Search

Lysine-based amphipathic nonapeptides, including homochiral peptides [Ac-(l-Lys-l-Lys-Xaa)3-NH2 (Xaa=Gly, Ala, Aib, Ac5c, or Ac6c) and Ac-(d-Lys-d-Lys-Aib)3-NH2], a heterochiral peptide [Ac-(l-Lys-d-Lys-Aib)3-NH2], and a racemic mixture of diastereomeric peptides [Ac-(rac-Lys-rac-Lys-Aib)3-NH2] were designed and synthesized to investigate the relationship between their preferred secondary structures and their antimicrobial activity. Peptide 5, [Ac-(l-Lys-l-Lys-Ac6c)3-NH2] formed a stable α-helical structure and exhibited strong activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa).

The zebrafish endoderm begins to develop at gastrulation stages as a monolayer of cells. The behaviour of the endoderm during gastrulation stages is well understood. However, knowledge of the morphogenic movements of the endoderm during somitogenesis stages, as it forms a mesenchymal rod, is lacking. Here we characterise endodermal development during somitogenesis stages, and describe the morphogenic movements as the endoderm transitions from a monolayer of cells into a mesenchymal endodermal rod. We demonstrate that, unlike the overlying mesoderm, endodermal cells are not polarised during their migration to the midline at early somitogenesis stages. Specifically, we describe the stage at which endodermal cells begin to leave the monolayer, a process we have termed ‘midline aggregation’. The planar cell polarity (PCP) signalling pathway is known to regulate mesodermal and ectodermal cell convergence towards the dorsal midline. However, a role for PCP signalling in endoderm migration to the midline during somitogenesis stages has not been established. In this report, we investigate the role for PCP signalling in multiple phases of endoderm development during somitogenesis stages. Our data exclude involvement of PCP signalling in endodermal cells as they leave the monolayer., Summary: Here we characterise endodermal morphogenesis during somitogenesis stages, and show that PCP signalling plays a mixed role in endoderm development during these stages.

Magainin 2 (Mag 2), which is isolated from the skin of frogs, is a representative antimicrobial peptide (AMP), exerts its antimicrobial activity via microbial membrane disruption. It has been reported that both the amphipathicity and helical structure of Mag 2 play an important role in its antimicrobial activity. In this study, we revealed that the sequence of 17 amino acid residues in Mag 2 (peptide 7) is required to exert sufficient activity. We also designed a set of Mag 2 derivatives, based on enhancement of helicity and/or amphipathicity, by incorporation of α,α-disubstituted amino acid residues into the Mag 2 fragment, and evaluated their preferred secondary structures and their antimicrobial activities against both Gram-positive and Gram-negative bacteria. As a result, peptide 11 formed a stable helical structure in solution, and possessed potent antimicrobial activities against both Gram-positive and Gram-negative bacteria without significant cytotoxicity.

Antimicrobial peptides (AMPs) have garnered much attention as novel therapeutic agents against infectious diseases. They exhibit antimicrobial activity through microbial membrane disruption based on their amphipathic properties. In this study, we rationally designed and synthesized a series of novel AMPs Block, Stripe, and Random, and revealed that Stripe exhibits potent antimicrobial activity against Gram-positive and Gram-negative microbes. Moreover, we also demonstrated that Stripe disrupts both Gram-positive and Gram-negative mimetic bacterial membranes. Finally, we investigated the hemolytic activity and cytotoxicity in human blood cells and human cell lines, and found that Stripe exhibited neither. These data indicated that Stripe is a promising antimicrobial reagent that does not display significant cytotoxicity.

A 12-year-old girl presented with a spinal epidural abscess (SEA), an unusual emergent infectious disease that often requires surgical intervention. Its nonspecific symptoms and rarity in pediatric patients frequently delay the diagnosis until neurologic symptoms develop. This patient had only fever without back pain or neurologic symptoms at the first visit. Whole-body CT later diagnosed a SEA, which was treated by antibiotics only. No neurologic deterioration was observed, and the fever and now-present pain gradually diminished. Early diagnosis of a SEA is important to avoid the development of symptoms. Despite its irradiation to the patient, CT might be useful for providing an early diagnosis in the absence of neurologic symptoms.

