Tracing the Origin of the RSPO2 Long-Hair Allele and Epistatic Interaction between FGF5 and RSPO2 in Sapsaree Dog.

Genetic analysis of the hair-length of Sapsaree dogs, a Korean native dog breed, showed a dominant mode of inheritance for long hair. Genome-Wide Association Study (GWAS) analysis and subsequent Mendelian segregation analysis revealed an association between OXR1, RSPO2, and PKHD1L1 on chromosome 13 (CFA13). We identified the previously reported 167 bp insertion in RSPO2 3' untranslated region as a causative mutation for hair length variations. The analysis of 118 dog breeds and wolves revealed the selection signature on CFA13 in long-haired breeds. Haplotype analysis showed the association of only a few specific haplotypes to the breeds carrying the 167 bp insertion. The genetic diversity in the neighboring region linked to the insertion was higher in Sapsarees than in other Asian and European dog breeds carrying the same variation, suggesting an older history of its insertion in the Sapsaree genome than in that of the other breeds analyzed in this study. Our results show that the RSPO2 3' UTR insertion is responsible for not only the furnishing phenotype but also determining the hair length of the entire body depending on the genetic background, suggesting an epistatic interaction between FGF5 and RSPO2 influencing the hair-length phenotype in dogs.

Genetic diversity and population structure of the Sapsaree, a native Korean dog breed.

BACKGROUND: The Sapsaree is a breed of dog (Canis familiaris) native to Korea, which became perilously close to extinction in the mid-1980s. However, with systematic genetic conservation and restoration efforts, this breed was rescued from extinction and population sizes have been gradually increasing over the past few decades. The aim of this study was to ascertain novel information about the genetic diversity, population structure, and demographic history of the Sapsaree breed using genome-wide single nucleotide polymorphism data. We characterized the genetic profile of the Sapsaree breed by comparison with seven foreign dog breeds with similar morphologies to estimate genetic differentiation within and among these breeds. RESULTS: The results suggest that Sapsarees have higher genetic variance compared with the other breeds analyzed. The majority of the Sapsarees in this study share a discrete genetic pattern, although some individuals were slightly different, possibly as a consequence of the recent restoration process. Concordant results from analyses of linkage disequilibrium, effective population size, genetic diversity, and population structural analyses illustrate a relationship among the Sapsaree and the Tibetan breeds Tibetan terrier and Lhasa Apso, and a small genetic introgression from European breeds. The effective population size of the Sapsaree has contracted dramatically over the past generations, and is currently insufficient to maintain long-term viability of the breed's genetic diversity. CONCLUSIONS: This study provides novel insights regarding the genetic diversity and population structure of the native Korean dog breed Sapsaree. Our results suggest the importance of a strategic and systematic approach to ensure the genetic diversity and the authenticity of the Sapsaree breed.

Whole-transcriptome analyses of the Sapsaree, a Korean natural monument, before and after exercise-induced stress.

BACKGROUND: The Sapsaree (Canis familiaris) is a Korean native dog that is very friendly, protective, and loyal to its owner, and is registered as a natural monument in Korea (number: 368). To investigate large-scale gene expression profiles and identify the genes related to exercise-induced stress in the Sapsaree, we performed whole-transcriptome RNA sequencing and analyzed gene expression patterns before and after exercise performance. RESULTS: We identified 525 differentially expressed genes in ten dogs before and after exercise. Gene Ontology classification and KEGG pathway analysis revealed that the genes were mainly involved in metabolic processes, such as programmed cell death, protein metabolic process, phosphatidylinositol signaling system, and cation binding in cytoplasm. The ten Sapsarees could be divided into two groups based on the gene expression patterns before and after exercise. The two groups were significantly different in terms of their basic body type (p ≤ 0.05). Seven representative genes with significantly different expression patterns before and after exercise between the two groups were chosen and characterized. CONCLUSIONS: Body type had a significant effect on the patterns of differential gene expression induced by exercise. Whole-transcriptome sequencing is a useful method for investigating the biological characteristics of the Sapsaree and the large-scale genomic differences of canines in general.

Genome analysis of the domestic dog (Korean Jindo) by massively parallel sequencing.

