The kpc-1 3'UTR facilitates dendritic transport and translation efficiency of mRNAs for dendrite arborization of a mechanosensory neuron important for male courtship.

A recently reported Schizophrenia-associated genetic variant in the 3'UTR of the human furin gene, a homolog of C. elegans kpc-1, highlights an important role of the furin 3'UTR in neuronal development. We isolate three kpc-1 mutants that display abnormal dendrite arborization in PVD neurons and defective male mating behaviors. We show that the kpc-1 3'UTR participates in dendrite branching and self-avoidance. The kpc-1 3'UTR facilitates mRNA localization to branching points and contact points between sibling dendrites and promotes translation efficiency. A predicted secondary structural motif in the kpc-1 3'UTR is required for dendrite self-avoidance. Animals with over-expression of DMA-1, a PVD dendrite receptor, exhibit similar dendrite branching and self-avoidance defects that are suppressed with kpc-1 over-expression. Our results support a model in which KPC-1 proteins are synthesized at branching points and contact points to locally down-regulate DMA-1 receptors to promote dendrite branching and self-avoidance of a mechanosensory neuron important for male courtship.

Two intrinsic timing mechanisms set start and end times for dendritic arborization of a nociceptive neuron.

Choreographic dendritic arborization takes place within a defined time frame, but the timing mechanism is currently not known. Here, we report that the precisely timed lin-4-lin-14 regulatory circuit triggers an initial dendritic growth activity, whereas the precisely timed lin-28-let-7-lin-41 regulatory circuit signals a subsequent developmental decline in dendritic growth ability, hence restricting dendritic arborization within a set time frame. Loss-of-function mutations in the lin-4 microRNA gene cause limited dendritic outgrowth, whereas loss-of-function mutations in its direct target, the lin-14 transcription factor gene, cause precocious and excessive outgrowth. In contrast, loss-of-function mutations in the let-7 microRNA gene prevent a developmental decline in dendritic growth ability, whereas loss-of-function mutations in its direct target, the lin-41 tripartite motif protein gene, cause further decline. lin-4 and let-7 regulatory circuits are expressed in the right place at the right time to set start and end times for dendritic arborization. Replacing the lin-4 upstream cis-regulatory sequence at the lin-4 locus with a late-onset let-7 upstream cis-regulatory sequence delays dendrite arborization, whereas replacing the let-7 upstream cis-regulatory sequence at the let-7 locus with an early-onset lin-4 upstream cis-regulatory sequence causes a precocious decline in dendritic growth ability. Our results indicate that the lin-4-lin-14 and the lin-28-let-7-lin-41 regulatory circuits control the timing of dendrite arborization through antagonistic regulation of the DMA-1 receptor level on dendrites. The LIN-14 transcription factor likely directly represses dma-1 gene expression through a transcriptional means, whereas the LIN-41 tripartite motif protein likely indirectly promotes dma-1 gene expression through a posttranscriptional means.

Sebacic acid, a royal jelly-containing fatty acid, decreases LPS-induced IL-6 mRNA expression in differentiated human THP-1 macrophage-like cells.

Macrophages produce many inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in innate immune responses. However, excess production of these mediators by activated macrophages triggers deleterious effects, leading to disorders associated with inflammation. Royal jelly (RJ), a milky-white substance secreted by worker bees, contains unique fatty acids, including 10-hydroxy-2-decenoic acid (10H2DA) and sebacic acid (SA). 10H2DA has been reported to have various biological functions, such as anti-inflammation. However, the anti-inflammatory effect of SA is not fully understood. In this study, we investigated the effects of SA on lipopolysaccharide (LPS)-induced cytokine expression using differentiated human THP-1 macrophage-like cells. SA dose-dependently decreased LPS-induced mRNA expression of IL-6, but not TNF-α and IL-1ß. SA suppressed the phosphorylation of signal transducers and activators of transcription 1 (STAT1) and STAT3, but hardly affected the activation of JNK, p38, or NF-κB. In addition, SA decreased LPS-induced interferon-ß (IFN-ß) expression, and the addition of IFN-ß restored the inhibition by SA of LPS-induced STAT activation and IL-6 expression. Furthermore, SA suppressed LPS-induced nuclear translocation of interferon regulatory factor 3 (IRF3), a transcription factor responsible for IFN-ß expression. Taken together, we conclude that SA selectively decreases LPS-induced expression of IL-6 mRNA through inhibition of the IRF3/IFN-ß/STAT axis.

Characterization of a murine model with arteritis induced by Nod1 ligand, FK565: A comparative study with a CAWS-induced model.