Background Transcranial magnetic stimulation (TMS), when applied over the primary motor cortex, elicits a motor-evoked potential (MEP) in electromyograms measured from peripheral muscles. MEP amplitude has often been observed to fluctuate trial to trial, even with a constant stimulus. Many factors cause MEP fluctuations in TMS. One of the primary factors is the weak stationarity and instability of cortical activity in the brain, from which we assumed MEP fluctuations originate. We hypothesized that MEP fluctuations are suppressed when TMS is delivered to the primary motor cortex at a time when several electroencephalogram (EEG) channels measured on the scalp are highly similar in the frequency domain. Objective We developed a TMS triggering system to suppress MEP fluctuations using EEG coherence analysis, which was performed to detect the EEG signal similarity between the 2 channels in the frequency domain. Methods Seven healthy adults participated in the experiment to confirm whether the TMS trigger system works adequately, and the mean amplitude and coefficient of the MEP variation were recorded and compared with the values obtained during the control task. We also determined the experimental time under each condition and verified whether it was within the predicted time. Results The coefficient of variation of MEP amplitude decreased in 5 of the 7 participants, and significant differences (P=.02) were confirmed in 2 of the participants according to an F test. The coefficient of variation of the experimental time required for each stimulus after threshold modification was less than that without threshold modification, and a significant difference (P Conclusions We found that MEP could be suppressed using the system developed in this study and that the TMS trigger system could also stabilize the experimental time by changing the triggering threshold automatically.

We propose an extension of distcloud-fs for achieving a global distributed POSIX file system in intercloud environment. The file system works in intercloud environment, that is, on the top of multiple independent cloud services. The important capability of the intercloud extension is that there is no need for users to take care of infrastructure layer, while the file system stores data into multiple cloud services, transparently and automatically. As the result, cloud users may have diversity about vendors and locations for data platform, as discussed in various intercloud research projects. In this paper, we describe the architecture of the distcloud-fs and its intercloud extension. We also describe design and implementation of the prototype and discuss some typical use cases of the file system with practical demonstrations.

The adenohypophysis (AH) consists of six distinct types of hormone-secreting cells. In zebrafish, although proper differentiation of all AH cell types has been shown to require Notch signaling within a period of 14-16 h postfertilization (hpf), the mechanisms underlying this process remain to be elucidated. Herein, we observed using the Notch inhibitor dibenzazepine (DBZ) that Notch signaling also contributed to AH cell specification beyond 16 hpf. Specification of distinct cell types was perturbed by DBZ treatment for different time frames, suggesting that AH cells are specified by Notch-dependent and cell-type-specific mechanisms. We also found that two hes-family genes, her4.1 and hey1, were expressed in the developing AH under the influence of Notch signaling. her4.1 knockdown reduced expression of proopiomelanocortin a (pomca), growth hormone (gh), and prolactin, whereas hey1 was responsible only for gh expression. Simultaneous loss of both Her4.1 and Hey1 produced milder phenotypes than that of DBZ-treated embryos. Moreover, DBZ treatment from 18 hpf led to a significant down-regulation of both gh and pomca genes only when combined with injection of a subthreshold level of her4.1-morpholino. These observations suggest that multiple downstream effectors, including Her4.1 and Hey1, mediate Notch signaling during AH cell specification.

Although body color pattern formation by pigment cells plays critical roles in animals, pigment cell specification has not yet been fully elucidated. In zebrafish, there are three chromatophores: melanophore, iridophore, and xanthophore, that are derived from neural crest cells (NCCs). A recent study has reported the differentially expressed genes between melanophores and iridophores. Based on transcriptome data, we identified that Gbx2 is required for iridophore specification during development. In support of this, iridophore formation is suppressed by gbx2 knockdown by morpholino antisense oligonucleotide, at 72 h post fertilization (hpf) in zebrafish. Moreover, gbx2 is expressed in sox10-expressing NCCs and guanine crystal plates-containing iridophores during development at 24 and 48 hpf, respectively. In gbx2 knockdown zebrafish embryos, apoptosis of sox10-expressing NCCs was detected at 24 hpf without any effect on the formation of melanophores and xanthophores at 48 hpf. We further observed that the N-terminal domain of Gbx2 is able to rescue the iridophore formation defect caused by gbx2 knockdown. Our study provides insights into the requirement of N-terminal domain of Gbx2 for iridophore specification in zebrafish.

