Combined Mesenchymal Stem Cells and Cartilage Acellular Matrix Injection Therapy for Osteoarthritis in Goats.

BACKGROUND: Human umbilical cord blood-derived MSCs (hUCB-MSCs) have been studied in osteoarthritis (OA) and cartilage regeneration. Our previous study demonstrated that hUCB-MSCs combined with cartilage acellular matrix injection (CAM Inj.) represent potential therapeutic agents for structural improvement and anti-inflammatory effects in a rabbit model of OA. METHODS: Based on a previous study, this study has evaluated the safety and efficacy of hUCB-MSCs combined with CAM Inj. in an anterior cruciate ligament transection (ACLT) with medial meniscectomy (MMx) in a goat model. In this study, 27 goats were divided into 5 groups: normal (n = 3), OA (n = 6), OA + CAM Inj. (n = 6), OA + hUCB-MSCs (n = 6), and OA + hUCB-MSCs + CAM Inj. (n = 6). Lameness and radiographic parameters were assessed 6 months after administration, and macroscopic and histological evaluations of the goat articular cartilage were performed 6 months after intervention. RESULTS: The results showed significant improvement in lameness score only in the OA + hUCB-MSCs group at 5 months after treatment (*p < 0.05), whereas the K&L score showed significant improvement only in the OA + hUCB-MSCs + CAM Inj. group 6 months after intervention (*p < 0.05). In addition, the gross findings showed significance in OA + CAM Inj. and OA + hUCB-MSCs + CAM Inj. groups 6 months after treatment (*p < 0.05 and **p < 0.01). CONCLUSION: In conclusion, treatment with a combination of hUCB-MSCs and CAM Inj. reduced OA symptoms and induced effective cartilage tissue repair in a goat model. We suggest the combination of hUCB-MSCs and CAM Inj. as an alternative therapy for OA.

Claudin-5a knockdown attenuates blood-neural barrier in zebrafish.

Mammalian claudin-5 (cldn5), a zebrafish cldn5a homolog, is essential to blood-brain barrier (BBB) integrity. Previously, the existence of an endothelial tight junction-based BBB with cldn5a expression in the cerebral microvessels was reported in zebrafish. However, the role of cldn5a in the cerebral microvessels of developing zebrafish has not been elucidated. Here, we further investigated the functional integrity of cldn5a in developing zebrafish by injecting cldn5a morpholinos. At 7 days post-fertilization, cldn5a immunoreactivity was detected on the brain surface, ventricular ependyma, and cerebral mircovessels but disappeared following cldna5a knockdown. Cldn5a morphants showed size-selective leakage of tracers through the BBB and downregulated expression of glucose transporter 1 (glut1) in the cerebral microvessels. In addition, leakiness in the blood-cerebrospinal fluid barrier was observed, implying the overall abnormal development of blood-neural barriers. The results of our study suggest that cldn5a is required for building and maintaining the blood-neural barrier during zebrafish development.

Human Leukocyte Antigen Class I Pseudo-Homozygous Mesenchymal Stem Cells Derived from Human Induced Pluripotent Stem Cells.

Mesenchymal stem cells (MSC) are an important type of cell that are highly recognized for their safety and efficacy as a cell therapy agent. In order to obtain MSC, primary tissues (adipose tissue, bone marrow, and umbilical cord blood) must be used; however, these tissues, especially umbilical cord blood, are difficult to obtain due to various reasons, such as the low birth rate trend. In addition, to maximize the safety and efficacy of MSC as allogenic cell therapeutic agents, it is desirable to minimize the possibility of an immune rejection reaction after in vivo transplantation. This study tried to establish a novel method for producing induced pluripotent stem cells (iPSC)-derived MSC in which the human leukocyte antigen (HLA)-class I gene is knocked out. To do so, dermal fibroblast originated iPSC generation using Yamanaka 4-factor, HLA class I gene edited iPSC generation using CRISPR/Cas9, and differentiation from iPSC to MSC using MSC culture medium was utilized. Through this, HLA-A, B, and C pseudo-homozygous iPSC-derived MSC (KO iMSC) were produced by monoallelically knocking out the polymorphic HLA-A, B, and C genes, which are the major causes of immune rejection during allogenic cell transplantation. Produced KO iMSC possesses multipotency and it was safe in vivo to be able to be differentiated to cartilage. In addition, it was not attacked by natural killer cells unlike HLA class I null cells. In conclusion, KO iMSC that do not induce immune rejection during allogenic cell transplantation can be produced. In the future, KO iMSC can be successfully utilized as allogenic cell therapeutic agents for many recipients through HLA screening.