OBJECTIVE: Kawasaki disease (KD) occurs via activation of the innate immune system. Nucleotide oligomerization domain-1 (NOD1) is a pattern recognition receptor regulating the innate immunity. We characterized histopathology of arteritis induced by FK565, a ligand for NOD1, in mice, compared with Candida albicans water-soluble fraction (CAWS)-induced model. METHODS: Vasculitis was induced by injection of FK565 or CAWS into C57BL6/J mice (n = 9 and n = 11, respectively). At 4 weeks, they were sacrificed, and plasma cytokines and chemokines were measured. RESULTS: FK565 injection induced vasculitis mainly involving bilateral coronary arteries whereas the aortic root was diffusely affected in CAWS mice. In FK565 animals, the abdominal aorta and its branching arteries also exhibited inflammation with atherosclerosis. IL-1α, IL-1ß, IL-5 and RANTES were increased in FK565 group whereas IL-6, IL-13, G-CSF, IFN-γ, and TNF-α were higher in CAWS animals (p < .05 for all variables). The total area of inflammation in FK565 mice appeared to correlate with IL-1ß levels (r = 0.71, p = .05). CONCLUSIONS: Histopathology of FK565-induced model demonstrated 'site-specific' coronary arteritis mimicking KD. This histopathological difference from CAWS model may be due to different cytokine expression profiles.

ERK signaling controls blastema cell differentiation during planarian regeneration.

The robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Here, using the planarian Dugesia japonica we found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, was specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic [RNA interference (RNAi)] approaches provided evidence that ERK activity was required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induced an increased level of ERK activity and rescued the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration.

Evaluation of Coronary Circulation by 13N-Ammonia Myocardial Perfusion Positron Emission Tomography in Patients with Right Coronary Artery Occlusion Due to Kawasaki Disease.

BACKGROUND: Although occlusion of the right coronary artery (RCA) is common in the remote stages of Kawasaki disease, revascularization of the RCA is challenging in children and is usually managed by observation without intervention. METHODS: Using adenosine-stress 13N-ammonia myocardial perfusion positron emission tomography, we evaluated coronary circulation in 14 patients (12 males) with RCA occlusion to identify ischemia (myocardial flow ratio < 2.0) in the RCA region and examined hemodynamics, cardiac function, and coronary aneurysm diameter. These variables were also compared in patients with/without RCA segmental stenosis (SS). RESULTS: There were five cases of ischemia in the RCA region. RCA myocardial blood flow (MBF) at rest was higher in patients with ischemia than in those without ischemia, but the difference was not significant (1.27 ± 0.21 vs. 0.82 ± 0.16 mL/min/g, p = 0.2053). Nine patients presented with RCA SS, and age at onset of Kawasaki disease tended to be lower in those with SS. The maximum aneurysm diameter of RCA was significantly smaller in patients with SS (10.0 ± 2.8 vs. 14.7 ± 1.6, p = 0.0239). No significant differences in other variables were observed between patients with/without ischemia and SS. CONCLUSIONS: At rest, MBF in the RCA region was relatively well preserved, even in patients with RCA occlusion, and there was no progressive deterioration in cardiac function. Adenosine stress showed microcirculatory disturbances in only half of the patients, indicating that it is reversible in children with Kawasaki disease.

High-fidelity fractionation of ssDNA fragments differing in size by one-base on a spiral-channel electrophoretic chip.

For the fractionation of fragments of interest from selective PCR products generated by high coverage gene expression profiling (HiCEP) analysis, high-resolution with the ability to discriminate and fractionate fragments differing by one base (base pair) in size is highly required. We report here on a new 4-inch diameter spiral-channel chip device for automatic high-fidelity fractionation. Overlapping DNA fragments of 180, 181 and 182 bases, with only one-base difference in size, were successfully fractionated. The collected fragments were PCR amplified, and then evaluated by size checking analysis, DNA sequencing, and homolog search. The high-resolution fractionation has been achieved because of the combined contributions of (i) the high-resolution separation using a 30 cm long spiral channel, (ii) a blocking technique to avoid contamination from unselected fragments during CE, and (iii) precise micro-scale target extraction. Contaminations due to unselected fractions have been greatly decreased to a negligible level by optimization of the extraction position and extraction time corresponding to the targeted segment only. This technique can be adapted to a wide range of applications, such as protein or cell collections where requirements for the high purity are more important than the amount of recovered fractionated material.

