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1.  Occlusal hypofunction causes periodontal atrophy and VEGF/VEGFR inhibition in tooth movement 
The Angle orthodontist  2012;83(1):48-56.
Objective
To examine changes in microvasculature and the expression of vascular endothelial growth factor A (VEGF-A) and VEGF receptor 2 (VEGFR-2) in rat hypofunctional periodontal ligament (PDL) during experimental tooth movement.
Materials and Methods
Twelve-week-old male Sprague-Dawley rats were divided into normal occlusion and occlusal hypofunction groups. After a 2-week bite-raising period, rat first molar was moved mesially using a 10-gf titanium-nickel alloy closed coil spring in both groups. On days 0, 1, 2, 3, and 7 after tooth movement, histologic changes were examined by micro–computed tomography and immunohistochemistry using CD31, VEGF-A, VEGFR-2, and the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method.
Results
Hypofunctional molars inclined more than normal molars and did not move notably after day 1 of tooth movement. Blood vessels increased on the tension side of the PDL in normal teeth. Immunoreactivities for VEGF-A and VEGFR-2 in normal teeth were greater than those in hypofunctional teeth during tooth movement. Compressive force rapidly caused apoptosis of the PDL and vascular endothelial cells in hypofunctional teeth, but not in normal teeth.
Conclusions
Occlusal hypofunction induces vascular constriction through a decrease in the expression of VEGF-A and VEGFR-2, and apoptosis of the PDL and vascular cells occurs during tooth movement.
doi:10.2319/011712-45.1
PMCID: PMC4037556  PMID: 22716278
Occlusal hypofunction; Tooth movement; VEGF; VEGFR
2.  Total-body low-dose irradiation of mice induces neither learning disability and memory impairment in Morris water maze test nor Alzheimer's disease-like pathogensis in the brain 
Journal of Radiation Research  2014;55(Suppl 1):i20-i21.
Purpose: Alzheimer's disease (AD) is the most common form of dementia, while its cause and progression are not well understood [ 1]. The possible cognitive and behavioral consequences induced by low-dose radiation are of great concern as humans are exposed to ionizing radiations from various sources including medical diagnosis [ 2]. A recent study in mice reported early transcriptional response in brain to low-dose X-rays (0.10 Gy) suggesting alterations of molecular networks and pathways associated with cognitive functions, advanced aging and AD. The present study is to investigate the late pathological, cognitive and behavioral consequences induced by low-dose radiation.
Materials and methods: C57BL/6J mice were total-body irradiated with an acute dose from X-rays (0.10 Gy) or carbon ions (0.05 or 0.10 Gy). The hippocampus was collected and the expression of 84 AD-related genes was analyzed. Morris water maze test was applied to the measurement of the learning ability and memory of the animals. Amyloid imaging with positron emission tomography were performed to detect the accumulation of fibrillary amyloid β peptide (Aβ), and characteristic pathologies of AD were examined with immunohistochemical staining of amyloid precursor protein (APP), Aβ, tau, and phosphorylated tau.
Results: For the transcriptional studies, results showed that a few genes out of 84 AD-related genes were significantly up-regulated at 4 h after irradiation and the other genes had no marked change; on the other hand, a few other genes showed a significant down-regulation, while the other genes had no marked change at 1 year after irradiation. For the behavioral studies, no significant difference on learning ability and memory was observed at 1 and 2 years after irradiation. Imaging and immunohistochemical staining showed no change in the accumulation of fibrillar amyloid and the expression of APP, Aβ, tau and phosphorylated tau were detectable in the animals 4 months and 2 years after irradiation.
Conclusion: These findings suggest that total-body irradiation at a dose of 0.10 Gy could hardly induce significant early or late transcriptional alterations in most of the AD-related genes in the hippocampus, learning disability and memory impairment, and AD-like pathological change in the brain in mice [ 3].
doi:10.1093/jrr/rrt189
PMCID: PMC3941512
total-body irradiation; low dose; Alzheimer's disease; Morris water maze test; Alzheimer's disease-like pathogenesis; mice
3.  Low-dose CT scan screening for lung cancer: comparison of images and radiation doses between low-dose CT and follow-up standard diagnostic CT 
SpringerPlus  2013;2:393.
