Expression analysis of ciliary rootlet coiled coil protein mRNA during Xenopus development / 대한수의학회지

Ciliary rootlet coiled coil protein (CROCC), the structural component that originates from the basal body at the proximal end of the ciliary rootlet, plays a crucial role in maintaining the cellular integrity of ciliated cells. In the current study, we cloned Xenopus CROCC and performed the expression analysis. The amino acid sequence of Xenopus laevis was related to those of Drosophila, cow, goat, horse, chicken, mouse and human. Reverse transcription polymerase chain reaction analysis revealed that CROCC mRNA encoding a coiled coil protein was present maternally, as well as throughout early development. In situ hybridization indicated that CROCC mRNA occurred in the animal pole of embryo during gastrulation and subsequently in the presumptive neuroectoderm at the end of gastrulation. At tailbud stages, CROCC mRNA expression was localized in the anterior roof plate of the developing brain, pharyngeal epithelium connected to gills, esophagus, olfactory placode, intestine and nephrostomes of the pronephric kidney. Our study suggests that CROCC may be responsible for control of the development of various ciliated organs.

Sox9 regulates development of neural crest and otic placode in a time- and dose-dependent fashion / 동물의과학연구지

Neural crest and placodes share a number of important features, pointing to a possible common evolutionary origin. They both arise from the neural plate border, which is the boundary between the non-neural ectoderm and neural plate. The transcription factor Sox9 has been implicated in neural crest and otic placode induction in several species. To investigate the differential regulation of neural crest and otic placode induction by Sox9, a gain of function assay was performed using a hormone-inducible version of the Sox9 construct at different doses and time periods. Sox9 was expressed in both neural crest and otic placode cell populations in the same stage embryos by in situ hybridization. Using a gain of function approach, increased expression of neural crest marker (Snail2) and otic placode marker (Pax8) in Sox9-overexpressed embryos was observed. Higher dose of Sox9 reduced or eliminated both neural crest and placode cells in the embryos. Interestingly, otic placodes cells were more strongly affected as compared to neural crest cells. So, optimal dosage and timing of Sox9 expression are important for the development of the neural crest and otic placode. The development of the neural crest and otic placode are affected by Sox9 in a time- and dose-dependent manner.

Na, K-ATPase beta2 isoform (atp1b2) expressed in the retina of Xenopus / 동물의과학연구지

The ubiquitous Na, K-ATPase is a membrane-bound ion pump located in the plasma membrane in all animal cells and plays an essential role in a variety of cellular functions. Studies in several organisms have shown that this protein regulates different aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Na, K-ATPase is an important factor for retinal development, and combinations of the isoforms of each of its subunits are expressed in different cell types and determine its functional properties. In this study, we performed RT-PCR assay to determine temporal expression and in situ hybridization to determine spatial expression of Na, K-ATPase beta2 isoform (atp1b2) in Xenopus laevis. Focusing on retinal expression to distinguish the specific expression domain, we used retinal marker genes sox4, sox11, vsx1, and . Xenopus atp1b2 was expressed from late gastrulation to the tadpole stage. Using whole mount in situ hybridization, we showed that Xenopus atp1b2 was expressed broadly in the eye, the whole surface ectoderm, and gills. In situ hybridization on sections revealed detailed and specific expression in the outer nuclear layer of the retina, which consists of two major classes of photoreceptors, rods and cones, surface ectoderm, pharyngeal epithelium, and gills. These findings indicate that atp1b2 may play an important role for the development of Xenopus retina.

Variation of brachiocephalic muscle in a dog / 동물의과학연구지

The brachiocephalic muscle in domestic mammals is formed as a homology of the sternocleidomastoid muscle and the clavicular part of the deltoid muscle since it results from reduction of the clavicle as a clavicular intersection. The cranial insertions of the brachiocephalic muscle vary among species in domestic mammals. In the dog, the brachiocephalic muscle comprises three parts, which arise from the clavicular intersection and insert at the humerus, the dorsal cervical raphe, and the mastoid process of the temporal bone. These three parts are named the cleidobrachial muscle, the cervical part of the cleidocephalic muscle, and the mastoid part of the cleidocephalic muscle, respectively. This complexity could confuse veterinarians and complicate surgical procedures in this area. Information about the normal structure of this muscle, and any variation therein, would help to avoid such situations. During dissections of a male cross-breed dog, we found that the brachiocephalic muscle had two bellies located on the mastoid part of the cleidocephalic muscle that extended from the clavicular intersection to the wing of the atlas and the mastoid process of the temporal bone. They were innervated by the accessory nerve and the ventral branches of the second, third, and fifth cervical nerves, and they were supplied by the ascending branch of the superficial cervical artery. These bellies were considered to be a rare variation of the muscle. This is the second report of a brachiocephalic muscle variation in a dog, in which the mastoid part of the cleidocephalic muscle was made of two bellies inserted independently. Such variations should be considered during anatomical dissections and surgical procedures.

Synthetic Prion Peptide 106-126 Resulted in an Increase Matrix Metalloproteinases and Inflammatory Cytokines from Rat Astrocytes and Microglial Cells

It has been shown that the accumulation of prion in the cytoplasm can result in neurodegenerative disorders. Synthetic prion peptide 106-126 (PrP) is a glycoprotein that is expressed predominantly by neurons and other cells, including glial cells. Prion-induced chronic neurodegeneration has a substantial inflammatory component, and an increase in the levels of matrix metalloproteinases (MMPs) may play an important role in neurodegenerative development and progression. However, the expression of MMPs in PrP induced rat astrocytes and microglia has not yet been compared. Thus, in this study, we examined the fluorescence intensity of CD11b positive microglia and Glial Fibrillary Acidic Protein (GFAP) positive astrocytes and found that the fluorescent intensity was increased following incubation with PrP at 24 hours in a dose-dependent manner. We also observed an increase in interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) protein expression, which are initial inflammatory cytokines, in both PrP induced astrocytes and microglia. Furthermore, an increase MMP-1, 3 and 11 expressions in PrP induced astrocytes and microglia was observed by real time PCR. Our results demonstrated PrP induced activation of astrocytes and microglia respectively, which resulted in an increase in inflammatory cytokines and MMPs expression. These results provide the insight into the different sensitivities of glial cells to PrP.

