Embryonic stem (ES) cells have potential for use in evaluation of developmental toxicity because they are generated in large numbers and differentiate into three germ layers following formation of embryoid bodies (EBs). In earlier study, embryonic stem cell test (EST) was established for assessment of the embryotoxic potential of compounds. Using EBs indicating the onset of differentiation of mouse ES cells, many toxicologists have refined the developmental toxicity of a variety of compounds. However, due to some limitation of the EST method resulting from species-specific differences between humans and mouse, it is an incomplete approach. In this regard, we examined the effects of several developmental toxic chemicals on formation of EBs using human ES cells. Although human ES cells are fastidious in culture and differentiation, we concluded that the relevancy of our experimental method is more accurate than that of EST using mouse ES cells. These types of studies could extend our understanding of how human ES cells could be used for monitoring developmental toxicity and its relevance in relation to its differentiation progress. In addition, this concept will be used as a model system for screening for developmental toxicity of various chemicals. This article might update new information about the usage of embryonic stem cells in the context of their possible ability in the toxicological fields.
Animals ; Embryoid Bodies ; Embryonic Stem Cells* ; Germ Layers ; Humans* ; Mass Screening ; Mice

Diabetes mellitus (DM) is becoming a lifestyle-related pandemic disease. Diabetic patients frequently develop electrolyte disorders, especially diabetic ketoacidosis or nonketotic hyperglycemic hyperosmolar syndrome. Such patients show characteristic potassium, magnesium, phosphate, and calcium depletion. In this review, we discuss a homeostatic mechanism that links calcium and DM. We also provide a synthesis of the evidence in favor or against this linking mechanism by presenting recent clinical indications, mainly from veterinary research. There are consistent results supporting the use of calcium and vitamin D supplementation to reduce the risk of DM. Clinical trials support a marginal reduction in circulating lipids, and some meta-analyses support an increase in insulin sensitivity, following vitamin D supplementation. This review provides an overview of the calcium and vitamin D disturbances occurring in DM and describes the underlying mechanisms. Such elucidation will help indicate potential pathophysiology-based precautionary and therapeutic approaches and contribute to lowering the incidence of DM.
Calcium Channels ; Calcium* ; Diabetes Mellitus* ; Diabetic Ketoacidosis ; Homeostasis* ; Humans ; Incidence ; Insulin Resistance ; Magnesium ; Pandemics ; Potassium ; Vitamin D

Alterations of genes are known to be critical for the induction of tumorigenesis, but the mechanism of ovarian carcinogenesis is little understood and remains to be elucidated. In this study, we investigated the roles of brca1, brca2 and p53 genes in the development of ovarian cancer using conditional knockout mice generated by a Cre-loxP recombinant system. Following the application of recombinant adenovirus expressing Cre in vitro, the proliferation of ovarian surface epithelium (OSE) was increased. For instance, a significant increase in cell growth was observed in OSE cells in vitro by conditional knockout isolated from the mice bearing concurrent floxed copies of brca1 and brca2/p53. However, the proliferative effect of the ovarian cells was not observed in concurrent brca1/brca2 or p53 knockout mice in vivo, indicating that we could not observe the direct evidence of the involvement of brca1, brca2, and p53 in ovarian carcinogenesis. Since morphological changes including tumor formation were not observed in mice bearing floxed copies of concurrent brca1/brca2 or p53, the inactivation of brca1/2 or p53 is not sufficient for the induction of tumor formation. Taken together, these results suggest that the deficiency of these genes may not be involved directly in the mechanism of ovarian carcinogenesis.
Animals ; BRCA1 Protein/*genetics/metabolism ; BRCA2 Protein/*genetics/metabolism ; Cell Proliferation ; Cell Transformation, Neoplastic/*genetics ; Epithelium/*pathology ; Extracellular Matrix Proteins/genetics ; Female ; Gene Silencing ; Mice ; Mice, Knockout ; Ovarian Neoplasms/*genetics ; Protein-Lysine 6-Oxidase/genetics ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53/*genetics/metabolism