Interleukin-1beta Inhibition Attenuates Vasculitis in a Mouse Model of Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD), a systemic vasculitis, is suspected to be related to abnormalities in innate immunity. Based on the important role of IL-1 signaling in innate immunity, we investigated the effects of an anti-IL-1ß antibody using a Candida albicans water-soluble fraction (CAWS)-induced mouse model of KD. METHODS: CAWS (0.5 mg/mouse) was injected intraperitoneally into 5-week-old DBA/2 mice on five consecutive days. An anti-Murine IL-1ß antibody (01BSUR) was administered at various doses (2.5, 5.0, and 10.0 mg/kg) and time points (2 days before, same day, and 2, 5, 7, and 14 days after CAWS administration). After 4 weeks, vasculitis in the aortic root was investigated histologically. Cytokines including IL-1ß, -6, -10, and TNF-α were also measured. RESULTS: Groups administered 01BSUR at all doses showed a significant reduction in the area of vasculitis. In addition, 01BSUR inhibited vasculitis until 7 days after CAWS administration. In the analysis of various time points, the level of IL-6 was lower in all groups compared to the CAWS only group, but the levels of IL-1ß, TNFα, and IL-10 were lower when 01BSUR was administered before CAWS. On the other hand, TNFα and IL-10 levels were restored when 01BSUR was administered after CAWS, suggesting that 01BSUR may have additional effects beyond blocking IL-1ß signaling. CONCLUSIONS: The anti-IL-1ß antibody significantly attenuated CAWS-induced vasculitis. The mechanism of inhibiting vasculitis is thought to include inhibition of the IL-1ß pathway and additional effects beyond blocking IL-1ß signaling.

Laminar specific gene expression reveals differences in postnatal laminar maturation in mouse auditory, visual, and somatosensory cortex.

Proper formation of laminar structures in sensory cortexes is critical for sensory information processing. Previous studies suggested that the timing of neuronal migration and the laminar position of cortical neurons differ among sensory cortexes. How they differ during postnatal development has not been systematically investigated. Here, identifying laminas using transcription factors, we examined postnatal changes in neuronal density and distribution in presumptive primary auditory (ACx), visual (VCx), and somatosensory cortexes (SCx) in a strain of mice using immunofluorescence techniques. Development of laminar thickness and its cortical proportion differed among the sensory cortexes. Layers 2-4 defined by Cut-like homeobox 1 (Cux1)-expressing neurons were narrower, and layer 5 was wider in ACx compared to those in VCx or SCx, while Forkhead-box protein P2 (Foxp2)-defined layer 6 was wider in SCx than the other two sensory cortexes throughout postnatal development. Meanwhile, thalamocortical input layers identified by Cux1-expressing neurons formed later in ACx than in the other two cortical regions. The cell densities of ETS-related protein 81-expressing neurons increased in both lower and upper layers but at distinct timing, while those of COUP-TF-interacting protein 2 expressing neurons in the lower layers changed bidirectionally (i.e., increased or decreased) both in layer- and cortical region-specific manners. Foxp2-expressing cells in layer 6 distributed differently and declined at different timing among the sensory cortexes. Overall, we demonstrate that the maturational timing of lamina differs among the sensory cortexes and that postnatal age-dependent changes in neuronal distribution are unique to each of the sensory cortexes.

Engulfing cells promote neuronal regeneration and remove neuronal debris through distinct biochemical functions of CED-1.

Two important biological events happen coincidently soon after nerve injury in the peripheral nervous system in C. elegans: removal of axon debris and initiation of axon regeneration. But, it is not known how these two events are co-regulated. Mutants of ced-1, a homolog of Draper and MEGF10, display defects in both events. One model is that those events could be related. But our data suggest that they are actually separable. CED-1 functions in the muscle-type engulfing cells in both events and is enriched in muscle protrusions in close contact with axon debris and regenerating axons. Its two functions occur through distinct biochemical mechanisms; extracellular domain-mediated adhesion for regeneration and extracellular domain binding-induced intracellular domain signaling for debris removal. These studies identify CED-1 in engulfing cells as a receptor in debris removal but as an adhesion molecule in neuronal regeneration, and have important implications for understanding neural circuit repair after injury.

Comprehensive gene expression analyses in pluripotent stem cells of a planarian, Dugesia japonica.