Paraburkholderia panacisoli sp. nov., a potentially antagonistic bacterium against the root rot fungal pathogen Cylindrocarpon destructans, isolated from ginseng cultivation soil.

A new bacterium, designated DCY113T, was isolated from ginseng cultivation soil in Gochang-gun, South Korea, and its taxonomic position identified by the polyphasic approach. 16S rRNA gene sequence analysis determined that this isolate belongs to the genus Paraburkholderia, and was closest to P. dipogonis DL7T (98.6%), P. phytofirmans PsJNT (98.5%), P. kirstenboschensis Kb15T (98.4%) and P. aromaticivorans BNT (98.1%). Strain DCY113T is Gram-reaction negative, strictly aerobic, rod-shaped, non-motile, and catalase and oxidase positive. The predominant isoprenoid quinone of DCY113T was ubiquinone Q-8. The major cellular fatty acids were C16:0, cyclo-C17:0 and the Summed feature 8 (C18:1ω7c and/or C18:1ω6c). The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and an unknown amino lipid (AL1). The G+C content of the genomic DNA was 62.2 mol%. Average nucleotide identity (ANI) between strain DCY113T and the related Paraburkholderia type strains were below the threshold value for species delineation. This low DNA relatedness in combination with phylogenetic and phenotypic tests indicates that strain DCY113T cannot be assigned to any recognized species. Strain DCY113T was also found to have antifungal activity against the pathogenic fungi Cylindrocarpon destructans. In conclusion, this study found DCY113T to be a novel species within the genus Paraburkholderia, for which the name P. panacisoli is proposed. The type strain is DCY113T (= KCTC 52951T = JCM 32098T).

Therapeutic Effects of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Combined with Cartilage Acellular Matrix Mediated Via Bone Morphogenic Protein 6 in a Rabbit Model of Articular Cruciate Ligament Transection.

Osteoarthritis (OA) is a general joint disease. Cartilage damage is associated with a decrease in the density of chondrocytes. Mesenchymal stem cells (MSCs) differentiate into adipocytes, osteocytes and chondrocytes, and are an excellent source of cell therapy. Cartilage-derived extracellular matrix (ECM) promotes chondrogenesis of MSCs. However, the role of MSCs stimulated by ECM is not well known in OA. The purpose of this study is to determine the role of specific factors generated by the application of ECM and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) in managing OA symptoms. Cartilage acellular matrix (CAM), which is a cartilage-derived ECM, was used to promote the chondrogenesis of UCB-MSCs. Induced MSCs were analyzed using chondrogenic markers (aggrecan, collagen type 2, and SOX9) and bone morphogenic protein 6 (BMP6). BMP6 is known to be involved in early chondrogenesis of MSCs. As a result, treatment with CAM significantly increased the expression of chondrogenic markers and BMP6 in UCB-MSCs. Treatment with recombinant human BMP6 also dramatically increased the levels of chondrogenic markers in UCB-MSCs. In addition, UCB-MSCs and CAM were used to evaluate OA symptom improvement in a rabbit articular cruciate ligament transection (ACLT) model. Application of UCB-MSCs and CAM enhanced not only the structure and synthesis of proteoglycan and collagen type 2 but also anti-inflammatory effects in both rabbit joint and synovial fluid. Moreover, the detection of human cells and involvement of BMP6 were confirmed in rabbit cartilage tissues. This study indicates that therapeutic potential of UCB-MSCs with CAM is mediated via BMP6 in OA.