Objectives
This study aim to compare image quality and radiation doses between low-dose CT and follow-up standard diagnostic CT for lung cancer screening.
Methods
In a single medical institution, 19 subjects who had been screened for lung cancer by low-dose CT before going through follow-up standard diagnostic CT were randomly selected. Both CT image sets for all subjects were independently evaluated by five specialized physicians.
Results
There were no significant differences between low-dose CT screening and follow-up standard diagnostic CT for lung cancer screening in all 11 criteria. The concordance rate for the diagnoses was approximately 80% (p < 0.001) for all categories. Agreement of the evaluation of all categories in the final diagnosis exceeded 94% (p < 0.001). Five physicians detecting and characterizing the pulmonary nodules did not recognized the difference between low-dose CT screening and follow-up standard diagnostic CT. With low-dose CT, the effective dose ranged between 1.3 and 3.4 mSv, whereas in the follow-up diagnostic CT, the effective dose ranged between 8.5 and 14.0 mSv.
Conclusion
This study suggests that low-dose CT can be effectively used as a follow-up standard diagnostic CT in place of standard-dose CT in order to reduce the radiation dose.
doi:10.1186/2193-1801-2-393
PMCID: PMC3755805  PMID: 24010047
Lung cancer; Screening CT; Llow-dose; WAZA-ARI; ImPACT
4.  Total body 100-mGy X-irradiation does not induce Alzheimer's disease-like pathogenesis or memory impairment in mice 
Journal of Radiation Research  2013;55(1):84-96.
The cause and progression of Alzheimer's disease (AD) are poorly understood. Possible cognitive and behavioral consequences induced by low-dose radiation are important because humans are exposed to ionizing radiation from various sources. Early transcriptional response in murine brain to low-dose X-rays (100 mGy) has been reported, suggesting alterations of molecular networks and pathways associated with cognitive functions, advanced aging and AD. To investigate acute and late transcriptional, pathological and cognitive consequences of low-dose radiation, we applied an acute dose of 100-mGy total body irradiation (TBI) with X-rays to C57BL/6J Jms mice. We collected hippocampi and analyzed expression of 84 AD-related genes. Mouse learning ability and memory were assessed with the Morris water maze test. We performed in vivo PET scans with 11C-PIB, a radiolabeled ligand for amyloid imaging, to detect fibrillary amyloid beta peptide (Aβ) accumulation, and examined characteristic AD pathologies with immunohistochemical staining of amyloid precursor protein (APP), Aβ, tau and phosphorylated tau (p-tau). mRNA studies showed significant downregulation of only two of 84 AD-related genes, Apbb1 and Lrp1, at 4 h after irradiation, and of only one gene, Il1α, at 1 year after irradiation. Spatial learning ability and memory were not significantly affected at 1 or 2 years after irradiation. No induction of amyloid fibrillogenesis or changes in APP, Aβ, tau, or p-tau expression was detected at 4 months or 2 years after irradiation. TBI induced early or late transcriptional alteration in only a few AD-related genes but did not significantly affect spatial learning, memory or AD-like pathological change in mice.
doi:10.1093/jrr/rrt096
PMCID: PMC3885129  PMID: 23908553
total-body X-irradiation; low dose; Alzheimer's disease; Morris water maze test; Alzheimer's disease-like pathogenesis; mice
5.  Size-Based Isolation of Circulating Tumor Cells in Lung Cancer Patients Using a Microcavity Array System 
PLoS ONE  2013;8(6):e67466.
Background
Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method.
Methods
Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer’s protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems.
Results
CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0–291 cells/7.5 mL) than by the CellSearch system (median 0, range 0–37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system.
Conclusions
The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.
doi:10.1371/journal.pone.0067466
PMCID: PMC3696066  PMID: 23840710
6.  Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in Caenorhabditis elegans 
PLoS Genetics  2012;8(10):e1002991.