The neoblasts are the only somatic stem cells in planarians possessing pluripotency, and can give rise to all types of cells, including germline cells. Recently, accumulated knowledge about the transcriptome and expression dynamics of various pluripotent somatic stem cells has provided important opportunities to understand not only fundamental mechanisms of pluripotency, but also stemness across species at the molecular level. The neoblasts can easily be eliminated by radiation. Also, by using fluorescence activated cell sorting (FACS), we can purify and collect many neoblasts, enabling identification of neoblast-related genes by comparison of the gene expression level among intact and X-ray-irradiated animals, and purified neoblasts. In order to find such genes, here we employed the high coverage expression profiling (HiCEP) method, which enables us to observe and compare genome-wide gene expression levels between different samples without advance sequence information, in the planarian D. japonica as a model organism of pluripotent stem cell research. We compared expression levels of ~17,000 peaks corresponding to independent genes among different samples, and obtained 102 peaks as candidates. Expression analysis of genes identified from those peaks by in situ hybridization revealed that at least 42 genes were expressed in the neoblasts and in neoblast-related cells that had a different distribution pattern in the body than neoblasts. Also, single-cell PCR analysis of those genes revealed heterogeneous expression of some genes in the neoblast population. Thus, using multidimensional gene expression analyses, we were able to obtain a valuable data set of neoblast-related genes and their expression patterns.

Electrophoretic chip for fractionation of selective DNA fragment.

A microchannel chip has been used to fractionate selected segments from an electrophoretic flow of separated fragments. A sample, which covers the size from 35 to 670 bp, was initially separated using an 8.8-cm-long channel at the electric field strength of 100 V/cm. The target fragment of 318 bp was selected and extracted from the separation channel. High-resolution fractionation was achieved by introducing new procedures for blocking, extraction, and segment transfer. Fractionation quality with and without blocking were compared using a 310 Genetic Analyzer (Applied Biosystems). The results show that no contamination was found in the sample, which was fractionated with blocking; however, a contamination by short segments was found in the sample, which was fractionated without blocking. Furthermore, fractionation by the chip was found to be of higher fidelity than that by the polyacrylamide slab gel, which displayed a small overlapped peak after the target peak. Compared with the traditional method, our chips enable faster and high-fidelity fractionation, thus providing a new tool for bioanalysis and other applications.

Rorschach Comprehensive System data for a sample of 190 Japanese nonpatient children at five ages.

This study is a part of our series of studies of nonpatient, Japanese children. In total, 346 children were administered the Rorschach. Thirty-two children had fewer than 10 responses, 112 children had more than 10 but fewer than 14 responses, and 12 children were maladjusted or mentally retarded, for a total of 156. In this study, we analyzed the remaining 190 children not excluded by the above criteria in the following age groups: 5 years (n = 24), 8 years (n = 43), 9 years (n = 42), 12 years (n = 42), and 14 years (n = 39). Japanese children showed few responses generally, with an average of 18 ~ 20 responses. In addition to having fewer responses, other features of Japanese children are high Lambda, low EA, very high X-%, and low X+%. The fact that the Rorschach test depends on verbal expression may be one factor in all these features, and we should consider this study in view of these Japanese cultural factors. Interrater reliability findings and Rorschach Comprehensive System (CS; Exner, 1995; Matsumoto, 2003; Matsumoto et al., 2002) data are presented.

Confirming the existence of five peaks in 129Xe rat head spectra.

A series of experiments were performed to investigate why two peaks (D and E) of the five dissolved phase peaks in hyperpolarized (129)Xe rat head spectra appeared inconsistently in previous work. Specifically, spectra were acquired under conditions of various shim states, anaesthetics, and arterial ligation. The shimming experiments showed that slice-shimming can be used to improve resolution of the dissolved phase peaks, but even so, subtle changes in the shim state that may dramatically alter the shape of peak E remain poorly understood. Also, the inability to shim gas spaces and tissue simultaneously may explain why inconsistent chemical shift values have been reported in the literature. A possible solution for this problem is suggested. The results of pre- and postligation spectra from the same animal indicated that two peaks (A and E) originate from brain. Changing the anaesthetic was found to have no effect on the number of dissolved peaks in xenon spectra.

More than 40,000 transcripts, including novel and noncoding transcripts, in mouse embryonic stem cells.

To study the transcriptome of embryonic stem cells, we used a new gene expression profiling method that can measure the expression levels of unknown and rarely expressed transcripts precisely. We detected a total of 33,136 signal peaks representing transcripts in mouse embryonic stem cells, E14. Subsequent random cloning of the peaks suggests that mouse embryonic stem cells express at least 40,000 transcripts, of which about 2,000 are still unknown. In addition, we identified 1,022 noncoding transcripts, several of which change depending on differentiation in gene expression. Our database provides a high-resolution expression profile of E14 cells and is applicable to other mouse embryonic stem cell analyses. It includes most transcription regulation factor-encoding genes and a significant number of unknown and noncoding transcripts.