Rhizobium panacihumi sp. nov., an isolate from ginseng-cultivated soil, as a potential plant growth promoting bacterium.

A novel bacterial strain designated DCY116T was isolated from ginseng-cultivated soil in Gochang-gun, Republic of Korea. Strain DCY116T, belongs to the genus Rhizobium, and is closely related to Rhizobium yantingense H66T (98.3%), Neorhizobium huautlense S02T (98.2%), Rhizobium soli DS-42T (98.1%), Rhizobium smilacinae PTYR-5T (97.9%), and Neorhizobium alkalisoli CCBAU 01393T (97.9%) based on 16S rRNA gene sequence analysis. Analysis of the housekeeping genes atpD, recA, and glnII showed low levels of sequence similarity (96.8%) between strain DCY116T and other closely related species. Strain DCY116T was Gram-stain negative, motile by peritrichous flagella, rod-shaped, strictly aerobic, catalase- and oxidase-positive. Q-10 was the predominant ubiquinone. The major cellular fatty acids were identified as C16:0 and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and an unknown lipid (L1-3). Genomic DNA G + C content of strain DCY116T was determined to be 57.2 mol%. DNA-DNA homology values between strain DCY116T and closely related species of the genus Rhizobium were lower than 40%. Strain DCY116T produced indole-3-acetic acid, siderophores, and was able to solubilize phosphate as a potential plant growth promoting bacterium. In conclusion, the results of this study support strain DCY116T as a novel species of the genus Rhizobium, for which the name Rhizobium panacihumi is proposed. The type strain is DCY116T (= KCTC 62017T = JCM 32251T).

Ornithinimicrobium panacihumi sp. nov., Antagonistic Bacteria Against Root Rot Fungal Pathogens, Isolated from Cultivated Ginseng Soil.

A Gram-positive bacterium (DCY118T) was isolated from ginseng-cultivated soil in Gochang-gun, Republic of Korea. This isolate was assigned to the genus Ornithinimicrobium and is closely related to Ornithinimicrobium kibberense K22-20T (98.8%), O. pekingense DSM 21552T (98.5%), O. algicola JC311T (98.2%), and O. humiphilum DSM 12362T (97.9%) based on 16S rRNA gene sequence analysis. However, strain DCY118T showed < 55% DNA-DNA homology with closely related reference strains. Cells were non-motile, non-sporulating, catalase- and oxidase-positive, aerobic, short rods, and cocci, and produced light-yellow, circular, and smooth colonies on TSA medium. MK-8(H4) was the predominant menaquinone. The major cellular fatty acids were iso-C15:0, anteiso-C15:0, and C16:0. The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI), an unknown phospholipid (PL1), an unknown amino lipid (AL1), and unidentified polar lipids (L1-5). The genomic DNA G+C content was 71.1 mol%. The peptidoglycan contained L-ornithine as the diagnostic diamino acid. Whole-cell sugars were composed of glucose, arabinose, and xylose. Overall, data collected from phenotypic and genotypic tests during this study indicated that strain DCY118T could not be assigned to a recognized species. Strain DCY118T showed antagonistic activity against the fungal pathogens causing root rot in ginseng, i.e., Fusarium solani (KACC 44891T) and Cylindrocarpon destructans (KACC 44660T). The results from this study confirm the DCY118T strain as a new species within the genus Ornithinimicrobium, for which the name Ornithinimicrobium panacihumi is proposed. The type strain is DCY118T (=KCTC 39962T=JCM 32156T).

Lysobacter panacihumi sp. nov., isolated from ginseng cultivated soil.