Transcranial magnetic stimulation (TMS) over the cerebellum facilitates the spinal reflex in healthy humans. The aim of this study was to investigate whether such cerebellar spinal facilitation (CSpF) appears in patients with spinocerebellar ataxia (SCA) presenting with atrophy in the cerebellar gray matter and dentate nucleus. One patient with SCA type 6 and another with SCA type 31 participated in this study. TMS over the right primary motor cortex was used to induce motor-evoked potentials in the right first dorsal interosseous muscle, which were detected using electromyography. Conditioning TMS using interstimulus intervals of 1-8 ms was performed over the right cerebellum as a test to measure cerebellar brain inhibition (CBI). To assess the H-reflex and the M-wave recruitment curve of the right soleus muscle, we performed electrical stimulation of the right tibial nerve. The stimulation intensity was set to that at the center of the H-reflex curve of the ascending limb. To measure CSpF, we delivered TMS over the right cerebellum 100, 110, 120, and 130 ms before the right tibial nerve stimulation. Voxel-based morphometry was used to verify the presence of atrophy in the cerebellar gray matter and dentate nucleus. CBI was absent in both cases. However, a significant facilitation of the H-reflex occurred with an interstimulus interval of 120 ms in both cases. These findings indicate that the pathways associated with the induction of CSpF and CBI are different, and that the cerebellar gray matter and dentate nucleus are not needed for the induction of CSpF. The possible origin of CSpF may be examined by stimulation of other cerebellar deep nuclei or the brainstem.

Background Long-term management of bronchial asthma based on the fractional exhaled nitric oxide (FeNO) value alone is not conclusive yet. Therefore, we combined FeNO testing and spirometry, a commonly used test in routine practice, to evaluate acute exacerbation and respiratory function in children with bronchial asthma. Objective We combined FeNO testing and spirometry, commonly used in routine practice, to evaluate acute exacerbations and respiratory function in children with bronchial asthma. Methods Subjects were school aged children 7 years and older with bronchial asthma who underwent FeNO testing in January 2015 to May 2016. We evaluated the changes in the frequency of acute exacerbations and respiratory function in the 30 subsequent months. Subjects were divided into 2 groups: those with initial FeNO levels ≥ 21 parts per billion (ppb) (high FeNO) and < 20 ppb (normal FeNO) groups. Results There were 48 children (33 boys) in the high FeNO group and 68 children (46 boys) in the normal FeNO group. Spirometry was conducted on 83 children (72%) prior to the initial FeNO test, revealing no difference in the ratio of detecting lung dysfunction between the 2 groups. The observation period was 25.8 ± 0.7 and 24.7 ± 0.6 months for the high and normal FeNO groups, respectively. The children in the high FeNO group with lung dysfunction in the initial FeNO test continued to exhibit lung dysfunction at the test at 30 months. In the normal FeNO group, even if lung dysfunction was observed at the initial FeNO, it improved within the 20-month point, and the improvement was maintained thereafter. Conclusion Children with bronchial asthma with high FeNO levels and lung dysfunction are at a higher risk of prolonged lung dysfunction.

Allergen sensitization differs according to countries and regions. Understanding the characteristics of allergen sensitization in one's own country is useful for the treatment of patients with allergic diseases. Recently, Japanese cedar pollen (JCP) sensitization is increasing in young Japanese children. Therefore, we evaluated allergen sensitization patterns in under 60-month-old Japanese recurrent wheezers using multiple allergen tests.Allergen tests for 204 Japanese recurrent wheezers between April 2010 and April 2016 were reviewed. Children were divided into 10 groups by age, in 6-month intervals, from 0 months and analyzed by age group.Seventy five percent of children were diagnosed as bronchial asthma. Children under 12-months showed sensitization to food allergens, such as egg white (13.3%) and cow's milk (3.3%), and no sensitization to inhaled allergens. However, house dust mite (HDM) and JCP sensitization increased significantly in children 1 to 2 year of age (HDM; 7.7-32.1%, JCP; 3.9-17.9%) and both sensitization increased to most frequent in children over 36 months of age. JCP sensitization was also frequently detected in children with HDM sensitization (47.1%).Sensitization to inhaled allergens was significantly increased in Japanese recurrent wheezers over 12 months of age and sensitization to HDM and JCP increased to most frequent over 36 months of age. JCP sensitization was established from early childhood and detected frequently in Japanese recurrent wheezers under 60 months of age. These characteristics are the same with HDM sensitization.