Mesenchymal stem cells are classified as multipotent stem cells, due to their capability to transdifferentiate into various lineages that develop from mesoderm. Their popular appeal as cell-based therapy was initially based on the idea of their ability to restore tissue because of their differentiation potential in vitro; however, the lack of evidence of their differentiation to target cells in vivo led researchers to focus on their secreted trophic factors and their role as potential powerhouses on regulation of factors under different immunological environments and recover homeostasis. To date there are more than 800 clinical trials on humans related to MSCs as therapy, not to mention that in animals is actively being applied as therapeutic resource, though it has not been officially approved as one. But just as how results from clinical trials are important, so is to reveal the biological mechanisms involved on how these cells exert their healing properties to further enhance the application of MSCs on potential patients. In this review, we describe characteristics of MSCs, evaluate their benefits as tissue regenerative therapy and combination therapy, as well as their immunological properties, activation of MSCs that dictate their secreted factors, interactions with other immune cells, such as T cells and possible mechanisms and pathways involved in these interactions.
Animals ; Dinoprostone ; Homeostasis ; Humans ; Immunomodulation* ; In Vitro Techniques ; Mesenchymal Stromal Cells* ; Mesoderm ; Multipotent Stem Cells ; Regeneration* ; Regenerative Medicine ; T-Lymphocytes ; Toll-Like Receptors

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein expressed in tissues in the intestine, uterus, placenta, kidney, pituitary gland and bone. Its exact function is unknown, but it is considered to regulate intracytoplasmic concentration and transport of free ions (Ca2+). CaBP-9k protein is involved in intestinal calcium absorption in the intestine and in the regulation of myometrial activity by intracellular calcium in the uterus. Renal CaBP-9k protein is expressed at the site of calcium re-absorption in the kidney and expressed in distal convoluted tubules, where it is thought to facilitate calcium re-absorption. Expression of the CaBP-9k gene has been explored in most mammalians except in a canine model. Presently, we elucidated the expression of CaBP-9k mRNA and protein in the duodenum, kidney and uterus in a canine model involving two adult (2.5-year-old) female beagles. To collect tissues, the dogs were euthanized and then the abdominal cavity was exposed by midline incision. The proximal duodenum, cortex of kidney and uterine horn were collected. Expression of CaBP-9k mRNA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. CaBP-9k protein expression and localization were ascertained by Western blot analysis and immunohistochemistry, respectively. CaBP-9k mRNA was detected in the duodenum, but not in the kidney and uterus. Its protein was expressed only in the enterocytes of the duodenum. Taken together, the results indicate that CaBP-9k mRNA and protein are highly expressed in the enterocytes of the duodenum of a canine model, consistent with findings in other mammalian species.
Animals ; Blotting, Western/veterinary ; Calcium-Binding Protein, Vitamin D-Dependent/*biosynthesis/genetics ; Dogs/*physiology ; Duodenum/*physiology ; Female ; Immunohistochemistry/veterinary ; Kidney/*physiology ; RNA, Messenger/biosynthesis/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Transcription, Genetic ; Uterus/*physiology

Ectonucleotide pyrophosphatase/phosphodiestrase 2 (Enpp2) isolated from the supernatant of human melanoma cells is a lysophospholipase D that transforms lysophosphatidylcholine into lysophospatidic acid. Although multiple analyses have investigated the function of Enpp2 in the hypothalamus, its role in the uterus during the estrous cycle is not well understood. In the present study, rat uterine Enpp2 was analyzed by RT-PCR, Western blotting, and immunohistochemistry. Quantitative PCR analysis demonstrated that uterine Enpp2 mRNA was decreased during estrus compared to proestrus and diestrus. To determine whether uterine Enpp2 expression is affected by sex steroid hormones, immature rats were treated with 17beta-estradiol (E2), progesterone, or both on postnatal days 14 to 16. Interestingly, the expression of Enpp2 mRNA and protein were down-regulated by E2 in the uterus during estrus but not during proestrus or diestrus, suggesting that Enpp2 may play a role in uterine function during estrus. Enpp2 is primarily localized in the stromal cells of the endometrium during proestrus and estrus. During diestrus, Enpp2 was highly expressed in the epithelial cells of the endometrium. Taken together, these results suggest that uterine Enpp2 may be regulated by E2 and plays a role in reproductive functions during female rat development.
Animals ; Estradiol/pharmacology ; Estrous Cycle/*physiology ; Female ; Gene Expression Regulation/*physiology ; Immunohistochemistry ; Mifepristone/pharmacology ; Phosphoric Diester Hydrolases/genetics/*metabolism ; Progesterone/pharmacology ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Uterus/*metabolism