A Gram-negative, non-motile, aerobic, catalase-, and oxidasepositive bacterial strain, designated DCY117T, was isolated from ginseng cultivated soil in Gochang-gun, Republic of Korea, and was characterized taxonomically using a multifaceted approach. 16S rRNA gene sequence analysis revealed that strain DCY117T showed highest similarity to Lysobacter ruishenii CTN-1T (95.3%). Phylogenetic analysis revealed that closely related relatives of strain DCY117T were L. aestuarii S2-CT (95.1%), L. daejeonensis GH1-9T (95.0%), and L. caeni BUT-8T (94.9%). Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) were the major polar lipids of strain DCY117T. The major isoprenoid quinone was Q-8. The major cellular fatty acids of strain DCY117T were iso-C15:0, iso-C16:0, and summed feature 9 (comprising iso-C17:1ω9c and/or 10-methyl-C16:0). Genomic DNA G + C content was 61.8 mol%. On the basis of our findings, strain DCY117T is a novel species in the genus Lysobacter. We propose the name Lysobacter panacihumi sp. nov., and the type strain is DCY117T (= KCTC 62019T = JCM 32168T).

Assessment of radical scavenging, whitening and moisture retention activities of Panax ginseng berry mediated gold nanoparticles as safe and efficient novel cosmetic material.

Panax ginseng berry extract possess remarkable pharmacological effects on skin treatment such as anti-aging, antioxidant, promotor of collagen synthesis and alleviation against atopic dermatitis. In recent years, gold nanoparticles have gained much attention due to their extensive range of applications in particular in the field of drug delivery as a result of their biological compatibility and low toxicity. In a previous study, we designed and developed biocompatible gold and silver nanoparticles based on phytochemical profile and pharmacological efficacy of P. ginseng berry extract, we were able to reduce gold ions to nanoparticles through the process of green synthesis. However, its potential as a cosmetic ingredient is still unexplored. The aim of the present study is to investigate the moisture retention, in-vitro scavenging and whitening properties of gold nanoparticles synthesized from P. ginseng berry in cosmetic applications. Our findings confirm that P. ginseng berry mediated gold nanoparticles exhibited moisture retention capacity. In addition, MTT assay results confirmed that P. ginseng berry mediated gold nanoparticles are non-toxic to human dermal fibroblast and murine melanoma skin cells, possess scavenging activity, protect and provide alleviation against injured caused by H2O2-induced damage. In addition, P. ginseng berry mediated gold nanoparticles, significantly reduced melanin content and suppress tyrosinase activity in α-MSH-stimulated B16BL6 cells. We conclude that P. ginseng berry mediated gold nanoparticles are biocompatible and environmental affable materials and can be a potential novel cosmetic ingredient.

Enhanced Cell Growth of Adipocyte-Derived Mesenchymal Stem Cells Using Chemically-Defined Serum-Free Media.

The multipotency and anti-inflammatory effects of mesenchymal stem cells (MSCs) make them attractive for cell therapy in regenerative medicine. A large number of MSCs is required for efficient therapy owing to the low homing efficiency of MSCs to target sites. Furthermore, owing to limitations in obtaining sufficient amounts of MSCs, in vitro expansion of MSCs that preserves their differentiation and proliferative potential is essential. The animal factor included in culture media also limits clinical application. In this study, adipose-derived MSCs showed a significantly higher proliferation rate in STK2, a chemically-defined medium, than in DMEM/FBS. The expression of MSC surface markers was increased in the culture using STK2 compared to that using DMEM/FBS. Tri-lineage differentiation analyses showed that MSCs cultured in STK2 were superior to those cultured in DMEM/FBS. In addition, MSCs cultured in STK2 showed a reduced senescence rate, small and homogenous cell size, and were more genetically stable compared to those cultured in DMEM/FBS. Furthermore, secretome analysis showed that the expression of factors related to proliferation/migration, anti-inflammation, and differentiation were increased in STK2 culture medium compared to DMEM/FBS. Taken together, these results suggest that culture using STK2 medium offers many advantages through which it is possible to obtain safer, superior, and larger numbers of MSCs.