Although pioneering sequencing projects have shed light on the boxer and poodle genomes, a number of challenges need to be met before the sequencing and annotation of the dog genome can be considered complete. Here, we present the DNA sequence of the Jindo dog genome, sequenced to 45-fold average coverage using Illumina massively parallel sequencing technology. A comparison of the sequence to the reference boxer genome led to the identification of 4 675 437 single nucleotide polymorphisms (SNPs, including 3 346 058 novel SNPs), 71 642 indels and 8131 structural variations. Of these, 339 non-synonymous SNPs and 3 indels are located within coding sequences (CDS). In particular, 3 non-synonymous SNPs and a 26-bp deletion occur in the TCOF1 locus, implying that the difference observed in cranial facial morphology between Jindo and boxer dogs might be influenced by those variations. Through the annotation of the Jindo olfactory receptor gene family, we found 2 unique olfactory receptor genes and 236 olfactory receptor genes harbouring non-synonymous homozygous SNPs that are likely to affect smelling capability. In addition, we determined the DNA sequence of the Jindo dog mitochondrial genome and identified Jindo dog-specific mtDNA genotypes. This Jindo genome data upgrade our understanding of dog genomic architecture and will be a very valuable resource for investigating not only dog genetics and genomics but also human and dog disease genetics and comparative genomics.

The effects of various antioxidants on the development of parthenogenetic porcine embryos.

The major objective of this study was to improve the development rate of parthenogenetic porcine embryos. In this study, the anti-oxidative and anti-apoptotic effects of three antioxidants, ß-mercaptoethanol (ß-ME), α-tocopherol, and extracellular superoxide dismutase (EC-SOD), were examined on the development of parthenogenetic porcine embryos. The development rate of parthenogenetic porcine embryos to the blastocyst stage was 8.1% for control; 19.1%, 14.6%, and 5.0% for 1, 3, and 5 µM ß-ME; 17.2% and 17.5% for 50 and 100 µM α-tocopherol and 12.0% and 4.0% for EC-SOD transgenic mouse embryonic fibroblast (Tg-MEF) and EC-SOD non-transgenic mouse embryonic fibroblast (NTg-MEF) conditioned medium at day 3, respectively. Here, ß-ME, α-tocopherol, and EC-SOD Tg-MEF conditioned medium increased the development rate of parthenogenetic porcine embryos to the blastocyst stage (P < 0.05). The average number of total cells and apoptotic cells at the blastocyst was analyzed at the optimal conditions of the three antioxidants. The three antioxidants increased the average number of total cells at the blastocyst, and they decreased apoptotic cells at the blastocyst as compared to control without supplementation (P < 0.05). When the reactive oxygen species levels in two-cell embryos after 1 µM ß-ME and 100 µM α-tocopherol treatment were examined, those were lower than control group (P < 0.05). In conclusion, it was found that the three antioxidants, ß-mercaptoethanol, α-tocopherol, and EC-SOD Tg-MEF, conditioned medium can play a role as a strong stimulator in the development of parthenogenetic porcine embryos.

Detection of low-abundant novel transcripts in mouse hematopoietic stem cells.

Gene expression profiles of hematopoietic stem cells (HSCs) provide clues for understanding molecular mechanisms of HSC behavior, including self-renewal and differentiation. We took advantage of serial analysis of gene expression (SAGE) to identify medium- and low-abundant transcripts expressed in HSCs/hematopoietic progenitor cells (HPCs). Among a total of 31,380 unique transcripts, 17,326 (55%) correspond to known genes and, 14,054 (45%) are low-copy transcripts that have no matches to currently known genes. Among the former class, 3,899 (23%) were alternatively spliced transcripts and 3,754 (22%) represent anti-sense transcripts from known genes. Mapping of the SAGE tags to the mouse genome showed that differences in gene expression exist among chromosomes. In addition, comparison of the HSCs/HPCs SAGE data to that of myeloid progenitor cells revealed that massive genetic reprogramming occurs in hematopoietic cell differentiation. Our results demonstrate a previously unrecognized complexity of gene expression in HSCs/HPCs, and indicate the need for further efforts to fully identify and characterize the transcripts expressed in this cell type.

Canine polydactyl mutations with heterogeneous origin in the conserved intronic sequence of LMBR1.