Pre–mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre–mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre–mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre–mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre–mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA–binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT–PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.
Author Summary
Muscle is a specialized organ with specialized contractile apparatuses. A number of genes encoding contractile apparatus-related proteins undergo muscle-specific pre–mRNA processing. However, the molecular mechanisms and consequences of muscle-specific alternative pre–mRNA processing remain largely unknown. In this study, we reveal regulation mechanisms of pre–mRNA processing of the unc-60 gene locus, encoding two tissue-specific isoforms of ADF/cofilin in C. elegans. The unc-60A and unc-60B genes share only the first exon, and UNC-60B protein is specifically expressed in muscle. We visualize the tissue-specific processing patterns of the unc-60 pre–mRNA with green and red fluorescent proteins in living worms. We provide genetic, biochemical, and immunohistochemical evidence that muscle-specific RNA–binding proteins ASD-2 and SUP-12 cooperatively bind to specific motifs in intron 1A to retain intron 1A, which leads to skipping of exon 2A through 5A and splicing between exon 1 and 2B. Consistently, disruption of the splicing factors leads to expression of UNC-60A in muscle and suppresses paralysis of an unc-60B-specific mutant. Our study raises a model of step-by-step execution of complex co-transcriptional pre–mRNA processing and provides insight into the fate decision of the entire transcript.
doi:10.1371/journal.pgen.1002991
PMCID: PMC3469465  PMID: 23071450
7.  The two actin-interacting protein 1 genes have overlapping and essential function for embryonic development in Caenorhabditis elegans 
Molecular Biology of the Cell  2011;22(13):2258-2269.
AIP1 is a conserved enhancer of ADF/cofilin-dependent actin dynamics. Caenorhabditis elegans has two AIP1 genes. They have overlapping functions, and ablation of both AIP1 isoforms causes embryonic lethality with strong defects in the assembly of muscle sarcomeres.
Disassembly of actin filaments by actin-depolymerizing factor (ADF)/cofilin and actin-interacting protein 1 (AIP1) is a conserved mechanism to promote reorganization of the actin cytoskeleton. We previously reported that unc-78, an AIP1 gene in the nematode Caenorhabditis elegans, is required for organized assembly of sarcomeric actin filaments in the body wall muscle. unc-78 functions in larval and adult muscle, and an unc-78–null mutant is homozygous viable and shows only weak phenotypes in embryos. Here we report that a second AIP1 gene, aipl-1 (AIP1-like gene-1), has overlapping function with unc-78, and that depletion of the two AIP1 isoforms causes embryonic lethality. A single aipl-1–null mutation did not cause a detectable phenotype. However, depletion of both unc-78 and aipl-1 arrested development at late embryonic stages due to severe disorganization of sarcomeric actin filaments in body wall muscle. In vitro, both AIPL-1 and UNC-78 preferentially cooperated with UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament disassembly but not with UNC-60A, a nonmuscle ADF/cofilin. AIPL-1 is expressed in embryonic muscle, and forced expression of AIPL-1 in adult muscle compensated for the function of UNC-78. Thus our results suggest that enhancement of actin filament disassembly by ADF/cofilin and AIP1 proteins is critical for embryogenesis.
doi:10.1091/mbc.E10-12-0934
PMCID: PMC3128528  PMID: 21551072
8.  Detection of a troponin I-like protein in non-striated muscle of the tardigrades (water bears) 
Bioarchitecture  2011;1(2):96-102.