Leukotriene receptor antagonists (LTRAs) are identified as a monotherapy for asthma and allergic rhinitis; however, their use in children for treatment of these diseases has not been examined. Accordingly, the present study investigated the efficacy of pranlukast dry syrup for children with both pollinosis and asthma. The subjects were children receiving treatment for asthma who were also diagnosed with cedar pollen allergy. Patients were divided into a group that received continuous treatment with pranlukast (group A; n=20) and a group that commenced add-on treatment for pollinosis following the onset of symptoms (group B; n=20). Patients in group B were randomly allocated to subgroup B1 (add-on treatment with pranlukast dry syrup) or subgroup B2 (add-on treatment with a second-generation antihistamine). In both groups, nasal and ocular symptoms were evaluated every day and recorded in a diary. Exacerbation of nasal obstruction was demonstrated in group B; however, not in group A. There was a significant difference in symptoms observed between the two groups during the late peak pollen period (P

Cell therapy using endothelial progenitor cells (EPCs) is a promising strategy for the treatment of ischemic diseases. Two types of EPCs have been identified: early EPCs and late EPCs. Late EPCs are able to form tube structure by themselves, and have a high proliferative ability. The functional marker(s) of late EPCs, which relate to their therapeutic potential, have not been fully elucidated. Here we compared the gene expression profiles of several human cord blood derived late EPC lines which exhibit different tube formation activity, and we observed that the expression of occludin (OCLN) in these lines correlated with the tube formation ability, suggesting that OCLN is a candidate functional marker of late EPCs. When OCLN was knocked down by transfecting siRNA, the tube formation on Matrigel, the S phase + G2/M phase in the cell cycle, and the spheroid-based sprouting of late EPCs were markedly reduced, suggesting the critical role of OCLN in tube formation, sprouting and proliferation. These results indicated that OCLN plays a novel role in neovascularization and angiogenesis. This article is protected by copyright. All rights reserved

Upon acquirement of pulmonary circulation, the ancestral heart may have been remodelled coincidently with, or accompanied by, the production and rearrangement of progenitor cells. However, the progenitor populations that give rise to the left ventricle (LV) and sinus venosus (SV) are still ambiguous. Here we show that the expression of Secreted frizzled-related protein Sfrp5 in the mouse identifies common progenitors for the outflow tract (OFT), LV, atrium and SV but not the right ventricle (RV). Sfrp5 expression begins at the lateral sides of the cardiac crescent, excluding early differentiating regions, and continues in the venous pole, which gives rise to the SV. Lineage-tracing analysis revealed that descendants of Sfrp5-expressing cells at E7.5 contribute not only to the SV but also to the LV, atria and OFT and are found also in the dorsal splanchnic mesoderm accompanied by the expression of the secondary heart field marker, Islet1. These findings provide insight into the arrangement of cardiac progenitors for systemic circulation., It is unclear which progenitors define different regions of the heart. Here, the authors find Secreted frizzled-related protein 5 is expressed in murine progenitor cells for the outflow tract, first heart field, and sinus venosus, but not the right ventricle, and Wnt inhibition prevents progenitor proliferation.

Although asymmetric nuclear positioning is observed during animal development, the regulation and significance of this nuclear positioning in cell differentiation remains poorly understood. Using zebrafish blastulae, we provide evidence that nuclear movement toward the yolk syncytial layer, which comprises extraembryonic tissue, occurs in the first endoderm specified cells during endoderm specification. Nodal signaling is essential for nuclear movement, whereas nuclear envelope proteins are involved in the movement through the microtubule formation. The positioning of the microtubule organizing center, which is proposed to be critical for nuclear movement, is regulated by Nodal signaling and nuclear envelope proteins. The non-Smad JNK signaling pathway, which is downstream of Nodal signaling, regulates nuclear movement independent of the Smad pathway, and this nuclear movement is associated with Smad signal transduction toward the nucleus. Our study provides insights into the function of nuclear movement in Smad signaling toward the nucleus, and could be applied to the control of Transforming Growth Factor-β signaling.