Aminoacyl-tRNA synthetase interacting multi-functional protein 1 attenuates liver fibrosis by inhibiting TGFß signaling.

The aminoacyl-tRNA synthetase interacting multi-functional protein 1 (AIMP1) participates in a variety of cellular processes, including translation, cell proliferation, inflammation and wound healing. Previously, we showed that the N-terminal peptide of AIMP1 (6-46 aa) induced ERK phosphorylation. Liver fibrosis is characterized by excessive deposition of extracellular matrix, which is induced by TGFß signaling, and activated ERK is known to induce the phosphorylation of SMAD, thereby inhibiting TGFß signaling. We assessed whether the AIMP1 peptide can inhibit collagen synthesis in hepatic stellate cells (HSCs) by activating ERK. The AIMP1 peptide induced phosphorylation of SMAD2 via ERK activation, and inhibited the nuclear translocation of SMAD, resulting in a reduction of the synthesis of type I collagen. The AIMP1 peptide attenuated liver fibrosis induced by CCl4, in a dose-dependent manner. Masson-Trichrome staining showed that the AIMP1 peptide reduced collagen deposition. Immunohistochemical staining showed that the levels of α-SMA, TGFß and type I collagen were all reduced by the AIMP1 peptide. Liver toxicity analysis showed that the AIMP1 peptide improved the levels of relevant biological parameters in the blood. These results suggest that AIMP1 peptide may have potential for development as a therapeutic agent to treat liver fibrosis.

Transplantation of human Wharton's jelly-derived mesenchymal stem cells highly expressing TGFß receptors in a rabbit model of disc degeneration.

INTRODUCTION: Mesenchymal stem cells (MSCs) are widely considered to hold promise for the treatment of intervertebral disc (IVD) degeneration. However, variation in the therapeutic efficacy of MSCs is a major problem and the derivation of MSCs for use in IVD regeneration has not been optimized. Additionally, no data are available on the efficacy of Wharton's Jelly-derived MSC (WJ-MSC) transplantation in an animal model of IVD degeneration. METHODS: This study evaluated the effectiveness of a cross-linked hyaluronic acid (XHA) scaffold loaded with human WJ-MSCs, according to their expression levels of transforming growth factor-ß receptor I/activin-like kinase receptor 5 (TßRI/ALK5) and TßRII, for IVD regeneration in a rabbit model. We compared the degree of IVD regeneration between rabbits transplanted with a XHA scaffold loaded with WJ-MSCs highly and lowly expressing TßRI/ALK5 and TßRII (MSC-highTR and MSC-lowTR, respectively) using magnetic resonance imaging (MRI) and histological analysis. RESULTS: At 12 weeks after transplantation, T2-weighted MRI analysis showed significant restoration of the disc water content in rabbits treated with a MSC-highTR-loaded XHA scaffold in comparison to rabbits treated with the scaffold alone or a MSC-lowTR-loaded XHA scaffold. In addition, morphological and histological analyses revealed that IVD regeneration was highest in rabbits transplanted with a MSC-highTR-loaded XHA scaffold. CONCLUSION: Taken together, our results suggest that a MSC-highTR-loaded XHA scaffold supports IVD regeneration more effectively than a MSC-lowTR-loaded XHA scaffold. This study supports the potential clinical use of MSC-highTR-loaded XHA scaffolds to halt IVD degeneration or to enhance IVD regeneration.

Poly-L-lysine increases the ex vivo expansion and erythroid differentiation of human hematopoietic stem cells, as well as erythroid enucleation efficacy.