Tardigrades, also known as water bears, have somatic muscle fibers that are responsible for movement of their body and legs. These muscle fibers contain thin and thick filaments in a non-striated pattern. However, the regulatory mechanism of muscle contraction in tardigrades is unknown. In the absence of extensive molecular and genomic information, we detected a protein of 31 kDa in whole lysates of tardigrades that cross-reacted with the antibody raised against nematode troponin I (TnI). TnI is a component of the troponin complex that regulates actin-myosin interaction in a Ca2+-dependent and actin-linked manner. This TnI-like protein was co-extracted with actin in a buffer containing ATP and EGTA, which is known to induce relaxation of a troponin-regulated contractile system. The TnI-like protein was specifically expressed in the somatic muscle fibers in adult animals and partially co-localized with actin filaments in a non-striated manner. Interestingly, the pharyngeal muscle did not express this protein. These observations suggest that the non-striated somatic muscle of tardigrades has an actin-linked and troponin-regulated system for muscle contraction.
doi:10.4161/bioa.1.2.16251
PMCID: PMC3158627  PMID: 21866271
troponin; actin; actin-linked regulation; non-striated muscle; tardigrades
9.  Interaction between activated chemokine receptor 1 and FcεRI at membrane rafts promotes communication and F-actin-rich cytoneme extensions between mast cells 
International Immunology  2010;22(2):113-128.
Chemokines play important regulatory roles in immunity, but their contributions to mast cell function remain poorly understood. We examined the effects of FcεRI–chemokine receptor (CCR) 1 co-stimulation on receptor localization and cellular morphology of bone marrow-derived mast cells. Whereas FcεRI and CCR1 co-localized at the plasma membrane in unsensitized cells, sensitization with IgE promoted internalization of CCR1 molecules. Co-stimulation of FcεRI and CCR1 with antigen and macrophage inflammatory protein-1α was more effective than FcεRI stimulation alone in causing leading edge formation, flattened morphology, membrane ruffles and ganglioside (GM1+) lipid mediator release. Co-stimulation resulted in phalloidin-positive cytoneme-like cellular extensions, also known as tunneling nanotubes, which originated at points of calcium accumulation. This is the first report of cytoneme formation by mast cells. To determine the importance of lipid rafts for mast cell function, the cells were cholesterol depleted. Cholesterol depletion enhanced degranulation in resting, sensitized and co-stimulated cells, but not in FcεRI-cross-linked cells, and inhibited formation of filamentous actin+ cytonemes but not GM1+ cytonemes. Treatment with latrunculin A to sequester globular-actin abolished cytoneme formation. The cytonemes may participate in intercellular communication during allergic and inflammatory responses, and their presence in the co-stimulated mast cells suggests new roles for CCRs in immunopathology.
doi:10.1093/intimm/dxp118
PMCID: PMC2825160  PMID: 20173038
co-stimulation; F/G actin, lipid raft; MIP-1α; tunneling nanotubes
10.  Actin-ADF/cofilin rod formation in Caenorhabditis elegans muscle requires a putative F-actin binding site of ADF/cofilin at the C-terminus 
Under a number of stress or pathological conditions, actin and actin depolymerizing factor (ADF)/cofilin form rod-like structures that contain abnormal bundles of actin filaments that are heavily decorated with ADF/cofilin. However, the mechanism of actin rod formation and the physiological role of actin rods are not clearly understood. Here, we report that overexpression of green fluorescent protein-fused UNC-60B, a muscle-specific ADF/cofilin isoform, in Caenorhabditis elegans body wall muscle induces formation of rod-like structures. The rods contained GFP-UNC-60B, actin-interacting protein 1 (AIP1), and actin, but not other major actin-associated proteins, thus resembling actin-ADF/cofilin rods found in other organisms. However, depletion or overexpression of AIP1 did not affect formation of the actin-GFP-UNC-60B rods, suggesting that AIP1 does not play a significant role in the rod assembly. Truncation of the C-terminal tail, a putative F-actin binding site, of UNC-60B abolished induction of the rod formation, strongly suggesting that stable association of UNC-60B with F-actin, which is mediated by its C-terminus, is required for inducing actin-ADF/cofilin rods. This study suggests that C. elegans can be a new model to study functions of actin-ADF/cofilin rods.