Background The types of exercise therapy that have an effect on functioning in early-stage amyotrophic lateral sclerosis patients are not well-known. Aim To investigate the types of exercise therapy that have an effect on functioning in early-stage amyotrophic lateral sclerosis patients. Methods The participants were consecutive amyotrophic lateral sclerosis patients who were prescribed exercise therapy at six institutions in Japan. All data (age, sex, site of onset and disease duration) were collected retrospectively from the patients' medical records. The amyotrophic lateral sclerosis functional rating scale-revised scores were collected to assess patients' functioning at two time-points, at baseline and during exercise therapy. Furthermore, details of the types of exercise therapy were investigated. The change in the amyotrophic lateral sclerosis functional rating scale-revised score was calculated using the score at two time-points. A multiple regression analysis was used to determine the effect of type of exercise therapy on the change in amyotrophic lateral sclerosis functional rating scale-revised scores. Results The analysis included 156 participants. The interaction term of performance factor (walking training, standing training, and activities of daily living task training) was significantly associated with the change in the amyotrophic lateral sclerosis functional rating scale-revised score, but the interaction term of the muscle function factor (resistance training and cycle ergometer training) was not. Conclusion The combination of performance task training appears to alleviate functional decline in early-stage amyotrophic lateral sclerosis patients.

We reported that cerebellar transcranial magnetic stimulation (TMS) facilitates spinal reflex in healthy humans. The aim of this study was to investigate whether the “cerebellar” spinal facilitation (CSpF) appears in patients with spinocerebellar ataxia (SCA) with atrophy in the cerebellar cortex and dentate nucleus (DN). Two patients with SCA (one was SCA6 and the other was SCA31), participated in this study. Cerebellar inhibition (CBI) was tested using paired-TMS-paradigm with interstimulus intervals (ISI) of 1–8 ms in the right first dorsal interosseous muscle. To measure CSpF, we delivered TMS over the right cerebellum 100–130 ms before right tibial nerve stimulation, and CSpF was calculated with obtained conditioned/unconditioned H-reflex amplitude in the right soleus muscle. Voxel-based morphometry was used to verify the atrophy in cerebellar cortex and the DN. The results showed absence of CBI, but there was a significant facilitation of the H-reflex which occurred in the 120 ms ISI in both patients. These findings indicate that the pathways associated with the induction of CSpF and CBI are different, and that the cerebellar cortex and the DN are not required for inducing CSpF. Possible generator of CSpF could be other deep cerebellar nuclei or the brain stem.

Balanced and precisely controlled processes between self-renewal and differentiation of hematopoietic stem cells (HSCs) into all blood lineages are critical for vertebrate definitive hematopoiesis. However, the molecular mechanisms underlying the maintenance and differentiation of HSCs have not been fully elucidated. Here, we show that zebrafish Ddx46, encoding a DEAD-box RNA helicase, is expressed in HSCs of the caudal hematopoietic tissue (CHT). The number of HSCs expressing the molecular markers cmyb or T-cell acute lymphocytic leukemia 1 (tal1) was markedly reduced in Ddx46 mutants. However, massive cell death of HSCs was not detected, and proliferation of HSCs was normal in the CHT of the mutants at 48 h postfertilization. We found that myelopoiesis occurred, but erythropoiesis and lymphopoiesis were suppressed, in Ddx46 mutants. Consistent with these results, the expression of spi1, encoding a regulator of myeloid development, was maintained, but the expression of gata1a, encoding a regulator of erythrocyte development, was downregulated in the mutants. Taken together, our results provide the first genetic evidence that zebrafish Ddx46 is required for the multilineage differentiation of HSCs during development, through the regulation of specific gene expressions.