Hematopoietic stem cells (HSCs) are continuously stimulated by physical interactions with bone marrow or umbilical cord niches as well as by chemical factors found within these niches. The niche can be mimicked by modification of the cytokine composition, elasticity, topography, and/or charge. This work employed cell culture plates coated with several concentrations of poly-L-lysine (PLL), a positively charged synthetic amino-acid chain. Culture substrates that employed relatively high initial coating concentrations of PLL significantly increased the total number of HSCs during ex vivo expansion of CD34(+) cells, as well as erythroid differentiation. Furthermore, the 0.01% PLL substrate stimulated enucleation of erythroid cells, leaving behind a number of extruded nuclei at the bottom of the culture plate, followed by an increase in the number of erythrocytes. Thus, PLL will likely prove useful to enhance the expansion of HSCs and erythroid cells, in addition to the generation of red blood cells.

AKAP12 regulates vascular integrity in zebrafish.

The integrity of blood vessels controls vascular permeability and extravasation of blood cells, across the endothelium. Thus, the impairment of endothelial integrity leads to hemorrhage, edema, and inflammatory infiltration. However, the molecular mechanism underlying vascular integrity has not been fully understood. Here, we demonstrate an essential role for A-kinase anchoring protein 12 (AKAP12) in the maintenance of endothelial integrity during vascular development. Zebrafish embryos depleted of akap12 (akap12 morphants) exhibited severe hemorrhages. In vivo time-lapse analyses suggested that disorganized interendothelial cell-cell adhesions in akap12 morphants might be the cause of hemorrhage. To clarify the molecular mechanism by which the cell-cell adhesions are impaired, we examined the cell-cell adhesion molecules and their regulators using cultured endothelial cells. The expression of PAK2, an actin cytoskeletal regulator, and AF6, a connector of intercellular adhesion molecules and actin cytoskeleton, was reduced in AKAP12-depleted cells. Depletion of either PAK2 or AF6 phenocopied AKAP12-depleted cells, suggesting the reduction of PAK2 and AF6 results in the loosening of intercellular junctions. Consistent with this, overexpression of PAK2 and AF6 rescued the abnormal hemorrhage in akap12 morphants. We conclude that AKAP12 is essential for integrity of endothelium by maintaining the expression of PAK2 and AF6 during vascular development.

Functional and developmental analysis of the blood-brain barrier in zebrafish.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and protecting the brain from toxic substances. Breakdown of this barrier results in severe brain pathologies, whereas impermeability of the BBB is a major obstacle for drug delivery to the brain. Despite its importance, our understanding of the maturation and modulation of the BBB is limited. Zebrafish (Danio rerio) has emerged as a useful model organism for studying vertebrate development and disease mechanisms, as well as for preclinical drug screening. However, the nature of the BBB has not yet been examined in teleost fish. In this paper, we report that with the exception of the circumventricular organs, the cerebral microvessels in zebrafish are impermeable to sulfo-NHS-biotin and horseradish peroxidase (HRP). Brain endothelial cells show immunoreactivity to Claudin-5 and Zonula Occludens-1 (ZO-1), implying the presence of tight junctions in these cells. The expression of Claudin-5 and ZO-1 was detected in cerebral microvessels from 3 days post-fertilization (dpf), concomitant with maturation of the BBB, as determined by restricted permeability to HRP and various fluorescent tracers. Real-time analysis of fluorescent tracer leakage in embryonic zebrafish suggests that they may be used as an in vivo model for BBB breakdown. Taken together, our results show that the endothelial tight junction-based BBB of zebrafish is similar to that of higher vertebrates and thus, zebrafish may be an excellent genetic and experimental model organism for studying development and maintenance of the BBB.

Meteorin regulates angiogenesis at the gliovascular interface.