Tight junctions (TJs) form continuous intercellular contacts in intercellular junctions. TJs involve integral proteins such as occludin (OCLN) and claudins (CLDNs) as well as peripheral proteins such as zona occludens-1 (ZO-1) and junctional adhesion molecules (JAMs). TJs control paracellular transportation across cell-to-cell junctions. Although TJs have been studied for several decades, comparison of the transcriptional-translational levels of these molecules in canine organs has not yet been performed. In this study, we examined uterine expression of CLDNs, OCLN, junction adhesion molecule-A, and ZO-1 in canine. Expression levels of canine uterine TJ proteins, including CLDN1, 2, 4, 5, JAM-A, ZO-1, and OCLN, were measured using reverse transcription PCR, real-time PCR, and Western blotting, whereas TJs distribution was determined by immunohistochemistry. The mRNA and protein expression levels of OCLN, CLDN-1, 4, JAM-1, and ZO-1 were identified in the uterus. Immunohistochemistry demonstrated that TJs were localized to the endometrium and/or myometrium of the uterus. Our results show that canine TJ proteins, including CLDNs, OCLN, JAM-A, and ZO-1, were expressed in the canine uterus. Taken together, these proteins may perform unique physiological roles in the uterus. Therefore, these findings may serve as a basis for further studies on TJ proteins and their roles in the physiological or pathological condition of the canine uterus.
Animals ; Blotting, Western ; Claudins ; Dogs ; Endometrium ; Female ; Herpes Zoster ; Immunohistochemistry ; Intercellular Junctions ; Junctional Adhesion Molecules ; Mice ; Myometrium ; Occludin ; Physiology ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Reverse Transcription ; RNA, Messenger ; Tight Junctions* ; Transportation ; Uterus*

BACKGROUND/AIMS: Hypothyroidism has been reported in 36~85% of patients treated with sunitinib for renal cell carcinoma or gastrointestinal stromal tumor. However, the mechanism behind this hypothyroidism is unclear. This study evaluated the effects of sunitinib, a multi-target tyrosine kinase inhibitor, on the survival and proliferation of thyrocytes using FRTL-5 rat thyroid cells. METHODS: We examined the effect of sunitinib on cell proliferation in the presence and absence of thyroid stimulating hormone (TSH) in a colorimetric assay. Effects on the cell cycle were evaluated by flow cytometry, and on apoptosis using an annexin V apoptosis assay kit and by immunoblotting for caspase-3. Immunoblotting was also used to evaluate changes in the levels of intracellular proteins associated with the G1-S phase of the cell cycle. RESULTS: Sunitinib suppressed the proliferation of FRTL-5 cells in a dose- and time-dependent manner. This suppressive effect was enhanced by the presence of TSH (1 mU/mL). Sunitinib was subsequently shown, in flow cytometric analyses, to arrest the cell cycle at the G1-S phase. Furthermore, it induced apoptosis at a high concentration (15 micrometer) by activating caspase-3. G1-S phase arrest was associated with the induction of p27(kip1) and p21(cip1), whose expression is suppressed by TSH under control conditions. Sunitinib also decreased intracellular levels of cyclin D1 and cyclin-dependent kinase 2 in FRTL-5 cells. CONCLUSIONS: Sunitinib induced apoptosis in and suppressed the proliferation of FRTL-5 cells. Its suppression of proliferation was further enhanced by the presence of TSH. Sunitinib arrested the cell cycle in the G1-S phase by inducing the expression of p27(kip1)/p21(cip1), which are suppressed by TSH under normal conditions. Collectively, these findings suggest that sunitinib may interfere with TSH signaling pathways in normal thyrocytes.
Animals ; Annexin A5 ; Apoptosis ; Carcinoma, Renal Cell ; Caspase 3 ; Cell Cycle ; Cell Proliferation ; Cyclin D1 ; Cyclin-Dependent Kinase 2 ; Flow Cytometry ; Gastrointestinal Stromal Tumors ; Humans ; Hypothyroidism ; Immunoblotting ; Indoles ; Protein-Tyrosine Kinases ; Proteins ; Pyrroles ; Rats ; Thyroid Gland ; Thyrotropin