The objective of this research is to realize fully robot-operated farming from tillage to harvest in large-scale agriculture such as cultivation of rice, wheat and soybean in Japan. For this purpose, three types of robot have been developed; the first is robot tractor, the second are rice transplanter robots, the third are combine harvester robots. RTK-GPS and inertia measurement unit (IMU), or GPS compass, are used for navigation system. Robots have Controller Area Network (CAN) bus that all sensors and PC can be connected in common among other robots such as tractors, rice transplanters, and combine harvesters. These robots could be done autonomous operation in paddy field. In addition, moving between the fields for effective operations and safe guideline for robot system were discussed.

In this paper, development and standardization of on-vehicle control and communication data network for small size farm machinery is described. There already exist an international standard and many compliant products in the western countries. But, they are too huge and complicated for the average Japanese style of farming, which is represented by the words of small tractors and small paddy fields. In order to achieve an advancement and cost reduction in this field, enough and sufficient low cost simple electronic devices and related software were developed. The communication method adopted basically depends on the ISO 11783 standard technology, and some new features needed for the domestic agricultural conditions are considered. At the same time, a new domestic simple standard for data exchange between ECU's on tractors and attached implements are discussed. From some experiments, it became clear that the developed hardware and software together are strong tools to realize the domestic standardized devices. It can be said that standardization of data communication for lower range of farm machinery just has been started.

We have developed a new Global Positioning System (GPS) guided rice transplanter. We developed GPS and IMU guided rice transplanter in previous researches. Those were guided with GPS position data and inertia measurement unit (IMU) direction data. New one is guided with GPS position data with tilt correction during straight driving and guided with IMU direction data during turning at the headland. The GPS antenna is set to 1.5m height and 0.4m front offset from vehicle front axle. The actuator control command and data communication protocols comply with through the controller area network (CAN) bus. Steering and transmission are controlled by electrical actuators according to the location in a field. We report results of experiment.

Age-related changes in DNA methylation have been demonstrated in mammals, but it remains unclear as to the generality of this phenomenon in vertebrates, which is a criterion for the fundamental cause of senescence. Here we showed that the zebrafish genome gradually and clearly lost methylcytosine in somatic cells, but not in male germ cells during aging, and that age-dependent hypomethylation preferentially occurred at a particular domain called the CpG island shore, which is associated with vertebrates’ genes and has been shown to be hypomethylated in humans with age. We also found that two CpG island shores hypomethylated in zebrafish oocytes were de novo methylated in fertilized eggs, which suggests that the zebrafish epigenome is reset upon fertilization, enabling new generations to restart with a heavily methylated genome. Furthermore, we observed an increase in cleavage of the zebrafish genome to an oligonucleosome length in somatic cells from the age of 12 months, which is suggestive of an elevated rate of apoptosis in the senescent stage.

Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) and causes a foodborne illness that affects more than 100 cases in Japan each year. We previously reported that the consumption of raw olive flounder meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhea and emesis through an unknown mechanism. Here, we demonstrate that K. septempunctata sporoplasm plays an important role in mediating the toxicity of K. septempunctata. When K. septempunctata spores were inoculated in Caco-2 human intestinal cells, K. septempunctata sporoplasms were released from spores, and they invaded the cells. Electron microscopic observations revealed that the sporoplasm invasion severely damaged the Caco-2 cells. The inoculation of K. septempunctata spores eliminated the transepithelial electrical resistance (TER) across the cell monolayer. Inhibiting the invasion of the sporoplasms prevented the observed loss in cell layer integrity, as illustrated by the rapid elimination of the TER. These results suggest that the invasion by sporoplasms severely damaged individual intestinal cells, resulting in a loss of cell monolayer integrity.

A 13-year-old girl was admitted to the hospital because of cardiomegaly found incidentally at regular medical checkup at her school. Although dilated cardiomyopathy was suspected, the girl showed no symptoms. chest x-ray film showed cardiomegaly (cardiothoracic ratio, 67%), and echocardiogram demonstrated a dilatation of the left ventricle without thinning of the wall. While contraction was diffusely decreased, estimated cardiac output was increased to 13l/minute. Abdominal ultrasonography conducted to clarify the cause of this high output showed a multicystic lesion (8×6×4 cm) at the back of the left common iliac artery. Color Doppler ultrasonography revealed abundant blood flow within the lesion, establishing the diagnosis of arteriovenous malformation. Aortography delineated the arteriovenous malformation being supplied from the left internal iliac artery and from the left third and fourth lumbar arteries and drainage into the left common iliac vein and the right internal iliac vein. Although it did not show the precise location of the feeding and draining vessels, color Doppler ultrasonography was useful for the diagnosis of arteriovenous malformation.