Canine preaxial polydactyly (PPD) in the hind limb is a developmental trait that restores the first digit lost during canine evolution. Using a linkage analysis, we previously demonstrated that the affected gene in a Korean breed is located on canine chromosome 16. The candidate locus was further limited to a linkage disequilibrium (LD) block of <213 kb composing the single gene, LMBR1, by LD mapping with single nucleotide polymorphisms (SNPs) for affected individuals from both Korean and Western breeds. The ZPA regulatory sequence (ZRS) in intron 5 of LMBR1 was implicated in mammalian polydactyly. An analysis of the LD haplotypes around the ZRS for various dog breeds revealed that only a subset is assigned to Western breeds. Furthermore, two distinct affected haplotypes for Asian and Western breeds were found, each containing different single-base changes in the upstream sequence (pZRS) of the ZRS. Unlike the previously characterized cases of PPD identified in the mouse and human ZRS regions, the canine mutations in pZRS lacked the ectopic expression of sonic hedgehog in the anterior limb bud, distinguishing its role in limb development from that of the ZRS.

Effect of proton beam on blood vessel formation in early developing zebrafish (Danio rerio) embryos.

Proton beam therapy can kill tumor cells while saving normal cells because of its specific energy delivery properties and so is used to various tumor patients. However, the effect of proton beam on angiogenesis in the development of blood vessels has not been determined. Here we used the zebrafish model to determine in vivo whether proton beam inhibits angiogenesis. Flk-1-GFP transgenic embryos irradiated with protons (35 MeV, spread out Bragg peak, SOBP) demonstrated a marked inhibition of embryonic growth and an altered fluorescent blood vessel development in the trunk region. When cells were stained with acridine orange to evaluate DNA damage, the number of green fluorescent cell death spots was increased in trunk regions of irradiated embryos compared to non-irradiated control embryos. Proton beam also significantly increased the cell death rate in human umbilical vein endothelial cells (HUVEC), but pretreatment with N-acetyl cystein (NAC), an antioxidant, reduced the proton-induced cell death rate (p<0.01). Moreover, pretreatment with NAC abrogated the inhibition of trunk vessel development and prevented the trunk malformation caused by proton irradiation. In conclusion, proton irradiation significantly inhibited in vivo vascular development possibly due to increased vascular cell death via reactive oxygen species formation.

Human erythropoietin induces lung failure and erythrocytosis in transgenic mice.

We have expressed human erythropoietin (EPO) in transgenic mice using a recombinant EPO cDNA combined with a partial TPO construct. The gene was microinjected using standard techniques and five mice were detected as transgenic by PCR and further used as founders. The life span of the transgenic founders was much shorter than that of their normal littermates. Most of the tissues of the transgenic founders contained human EPO transcripts as judged by RT-PCR. Especially high expression levels were seen in the liver and lung. EPO protein levels in serum were examined by ELISA and ranged from 266, 414 mIU/ml. The number of red blood cell, white blood cell and hemoglobin in the hEPO transgenic mice was higher than in normal mice. These results indicate that overexpression of hEPO is deleterious and can provoke lung failure and erythrocytosis.

Effect of conditioned medium of mouse embryonic fibroblasts produced from EC-SOD transgenic mice in nuclear maturation of canine oocytes in vitro.

The rate of in vitro maturation (IVM) of canine oocytes has not improved in comparison to that of other mammalian species. This study aims to improve the efficiency of canine oocytes IVM using the antioxidant, extracellular superoxide dismutase (EC-SOD). Thus, the effect of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts cultured with MEF culture medium (DMEM + 5% FBS) for in vitro nuclear maturation in canine oocytes was investigated. In experiment I, oocytes were collected from the ovaries of domestic bitches, which were allotted to one of two groups: (1) TCM199 + 1% FBS (n = 108) or (2) DMEM + 5% FBS (n = 112), cultured for 48 h and investigated for in vitro nuclear maturation of canine oocytes using Hoechst staining. Meiotic progression to metaphase II in group 1 was 1.8% compared to 1.8% in group 2. In experiment II, EC-SOD levels were examined in NTg-CMEF and Tg-CMEF at 0, 2 and 4 days obtained from EC-SOD transgenic mice generated in our laboratory. The concentration of EC-SOD in Tg-CMEF at day 2 (371.7 +/- 3.1 ng/ml) was the highest for all groups (P < 0.05). EC-SOD levels in Tg-CMEF were higher than in NTg-CMEF; therefore, the efficiency of Tg-CMEF for IVM was investigated. In experiment III, oocytes were allotted to one of three groups: (1) Tg-CMEF at day 0 (n = 84), (2) Tg-CMEF at day 2 (n = 92) or (3) Tg-CMEF at day 4 (n = 98), cultured for 48 h and the IVM of canine oocytes investigated. The mean percentage of MII oocytes in IVM was 2.4, 4.4 and 2.0% for groups 1, 2 and 3, respectively. In experiment IV, the effects of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts (Tg-CMEF) cultured in MEF culture medium were compared with conditioned medium acquired from non-transgenic mouse embryonic fibroblasts (NTg-CMEF) on IVM of canine oocytes. In this experiment, meiotic progression to metaphase II was 7.1% in Tg-CMEF versus 0% in NTg-CMEF (P < 0.05). Tg-CMEF was more effective than NTg-CMEF. In conclusion, it was verified that canine oocytes were able to effectively progress to metaphase II in IVM when cultured in Tg-CMEF.