Regulation of IGF System in Rats after Experimental Myocardial Infarction / 체질인류학회지

Insulin-like growth factor system (IGF system) has been reported to be associated with the variety of disorders of myocardial function. However, the effect of myocardial infarction (MI) on the IGF system has not been fully described. Thus, the present study was to investigate in more detail the changes of IGF system in the male rat following myocardial infarction (MI). Ligation of the left coronary artery was performed in male Sprague-Dawley male rats at 60 days of age. Control rats were obtained sham-operated animals. MI rats were sacrificed at 1, 3, 7, 14, and 30 day after ligation of left anterior descending coronary artery. Control rats were sacrificed on 30 day after thoracotomy. Myocardial infarct size was assessed by planimetry and perimetry. Serum and heart concentrations of IGF-I and -II were determined by radioimmunoassay. Serum levels of IGF-binding protein (IGFBP)-1 and IGFBP-3 were analyzed with a two-site immunoradiometric assay. Mean infarct size was 35.2~42.3% of the left ventricle after coronary occlusion in experimental groups. Serum levels of IGF-I were markedly reduced, but the levels of IGF-II were not altered in MI rats compared with shamligated controls. Serum IGFBP-I levels in MI rats were significantly increased at 1 and 3 day compared with sham rats. The levels of serum IGFBP-3 were significantly higher in the ligated rats. IGF-I levels of the infarct/periinfarct area of the left ventricle were significantly decreased in rats with myocardial infarction, whereas the levels of IGF-II remained unchanged. These results demonstrate that the IGF system is altered in the myocardial infarction and suggest that the IGF system plays an important role in the response of the heart to myocardial infarction.

Effect of Pdx1 Expression after Sox9 Depletion in the Early Pancreatic Development in African Clawed Frog (Xenopus laevis) / 대한해부학회지

The pancreas is a mixed exocrine and endocrine gland involved in the control of many homeostatic functions.During embryogenesis,the pancreas arises from dorsal and ventral evaginations of the foregut,which subse- quently fuse into a single organ.The characterization of early genes expressed in the developing pancreas is critical to understand its specification and differentiation.Pdx1 is one of the earliest markers of pancreatic development and a key molecule in its development.Sox proteins form a large class of transcriptional regulators implicated in the control of a variety of developmental processes.One member of this family,Sox9,is expressed in the developing pancreas, but little is known about the function of Sox9 in the developing pancreas.We further investigated Sox9 function during pancreatic development in Xenopus .Using a hormone-inducible inhibitory mutant of Sox9 ,we found that Pdx1 expres- sion was reduced in the ventral pancreatic buds in Sox9-depleted embryos.We suggest that Sox9 gene expression may be involved in pancreatic development in Xenopus.

A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound-healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.

Effect of Retinoic Acid on the Expression of PDGF Receptors During Palatogenesis / 대한해부학회지

Although cleft lip and palate are one of the most common craniofacial malformation, little is still known about the mechanism of the palate formation. Retinoic acid (RA) is known a teratogen, and cleft palate is induced by retinoic acid administration in the secondary palate formation stage. Many growth factors and their receptors are involved in the formation of the secondary palate. Here, we investigated the expression of PDGFR-alpha, and PDGFR-beta during palatogenesis after retinoid acid administration in mice by RT-PCR and immunohistochemistry. At E15.5, the opposing palatal shelves fused with one another in the control group, but the palatal shelves were not elevated and cleft palate was induced in the RA-treated group. In RT-PCR, PDGFR-beta was downregulated during palatogenesis after RA administration. In immunohistochemical experiment, PDGFR-alpha and PDGFR-beta were reduced in RA-induced group. Taken together, we suggest that PDGF receptors may be molecules involved in palate formation.

CEA and CA19-9 in the Tissue, Portal, and Peripheral Blood of Gastric Cancer Patients

BACKGROUND: To clarify the clinical significance of CEA and CA19-9 in patients with gastric cancer, we evaluated the correlation between tissue expression, the peripheral and the portal levels of these tumor markers, and ten clinicopathological factors, as well as the prognosis. METHODS: Surgical specimens from 40 patients with gastric cancer were examined by using immunohistochemical staining with anti-CEA and anti-CA19-9 monoclonal antibodies. Serum levels of CEA and CA19-9 in the portal and the peripheral blood were measured by using immunoradiometric assays. RESULTS: Positive values of the portal venous CEA were more common in patients with lymph-node metastasis, distant metastasis, and lymphatic invasion than in those without these factors. Curative surgery was performed in 50.5% of the patients with high portal CEA levels and in 90.6% of the patients with low portal CEA levels. Positive values of the peripheral venous CEA were significantly higher in cases with lymph-node metastasis. The positive rate of CA19-9 immunohistochemistry was significantly higher in patients with distant metastasis and in non-curative surgery. The positive rate of peripheral venous CA19-9 was higher in cases with distant metastasis. The three-year survival rate of patients with negative tissue CEA was significantly higher than that of patients with a positive result. The peripheral venous levels of CEA and CA19-9 reflected the portal venous levels accurately. CONCLUSIONS: These results suggest that immunohistochemical examination of CEA in patients with gastric cancer is useful for the evaluation of the biological aggressiveness and progression of the disease and can be used for making a prognosis.