The human cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI) anchored membrane glycoprotein with two N-glycosylation sites at residues 181 and 197. This protein migrates in several bands by Western blot analysis (WB). Interestingly, PNGase F treatment of human brain homogenates prior to the WB, which is known to remove the N-glycosylations, unexpectedly gives rise to two dominant bands, which are now known as C-terminal (C1) and N-terminal (N1) fragments. This resembles the β-amyloid precursor protein (APP) in Alzheimer disease (AD), which can be physiologically processed by α-, β-, and γ-secretases. The processing of APP has been extensively studied, while the identity of the cellular proteases involved in the proteolysis of PrP(C) and their possible role in prion biology has remained limited and controversial. Nevertheless, there is a strong correlation between the neurotoxicity caused by prion proteins and the blockade of their normal proteolysis. For example, expression of non-cleavable PrP(C) mutants in transgenic mice generates neurotoxicity, even in the absence of infectious prions, suggesting that PrP(C) proteolysis is physiologically and pathologically important. As many mouse models of prion diseases have recently been developed and the knowledge about the proteases responsible for the PrP(C) proteolysis is accumulating, we examine the historical experimental evidence and highlight recent studies that shed new light on this issue.

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis.

The control of organ size and position relies, at least in part, upon appropriate regulation of the signals that specify organ progenitor fields. Pancreatic cell fates are specified by retinoic acid (RA), and proper size and localization of the pancreatic field are dependent on tight control of RA signaling. Here we show that the RA-degrading Cyp26 enzymes play a critical role in defining the normal anterior limit of the pancreatic field. Disruption of Cyp26 function causes a dramatic expansion of pancreatic cell types toward the anterior of the embryo. The cyp26a1 gene is expressed in the anterior trunk endoderm at developmental stages when RA is signaling to specify pancreas, and analysis of cyp26a1/giraffe ( gir ) mutant zebrafish embryos confirms that cyp26a1 plays the primary role in setting the anterior limit of the pancreas. Analysis of the gir mutants further reveals that cyp26b1 and cyp26c1 function redundantly to partially compensate for loss of Cyp26a1 function. We used cell transplantation to determine that Cyp26a1 functions directly in endoderm to modulate RA signaling and limit the pancreatic field. Taken together with our finding that endodermal expression of cyp26 genes is subject to positive regulation by RA, our data reveal a feedback loop within the endoderm. Such feedback can maintain consistent levels of RA signaling, despite environmental fluctuations in RA concentration, thus ensuring a consistent size and location of the pancreatic field.

During vertebrate gastrulation, both mesodermal and endodermal cells internalize through the blastopore beneath the ectoderm. In zebrafish, the internalized mesodermal cells move towards the dorsal side of the gastrula and, at the same time, they extend anteriorly by convergence and extension(C&E) movements. Endodermal cells showing characteristic filopodia then migrate into the inner layer within the hypoblast next to the yolk syncytial layer (YSL). However, little is known about how the movement of endodermal cells is regulated during gastrulation. Here we show that sdf1a- and sdf1b-expressing mesodermal cells control the movements of the cxcr4a-expressing endodermal cells. The directional migration of endodermal cells during gastrulation is inhibited by knockdown of either cxcr4a or sdf1a/sdf1b (sdf1). We also show that misexpressed Sdf1 acts as a chemoattractant for cxcr4a-expressing endodermal cells. We further found, using the endoderm-specific transgenic line Tg(sox17:EGFP), that Sdf1/Cxcr4 signaling regulates both the formation and orientation of filopodial processes in endodermal cells. Moreover, the accumulation of phosphoinositide 3,4,5-trisphosphate (PIP3), which is known to occur at the leading edge of migrating cells, is not observed at the filopodia of endodermal cells. Based on our results, we propose that sdf1-expressing mesodermal cells, which overlie the endodermal layer, guide the cxcr4a-expressing endodermal cells to the dorsal side of the embryo during gastrulation, possibly through a PIP3-independent pathway.