The parthenogenetic activation of canine oocytes with Ca-EDTA by various culture periods and concentrations.

In the present study, canine oocytes were exposed to various concentrations of and durations of exposure to EDTA saturated with Ca(2+) (Ca-EDTA), a cell membrane-impermeable metal ion chelator, to determine if parthenogenetic activation could be induced. When oocytes were cultured for 48 or 72 h in parthenogenetic activation medium (PAM) without Ca-EDTA (control) or PAM supplemented with 1 or 5mM Ca-EDTA, the highest rate of pronuclear formation (PN) was obtained in oocytes cultured in 1mM Ca-EDTA for 48 h (8.0%; P<0.05). There was no pronuclear formation in the control group (PAM without Ca-EDTA). Oocytes treated with 5mM Ca-EDTA for 48 h or 1mM Ca-EDTA for 72 h formed a parthenogenetic pronucleus (3.1 and 4.5, respectively). However, there was no pronuclear formation in oocytes treated with 5mM Ca-EDTA for 72 h. In summary, exposure to Ca-EDTA can induce pronuclear formation in canine oocytes.

Characterization of a brain tumor cell line established from transgenic mice expressing the vasopressin SV-40 T antigen.

We previously reported that transgenic mice produced with a transgene consisting of the SV40 T antigen and vasopressin without the 3'-flanking region exhibit brain tumors and lymphoma. In this study, transgenic mice were produced with the fusion gene containing the SV40 T antigen and the whole vasopressin gene with the 3'-flanking region. Six transgenic mice were generated, five which died after 2-6 weeks. The remaining founder mouse was investigated for fusion gene expression and tumor progression at the age of 6 weeks. Brain tumor cells were characterized for phenotypes and transgene expression. During in vitro cell cultures, the phenotypic appearances at 10, 20, and 30 passages were as a uniform monolayer with similar growth rates. The site of SV40 T antigen integration was in the A2 region of chromosome 11, and SV40 T antigen was expressed at the same level in cells of both earlier and later passages. Thirty passages were probably insufficient to reach crisis and immortalization. These cells enriched brain tumor cell compositions with astrocytes and neuronal cells.

Calcium-binding protein calretinin immunoreactivity in the dog superior colliculus.

We studied calretinin-immunoreactive (IR) fibers and cells in the canine superior colliculus (SC) and studied the distribution and effect of enucleation on the distribution of this protein. Localization of calretinin was immunocytochemically observed. A dense plexus of anti--calretinin-IR fibers was found within the upper part of the superficial gray layer (SGL). Almost all of the labeled fibers were small in diameter with few varicosities. The intermediate and deep layers contained many calretinin-IR neurons. Labeled neurons within the intermediate gray layer (IGL) formed clusters in many sections. By contrast, labeled neurons in the deep gray layer (DGL) did not form clusters. Calretinin-IR neurons in the IGL and DGL varied in morphology and included round/oval, vertical fusiform, stellate, and horizontal neurons. Neurons with varicose dendrites were also labeled in the IGL. Most of the labeled neurons were small to medium in size. Monocular enucleation produced an almost complete reduction of calretinin-IR fibers in the SC contralateral to the enucleation. However, many calretinin-IR cells appeared in the contralateral superficial SC. Enucleation appeared to have no effect on the distribution of calretinin-IR neurons in the contralateral intermediate and deep layers of the SC. The calretinin-IR neurons in the superficial dog SC were heterogeneous small- to medium-sized neurons including round/oval, vertical fusiform, stellate, pyriform, and -horizontal in shape. Two-color immunofluorescence revealed that no cells in the dog SC -expressed both calretinin and GABA. Many horseradish peroxidase (HRP)-labeled retinal ganglion cells were seen after injections into the superficial layers. The vast majority of the double-labeled cells (HRP and calretinin) were small cells. The present results indicate that antibody to calretinin labels subpopulations of neurons in the dog SC, which do not express GABA. The results also suggest that the calretinin-IR afferents in the superficial layers of the dog SC originate from small class retinal ganglion cells. The expression of calretinin might be changed by the cellular activity of selective superficial collicular neurons. These results are valuable in delineating the basic neurochemical architecture of the dog visual system.