Brain microvasculature requires a coordinated interaction between endothelial cells and astrocytes at the gliovascular interface. However, the role of the factors involved in that interaction and expressed by these cells is poorly understood. In this study, we demonstrate that Meteorin is highly expressed in astrocytes of the brain and retina during the late embryonic and postnatal stages of mouse development. Most notably, Meteorin is localized to the astrocyte endfeet that surround the blood vessels. To investigate the role of Meteorin in perivascular astrocytes, we depleted endogenous levels of Meteorin in cultured astrocytes using siRNA, and found that Meteorin attenuates angiogenic activity indirectly via astrocyte-derived thrombospondin-1/-2 (TSP-1/-2). Exogenous treatment of astrocytes with Meteorin protein also promotes astrocyte expression and secretion of TSP-1/-2. The conditioned media from the Meteorin-treated astrocytes attenuated angiogenic activity of microvascular endothelial cells. This activity was reversed by inhibiting the binding of TSP-1/-2 to its receptor. Furthermore, we found that TSP-1/-2 was co-localized with Meteorin in the developing brain. Therefore, our data strongly suggests that Meteorin is expressed and secreted by perivascular astrocytes and the secreted protein upregulates TSP-1/-2 to attenuate angiogenesis in the surrounding endothelial cells and to promote vascular maturation.

Cloning and characterization of zebrafish CTCF: Developmental expression patterns, regulation of the promoter region, and evolutionary aspects of gene organization.

CTCF is a nuclear phosphoprotein capable of using different subsets of its 11 Zn fingers (ZF) for sequence-specific binding to many dissimilar DNA CTCF-target sites. Such sites were identified in the genomic DNA of various multicellular organisms, in which the CTCF gene was cloned, including insects, birds, rodents, and primates. CTCF/DNA-complexes formed in vivo with different 50-bp-long sequences mediate diverse functions such as positive and negative regulation of promoters, and organization of all known enhancer-blocking elements ("chromatin insulators") including constitutive and epigenetically regulated elements. Abnormal functions of certain CTCF sites are implicated in cancer and in epigenetic syndromes such as BWS and skewed X-inactivation. We describe here the cloning and characterization of the CTCF cDNA and promoter region from zebrafish, a valuable vertebrate model organism. The full-length zebrafish CTCF cDNA clone is 4244 bp in length with an open reading frame (ORF) of 2391 bp that encodes 797 amino acids. The zebrafish CTCF amino acid sequence shows high identity (up to 98% in the zinc finger region) with human CTCF, and perfect conservation of exon-intron organization. Southern blot analyses indicated that the zebrafish genome contains a single copy of the CTCF gene. In situ hybridization revealed the presence of zebrafish CTCF transcripts in all early stages of embryogenesis. Transfection assays with luciferase reporter-constructs identified a core promoter region within 146 bp immediately upstream of the transcriptional start site of zebrafish CTCF that is located at a highly conserved YY1/Initiator element.

Effects of a Korean herbal formulation, Silsosangami, consisting of seven medicinal herbs, and its seven herbs on endotoxin-induced experimental thrombosis in rats.

A traditional Korean medicine, Silsosangami (SSG), consisting of seven different herbs of Typhae Pollen, Pteropi Faeces, Paeoniae Radicis rubra, Cnidii Rhizoma, Persicae Semen, Carthami Flos and Curcumae Tuber, has been reported to have a hypolipidemic effect in human subjects. In the present study, the inhibitory effects of SSG on a thrombosis in rats, induced by endotoxin treatment were examined. The anti-thrombic properties of SSG were also investigated with respect to blood parameters. The extracts of SSG and five of the seven herbs, except Cnidii Rhizoma and Carthami Flos, inhibited both endotoxin-induced disseminated intravascular coagulation (DIC) and thrombosis in rats. The extract also inhibited the endotoxin-induced decrease in blood platelets and fibrinogen, and the endotoxin-induced increase in fibrin degradation products (FDP) on disseminated intravascular coagulation in normal rats. In conclusion, the artificially induced, protective effects of SSG on ischemic infarction might be related to their inhibitory effects on DIC, platelet coagulation and thrombotic action.