Previously, we established a method to produce recombinant proteins (r-proteins) in cocoons of germline transgenic silkworms, and showed that a step(s) in post-transcription processes was rate-limiting in obtaining a high yield of r-proteins. In this study, we examined whether the 5'-untranslated region (5'-UTR) of the polyhedrin gene (pol) of nucleopolyhedrovirus (NPV) has a translational enhancer activity in the r-protein expression by middle silk gland (MSG) cells of silkworm Bombyx mori (Bm). Sericin 1 gene (ser1) promoter-driven transformation vectors were constructed in which pol5'-UTRs of NPVs isolated from four different species, Bm, Spodoptera frugiperda, Ectropis oblique, and Malacosoma neustria, were each placed upstream of a reporter gene. Transient expression assays in MSGs showed that these pol5'-UTRs all enhanced the protein expression of reporter genes, and the pol5'-UTR of Bm NPV (pol5'-UTR/Bm) was the most effective among them. Thus, transgenic silkworms were generated, which bore the ser1 promoter-driven His-tagged secretory EGFP (sEGFP-His) gene under the control of pol5'-UTR/Bm. The synthesis of sEGFP-His proteins in MSGs of the transgenic worms was approximately 1.5-fold higher than that in those bearing null vectors. However, its mRNA expression levels were 67% of the control worms, indicating that the pol5'-UTR/Bm specifically enhanced the translational level. In conclusion, pol5'-UTR/Bm increased the yield of r-protein production in transgenic silkworms by enhancing the translational activity and this 5'-UTR could be useful for the mass production of r-proteins in germline transgenic silkworms.

Embryonic stem (ES) cells are thought to have unique chromatin structures responsible for their capacity for self-renewal and pluripotency. To examine this possibility, we sought nuclear proteins in mouse ES cells that specifically bind to histones using a pull-down assay with synthetic peptides of histone H3 and H4 tail domain as baits. Nuclear proteins preferentially bound to the latter. We identified 45 proteins associated with the histone H4 tail and grouped them into four categories: 10 chromatin remodeling proteins, five histone chaperones, two histone modification-related proteins, and 28 other proteins. mRNA expression levels of 20 proteins selected from these 45 proteins were compared between undifferentiated and retinoic acid (RA)-induced differentiated ES cells. All of the genes were similarly expressed in both states of ES cells, except nucleoplasmin 3 (NPM3) that was expressed at a higher level in the undifferentiated cells. NPM3 proteins were localized in the nucleoli and nuclei of the cells and expression was decreased during RA-induced differentiation. When transfected with NPM3 gene, ES cells significantly increased their proliferation compared with control cells. The present study strongly suggests that NPM3 is a chromatin remodeling protein responsible for the unique chromatin structure and replicative capacity of ES cells.

The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI-anchorless PrP (GPI− PrPSV), was unglycosylated and soluble in non-ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non-neuronal tissues. When long-term passaged T98G cells were placed in a low-oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI− PrPSV expression in T98G cells.

The homeobox transcription factor Mtx2 is essential for epiboly, the first morphogenetic movement of gastrulation in zebrafish. Morpholino knockdown of Mtx2 results in stalling of epiboly and lysis due to yolk rupture. However, the mechanism of Mtx2 action is unknown. The role of mtx2 is surprising as most mix/bix family genes are thought to have roles in mesendoderm specification. Using a transgenic sox17-promoter driven EGFP line, we show that Mtx2 is not required for endoderm specification but is required for correct morphogenetic movements of endoderm and axial mesoderm. During normal zebrafish development, mtx2 is expressed at both the blastoderm margin and in the zebrafish equivalent of visceral endoderm, the extra-embryonic yolk syncytial layer (YSL). We show that formation of the YSL is not Mtx2 dependent, but that Mtx2 directs spatial arrangement of YSL nuclei. Furthermore, we demonstrate that Mtx2 knockdown results in loss of the YSL F-actin ring, a microfilament structure previously shown to be necessary for epiboly progression. In summary, we propose that Mtx2 acts within the YSL to regulate morphogenetic movements of both embryonic and extra-embryonic tissues, independently of cell fate specification.