doi:10.1002/cm.20383
PMCID: PMC2733252  PMID: 19459188
Actin filaments; nematode; myofibrils; stress; overexpression
11.  Preliminary Trial of Rebamipide for Prevention of Low-Dose Aspirin-Induced Gastric Injury in Healthy Subjects: A Randomized, Double-Blind, Placebo-Controlled, Cross-Over Study 
Although low-dose aspirin is widely used, since it is a cheap and effective means of prevention of cardiovascular events, it can cause hemorrhagic gastrointestinal complications. The aim of this study was to evaluate the efficacy of rebamipide in preventing low-dose aspirin-induced gastric injury. A randomized, double-blind, placebo-controlled, crossover trial was performed in twenty healthy volunteers. Aspirin 81 mg was administered with placebo or rebamipide 300 mg three times daily for 7 consecutive days. The rebamipide group exhibited significant prevention of erythema in the antrum compared with the placebo group (p = 0.0393, respectively). Results for the body and fornix did not differ significantly between the placebo and rebamipide groups. In conclusion, short-term administration of low-dose aspirin induced slight gastric mucosal injury in the antrum, but not in the body or fornix. Rebamipide may be useful for preventing low-dose aspirin-induced gastric mucosal injury, especially which confined to the antrum.
doi:10.3164/jcbn.09-24
PMCID: PMC2735640  PMID: 19794936
low-dose aspirin; healthy subject; rebamipide; prevention
12.  Essential role of ADF/cofilin for assembly of contractile actin networks in the C. elegans somatic gonad 
Journal of cell science  2008;121(Pt 16):2662-2670.
The somatic gonad of the nematode Caenorhabditis elegans has the myoepithelial sheath that surrounds oocytes and provides contractile forces during ovulation. Contractile apparatuses of the myoepithelial sheath cells are non-striated and similar to those of smooth muscle. We report identification of a specific isoform of actin depolymerizing factor (ADF)/cofilin as an essential factor for assembly of contractile actin networks in the gonadal myoepithelial sheath. Two ADF/cofilin isoforms, UNC-60A and UNC-60B, are expressed from the unc-60 gene by alternative splicing. RNA interference of UNC-60A caused disorganization of the actin networks in the myoepithelial sheath. UNC-60B, which is known to function in the body wall muscle, was not necessary or sufficient for actin organization in the myoepithelial sheath. However, mutant forms of UNC-60B with reduced actin filament severing activity rescued the UNC-60A-depletion phenotype. UNC-60A has much weaker filament severing activity than UNC-60B, suggesting that an ADF/cofilin with weak severing activity is optimal for assembly of actin networks in the myoepithelial sheath. In contrast, strong actin filament severing activity of UNC-60B was required for assembly of striated myofibrils in the body wall muscle. Our results suggest that an optimal level of actin filament severing activity of ADF/cofilin is required for assembly of actin networks in the somatic gonad.
doi:10.1242/jcs.034215
PMCID: PMC2572110  PMID: 18653537
Actin dynamics; severing; contraction; ovulation; myoepithelial cells
13.  Caenorhabditis elegans Expresses Three Functional Profilins in a Tissue-Specific Manner 
Profilins are actin binding proteins, which also interact with polyphosphoinositides and proline-rich ligands. On the basis of the genome sequence, three diverse profilin homologues (PFN) are predicted to exist in Caenorhabditis elegans. We show that all three isoforms PFN-1, PFN-2, and PFN-3 are expressed in vivo and biochemical studies indicate they bind actin and influence actin dynamics in a similar manner. In addition, they bind poly(l-proline) and phosphatidylinositol 4,5-bisphosphate micelles. PFN-1 is essential whereas PFN-2 and PFN-3 are nonessential. Immunostainings revealed different expression patterns for the profilin isoforms. In embryos, PFN-1 localizes in the cytoplasm and to the cell–cell contacts at the early stages, and in the nerve ring during later stages. During late embryogenesis, expression of PFN-3 was specifically detected in body wall muscle cells. In adult worms, PFN-1 is expressed in the neurons, the vulva, and the somatic gonad, PFN-2 in the intestinal wall, the spermatheca, and the pharynx, and PFN-3 localizes in a striking dot-like fashion in body wall muscle. Thus the model organism Caenorhabditis elegans expresses three profilin isoforms and is the first invertebrate animal with tissue-specific profilin expression.
doi:10.1002/cm.20102
PMCID: PMC2575421  PMID: 16317718
Profilin; Caenorhabditis elegans; actin cytoskeleton; poly(l-proline); polyphospho-inositides
14.  Elucidation of the mode of interaction in the UP1–telomerase RNA–telomeric DNA ternary complex which serves to recruit telomerase to telomeric DNA and to enhance the telomerase activity 
Nucleic Acids Research  2008;36(21):6816-6824.