Antitumor effect of the cinnamaldehyde derivative CB403 through the arrest of cell cycle progression in the G2/M phase.

Cinnamaldehydes have been shown to have inhibitory effects on farnesyl protein transferase (FPTase; EC 2.5.1.29) in vitro, angiogenesis, cell-cell adhesion, and tumor cell growth and to be immunomodulators. However, the mechanisms responsible for these effects remain unknown. To elucidate the molecular mechanism of the cinnamaldehyde derivative CB403 for growth inhibition, CB403 was synthesized from 2'-hydroxycinnamaldehyde. CB403-treated cells were weakly adherent to the culture dishes. In addition, CB403 inhibited tumor growth in these cells in a concentration-dependent manner. FACS analysis using human cancer cells treated with this compound showed cell cycle arrest in mitosis, which was correlated with a marked increase in the amount of cyclin B1. Furthermore, CB403 blocked in vivo growth of human colon and breast tumor xenografts without loss of body weight in nude mice. These results support the hypothesis that the cinnamaldehyde derivative CB403 exerts cytostatic properties by inducing mitotic arrest in cancer cells.

Biological effects of G1 phase arrest compound, sesquicillin, in human breast cancer cell lines.

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.

Identification of genes differentially expressed between gastric cancers and normal gastric mucosa with cDNA microarrays.

To identify genes whose alterations lead to gastric cancer, gene expression profiles have been obtained from 22 gastric cancer tissues and their surrounding gastric mucosa tissues. A total of 16 genes were differentially expressed in more than 50% of gastric cancer tissues compared with surrounding gastric mucosa tissues. Genes such as HMG-Y, fibroblast collagenase inhibitor, and osteopontin are among those that are overexpressed in over 50% of the gastric cancer tissues. Dihydrodiol dehydrogenase, ribonuclease A, and glutathione peroxidase are among those genes that are underexpressed in over 50% of the gastric cancer tissues. We identified genes that are associated with clinical phenotypes of patients with gastric cancers. Alpha-II spectrin, Na/K-ATPase and KIAA0111 are those that are enhanced in intestinal type of gastric cancer. Gene such as platelet-endothelial tetraspan antigen 3 was enhanced in highly metastatic gastric cancer tissues.

MAPK signaling is involved in camptothecin-induced cell death.

Camptothecin, a topoisomerase I inhibitor, is a well-known anticancer drug. However, its mechanism has not been well studied in human gastric cancer cell lines. Camptothecin induced apoptotic cell death in human gastric cancer cell line AGS. Z-VAD-fmk, pan-caspase inhibitor, blocked apoptotic phenotypes induced by camptothecin suggesting that caspases are involved in camptothecin-induced cell death. An inhibitor of caspase-6 or -8 or -9 did not prevent cell death by camptothecin. Various protease inhibitors failed to prevent camptothecin-induced cell death. These results suggest that only few caspases are involved in camptothecin-induced cell death. Camptothecin induced phosphorylation of ERK1/2, JNK, and p38 MAPK, in a dose and time-dependent manner in AGS. Z-VAD-fmk did not affect MAPK signaling induced by camptothecin suggesting that caspase signaling occurs downstream of MAPK signaling. Blocking of p38 MAPK, but not ERK1/2, resulted in partial inhibition of cell death and PARP cleavage by camptothecin in AGS. Taken together, MAPK signaling is associated with apoptotic cell death by camptothecin.