (1.0 mg), a mixed agonist-antagonist opiate, on the neurological signs, symptoms, and vital signs in 19 patients with acute ischemic stroke. A temporary improvement of hemiplegia or hemiparesis was observed within several minutes after levallorphan injection in 13 of the patients. There were no significant alterations in blood pressure or pulse rate after injection. The findings indicate that levallorphan may have a temporary improving effect on neurological deficits in acute ischemic stroke. In addition, observation of the response to levallorphan may serve to predict the prognosis of the final neurological outcome in this type of patient.