We found that UP1, a proteolytic product of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), both enhances and represses the telomerase activity. The formation of the UP1–telomerase RNA–telomeric DNA ternary complex was revealed by a gel retardation experiment. The interactions in the ternary and binary complexes were elucidated by NMR. UP1 has two nucleic acid-binding domains, BD1 and BD2. In the UP1–telomerase RNA binary complex, both BD1 and BD2 interact with telomerase RNA. Interestingly, when telomeric DNA was added to the binary complex, telomeric DNA bound to BD1 in place of telomerase RNA. Thus, BD1 basically binds to telomeric DNA, while BD2 mainly binds to telomerase RNA, which resulted in the formation of the ternary complex. Here, UP1 bridges telomerase and telomeric DNA. It is supposed that UP1/hnRNP A1 serves to recruit telomerase to telomeric DNA through the formation of the ternary complex. A model has been proposed for how hnRNP A1/UP1 contributes to enhancement of the telomerase activity through recruitment and unfolding of the quadruplex of telomeric DNA.
doi:10.1093/nar/gkn767
PMCID: PMC2588520  PMID: 18953025
15.  Structural Components of the Nonstriated Contractile Apparatuses in the Caenorhabditis elegans Gonadal Myoepithelial Sheath and Their Essential Roles for Ovulation 
Ovulation in the nematode Caenorhabditis elegans is regulated by complex signal transduction pathways and cell–cell interactions. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells that provide contractile forces to push a mature oocyte into the spermatheca for fertilization. Although several genes that regulate sheath contraction have been characterized, basic components of the contractile apparatuses of the myoepithelial sheath have not been extensively studied. We identified major structural proteins of the contractile apparatuses of the myoepithelial sheath and characterized their nonstriated arrangement. Of interest, integrin and perlecan were found only at the dense bodies, whereas they localized to both dense bodies and M-lines in the striated body wall muscle. RNA interference of most of the myofibrillar components impaired ovulation in a soma-specific manner. Our results provide basic information that helps understanding the mechanism of sheath contraction during ovulation and establishing a new model to study morphogenesis of nonstriated muscle.
doi:10.1002/dvdy.21091
PMCID: PMC1994093  PMID: 17326220
actin; myosin; integrin; myofibrils; muscle; ovulation; contraction
16.  The RNA-binding Protein SUP-12 Controls Muscle-Specific Splicing of the ADF/cofilin Pre-mRNA in C. elegans 
The Journal of cell biology  2004;167(4):639-647.
Tissue-specific alternative pre-mRNA splicing is essential for increasing diversity of functionally different gene products. In Caenorhabditis elegans, UNC-60A and UNC-60B, nonmuscle and muscle isoforms of actin depolymerizing factor (ADF)/cofilin, are expressed by alternative splicing of unc-60 and regulate distinct actin-dependent developmental processes. We report that SUP-12, a member of a new family of RNA recognition motif (RRM) proteins, including SEB-4, regulates muscle-specific splicing of unc-60. In sup-12 mutants, expression of UNC-60B is decreased, whereas UNC-60A is up-regulated in muscle. sup-12 mutations strongly suppress muscle defects in unc-60B mutants by allowing expression of UNC-60A in muscle that can substitute for UNC-60B, thus unmasking their functional redundancy. SUP-12 is expressed in muscle and localized to the nuclei in a speckled pattern. The RRM domain of SUP-12 binds to several sites of the unc-60 pre-mRNA including the UG repeats near the 3′-splice site in the first intron. Our results suggest that SUP-12 is a novel tissue-specific splicing factor and regulates functional redundancy among ADF/cofilin isoforms.
doi:10.1083/jcb.200407085
PMCID: PMC1781344  PMID: 15545320
17.  Caenorhabditis elegans Kettin, a Large Immunoglobulin-like Repeat Protein, Binds to Filamentous Actin and Provides Mechanical Stability to the Contractile Apparatuses in Body Wall Muscle 
Molecular Biology of the Cell  2006;17(6):2722-2734.
Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with α-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction.
doi:10.1091/mbc.E06-02-0114
PMCID: PMC1474806  PMID: 16597697
18.  Enhancement of Actin-depolymerizing Factor/Cofilin-dependent Actin Disassembly by Actin-interacting Protein 1 Is Required for Organized Actin Filament Assembly in the Caenorhabditis elegans Body Wall MuscleD⃞ 
Molecular Biology of the Cell  2006;17(5):2190-2199.
Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal β-propeller, whereas one residue is located in the C-terminal β-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.
doi:10.1091/mbc.E05-11-1016
PMCID: PMC1446098  PMID: 16525019
19.  The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans 
The Journal of Cell Biology  2004;167(4):639-647.
Tissue-specific alternative pre-mRNA splicing is essential for increasing diversity of functionally different gene products. In Caenorhabditis elegans, UNC-60A and UNC-60B, nonmuscle and muscle isoforms of actin depolymerizing factor (ADF)/cofilin, are expressed by alternative splicing of unc-60 and regulate distinct actin-dependent developmental processes. We report that SUP-12, a member of a new family of RNA recognition motif (RRM) proteins, including SEB-4, regulates muscle-specific splicing of unc-60. In sup-12 mutants, expression of UNC-60B is decreased, whereas UNC-60A is up-regulated in muscle. sup-12 mutations strongly suppress muscle defects in unc-60B mutants by allowing expression of UNC-60A in muscle that can substitute for UNC-60B, thus unmasking their functional redundancy. SUP-12 is expressed in muscle and localized to the nuclei in a speckled pattern. The RRM domain of SUP-12 binds to several sites of the unc-60 pre-mRNA including the UG repeats near the 3′-splice site in the first intron. Our results suggest that SUP-12 is a novel tissue-specific splicing factor and regulates functional redundancy among ADF/cofilin isoforms.
doi:10.1083/jcb.200407085
PMCID: PMC1781344  PMID: 15545320
20.  TetraThymosinβ Is Required for Actin Dynamics in Caenorhabditis elegans and Acts via Functionally Different Actin-binding Repeats 
Molecular Biology of the Cell  2004;15(10):4735-4748.
Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinβ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinβ has four repeats, each related to the actin monomer-sequestering protein thymosinβ 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinβ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinβ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinβ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinβ is of crucial importance at specific developmental stages requiring actin polymerization.
doi:10.1091/mbc.E04-03-0225
PMCID: PMC519163  PMID: 15269284
21.  Tropomyosin and Troponin Are Required for Ovarian Contraction in the Caenorhabditis elegans Reproductive SystemV⃞ 
Molecular Biology of the Cell  2004;15(6):2782-2793.
Ovulation in the nematode Caenorhabditis elegans is coordinated by interactions between the somatic gonad and germ cells. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells, but the regulatory mechanism of their contraction is unknown. We show that contraction of the ovarian muscle requires tropomyosin and troponin, which are generally major actin-linked regulators of contraction of striated muscle. RNA interference of tropomyosin or troponin C caused sterility by inhibiting ovarian contraction that is required for expelling mature oocytes into the spermatheca where fertilization takes place, thus causing accumulation of endomitotic oocytes in the ovary. Tropomyosin and troponin C were associated with actin filaments in the myoepithelial sheath, and the association of troponin C with actin was dependent on tropomyosin. A mutation in the actin depolymerizing factor/cofilin gene suppressed the ovulation defects by RNA interference of tropomyosin or troponin C. These results strongly suggest that tropomyosin and troponin are the actin-linked regulators for contraction of ovarian muscle in the C. elegans reproductive system.
doi:10.1091/mbc.E04-03-0179
PMCID: PMC420102  PMID: 15064356
22.  Y chromosome microchimerism in rheumatic autoimmune disease 
Annals of the Rheumatic Diseases  2000;59(8):655-656.
doi:10.1136/ard.59.8.654b
PMCID: PMC1753213  PMID: 10991761
23.  Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics 
The Journal of Cell Biology  2002;156(6):1065-1076.
Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B–induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.
doi:10.1083/jcb.200110013
PMCID: PMC2173459  PMID: 11901171
myofibrils; thin filaments; actin; ADF/cofilin; tropomyosin
24.  Vertical and Oblique Saccade Disconjugacy in Strabismus 
Purpose.
Previous studies have shown that horizontal saccades are disconjugate in humans and monkeys with strabismus. The present study was designed to extend these results to vertical and oblique saccades. A major goal was to assess the conjugacy in terms of both amplitude and direction.
Methods.
Saccadic eye movements were recorded binocularly in three adult monkeys. One had normal eye alignment, one had exotropia resulting from a bilateral medial rectus tenotomy in the first week of life, and one had esotropia resulting from prism rearing during the first 3 months of life. We assessed the conjugacy of saccades in various directions by comparing both amplitude and direction.
Results.
Saccades in the strabismic monkeys were disconjugate in terms of both amplitude and direction. These effects were as large for vertical and oblique saccades as for horizontal ones. However, the pattern of disconjugacy often varied as a function of saccade direction. In some cases, saccades that appeared to be conjugate in terms of amplitude differed substantially when direction was taken into account.
Conclusions.
These data indicate that the assessment of saccade disconjugacy in strabismus may yield misleading results if direction is not considered. The complex pattern of disconjugacy suggests that strabismus is associated with substantial abnormalities within the circuitry controlling saccades. Neurophysiological studies are needed to identify the specific neural substrates for these behavioral effects.
We quantified the conjugacy of saccades in various directions in three monkeys (one normal, one exotrope, and one esotrope). For both strabismic animals, saccades in the two eyes often differed notably, in terms of both amplitude and direction.
doi:10.1167/iovs.13-13473
PMCID: PMC3891270  PMID: 24346173
saccade; strabismus; exotropia; esotropia; eye movement
25.  Suppressive effect of administration of recombinant human thioredoxin on cutaneous  inflammation caused by UV 
Bioengineered  2013;4(4):254-257.
Thioredoxin (TRX) is small ubiquitous protein, which regulates cellular redox status and scavenges reactive oxygen species (ROS). TRX has been shown to exert suppressive effect on skin inflammation where oxidative stress is involved in its pathogenesis. We investigated the effect of TRX on UVB response. Ear swelling after UVB irradiation was significantly reduced in TRX-transgenic mouse compared with wild-type mouse. Furthermore, we have demonstrated that intraperitoneal administration of recombinant human thioredoxin (rhTRX) also reduced acute skin inflammatory reaction, such as skin erythema and edema. Histologically, inflammatory cells including neutrophils and lymphocytes were significantly reduced and average size of the caliber of blood vessels were also reduced in rhTRX-injected mice. The number of apoptotic keratinocytes, were significantly reduced in rhTRX-injected mice. Immunohistochemical intensity of 8-hydroxy-2'-deoxyguanosine was strikingly reduced in rhTRX-injected mouse. Western blotting showed that administration of rhTRX inhibited phosphorylation of p38 mitogen-activated protein kinases and c-Jun NH2-terminal kinase, which play important roles in inflammatory and apoptotic signaling. These findings indicated that rhTRX attenuated inflammatory and apoptotic responses by UVB. Possible mechanisms for this might be via redox regulation of stress signaling and reduction of reactive oxygen species. We discussed the future use of TRX for sedative use of skin inflammation.
doi:10.4161/bioe.23612
PMCID: PMC3728197  PMID: 23328539
UVB; thioredoxin; oxidative stress; cytokine; nerutrophil; JNK; 8-OHdG; p38 MAPK; redox

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