Claudins, which are known as transmembrane proteins play an essential role in tight junctions (TJs) to form physical barriers and regulate paracellular transportation. To understand equine diseases, it is helpful to measure the tissue-specific expression of TJs in horses. Major equine diseases such as colic and West Nile cause damage to TJs. In this study, the expression level and distribution of claudin-1, -2, -4, and -5 in eight tissues were assessed by Western blotting and immunohistochemistry methods. Claudin-1 was primarily identified in the lung, duodenum, and uterus, claudin-2 was evenly observed in equine tissues, claudin-4 was abundantly detected in the liver, kidney and uterus, and claudin-5 was strongly expressed in the lung, duodenum, ovary, and uterus, as determined by Western blotting method. The localization of equine claudins was observed by immunohistochemistry methods. These findings provide knowledge regarding the expression patterns and localization of equine claudins, as well as valuable information to understand tight junction-related diseases according to tissue specificity and function of claudins in horses.
Animals ; Architectural Accessibility ; Blotting, Western ; Claudin-1* ; Claudin-2 ; Claudin-4 ; Claudin-5 ; Claudins ; Colic ; Duodenum ; Female ; Horse Diseases ; Horses ; Immunohistochemistry ; Kidney ; Liver ; Lung ; Methods ; Organ Specificity ; Ovary ; Tight Junctions ; Transportation ; Uterus

PURPOSE: Salivary fluid formation is primarily driven by Ca2+-activated, apical efflux of chloride into the lumen of the salivary acinus. The anoctamin1 protein is an anion channel with properties resembling the endogenous calcium-activated chloride channels. In order to better understand the role of anoctamin proteins in salivary exocrine secretion, the expression of the ten members of the anoctamin gene family in the mouse submandibular gland was studied. METHODS: Total RNA extracted from mouse submandibular salivary glands was reverse transcribed using primer pairs to amplify the full-length coding regions of each anoctamin gene and was subcloned into plasmid vectors for DNA sequencing. Alternative splice variants were also screened by polymerase chain reaction using primer pairs that amplified six overlapping regions of the complementary DNA of each anoctamin gene, spanning multiple exons. RESULTS: Multiple anoctamin transcripts were found in the mouse submandibular salivary gland, including full-length transcripts of anoctamin1, anoctamin3, anoctamin4, anoctamin5, anoctamin6, anoctamin9, and anoctamin10. Exon-skipping splicing in the N-terminal exons of the anoctamins1, anoctamin5, and anoctamin6 genes resulted in multiple alternative splice variants. No expression of anoctamin2, anoctamin7, or anoctamin8 was found. CONCLUSIONS: The predominant anoctamin transcript expressed in the mouse submandibular gland is anoctamin1ac. The chloride channel protein produced by anoctamin1ac is likely responsible for the Ca2+-activated chloride efflux, which is the rate-limiting step in salivary exocrine secretion.
Alternative Splicing ; Animals ; Chloride Channels ; Clinical Coding ; DNA, Complementary ; Exons ; Humans ; Mice* ; Plasmids ; Polymerase Chain Reaction ; RNA ; Salivary Glands* ; Sequence Analysis, DNA ; Submandibular Gland