ABSTRACT
BACKGROUND: In the livestock industry, Foreign Body Syndrome is a devastating disease condition. Feeding management, lacking of food discrimination, and eating chopped food increase the risk of swallowing sharp foreign bodies in bovine species. In addition to the honeycomb cells shape of the reticulum, the contractions of the reticular wall, gravid uterine pressure, and parturition efforts, foreign bodies can penetrate the reticular wall, causing cascade of problems including traumatic reticulitis, traumatic reticuloperitonitis, and traumatic pericarditis. The present study was carried out to evaluate the diagnostic significance of cardiac troponin I rapid test cassette and other cardiac biomarkers including serum cardiac troponin I (cTn I), creatine kinase-myocardial band (CK-MB), lactate dehydrogenase (LDH), and aspartate aminotransferase enzyme (AST), in confirmed cases of traumatic pericarditis (TP) and/or traumatic reticuleoperitonitis (TRP) in cattle and buffaloes. METHODS: A total number of 30 animals (22 cattle and 8 buffaloes) with different signs such as anorexia, jugular distension, brisket edema, and signs of pain (reluctance to move, arching back, and abduction of the forelimbs) were included in the present study. Based on case history, clinical signs, ferroscopic, pericardiocentesis, radiographic and ultrasonographic examinations, TP were confirmed in cattle (n = 10) and buffaloes (n = 8) while TRP were confirmed only in cattle (n = 12). Additionally, 20 clinically healthy animals (n = 10 cattle and 10 buffaloes) were used as a control group. Blood samples were collected for determination of blood level of Tn-I, and activity of CK-MB, LDH, and AST. RESULTS: The obtained results revealed a highly significant increase in serum cTn I in diseased cattle with TP and TRP (P = 0.00), while buffaloes with TP showed no significant changes in serum cTn I (P = 0.111). Both diseased cattle and buffaloes showed increased serum activities of CK-MB, AST, and LDH enzyme. On the other hand, cardiac troponin I rapid test cassette failed to detect cTn I in diseased animals. CONCLUSION: The study concluded that the cardiac troponin I rapid test cassette did not have a diagnostic significance and could not be used as a point-of-care under field condition for diagnosis of TP and TRP in large ruminants. However, the serum troponin I level is helpful in diagnosis of TP and TRP in cattle. Although cardiac biomarkers have some diagnostic values in TP and TRP, the traditional diagnostic methods (clinical, radiography and ultrasonography examinations) are crucial for thorough evaluation of TP/TRP cases in bovine.
Subject(s)
Biomarkers , Buffaloes , Cattle Diseases , Creatine Kinase, MB Form , Pericarditis , Reticulum , Troponin I , Animals , Pericarditis/veterinary , Pericarditis/diagnosis , Pericarditis/blood , Cattle , Biomarkers/blood , Troponin I/blood , Cattle Diseases/diagnosis , Cattle Diseases/blood , Creatine Kinase, MB Form/blood , Female , Peritonitis/veterinary , Peritonitis/diagnosis , Peritonitis/blood , L-Lactate Dehydrogenase/blood , Aspartate Aminotransferases/blood , Male , Foreign Bodies/veterinary , Foreign Bodies/complications , Foreign Bodies/diagnosisABSTRACT
Ingestion of metallic and/or sharp foreign bodies triggers cases of traumatic reticuloperitonitis and its sequelae in cattle. Among these sequelae, we can highlight traumatic reticulosplenitis, that has high mortality, although its frequency in the ruminant medicine is low. Therefore, based on the scarcity of information on this disease, the current study aimed to evaluate the clinical, laboratory, ultrasonographic, and pathological findings of 30 adult cattle diagnosed with traumatic reticulosplenitis. Clinical, ultrasound, and anatomopathological findings were analyzed using descriptive statistics and laboratory data were evaluated using measures of central tendency. Clinically the animals presented dehydration and alterations in behavior, appetite, and ruminal motility. Hematological findings revealed neutrophilic leukocytosis (37077.17±25004.59cell/µL) with regenerative left shift and hyperfibrinogenemia (1130±364.98mg/dL). The ultrasound examination enabled visualization of mobile and echogenic filaments that corresponded to the presence of fibrin adhesions. Displacement of the reticulum and irregularity in its contour, as well as alterations in the quantity, pattern, and amplitude of reticular contractions were also observed. Splenic alterations such as abscesses were found, characterized as circular structures of varying sizes delimited by capsules containing variable echogenicity. Splenic vein thrombosis and spleen folding were also observed. The results obtained in the current study indicated that traumatic reticulosplenitis causes nonspecific clinical signs, severe laboratory alterations and, mainly, that ultrasound is an efficient method for the diagnosis of this disease, since the anatomopathological lesions confirmed the ultrasound findings.(AU)
A ingestão de corpos estranho de origem metálica e/ou pontiagudos desencadeia em bovinos, quadros de Reticuloperitonite Traumática e suas sequelas. Dentre as quais podemos destacar a retículo esplenite traumática cuja letalidade é elevada, embora a mesma apresente uma baixa frequência na clínica de ruminantes. Portanto, baseado na escassez de informações sobre esta enfermidade, este trabalho teve por objetivo avaliar os achados clínicos, laboratoriais, ultrassonográficos e anatomopatológicos de 30 bovinos adultos diagnosticados com retículo esplenite traumática. Os achados clínicos, ultrassonográfico e anatomopatológico foram analisados por meio de estatística descritiva, e os dados laboratoriais foram avaliados utilizando-se as medidas de tendência central. Clinicamente os animais apresentaram desidratação e alterações no comportamento, apetite e na motilidade ruminal. Os achados hematológicos revelaram leucocitose (37077.17±25004.59cell/µL) por neutrofilia com desvio à esquerda regenerativo e hiperfibrinogenemia (1130±364.98mg/dL). O exame ultrassonográfico possibilitou a visualização de filamentos móveis e ecogênicos que corresponderam à presença de aderências fibrinosas, observou-se também, deslocamento do retículo e irregularidade no seu contorno além das alterações na quantidade, padrão e amplitude das contrações reticulares. Permitiu ainda, a constatação de alterações esplênicas como abscessos que foram caracterizados como estruturas circulares de variados tamanhos delimitada por capsula contendo no seu interior conteúdo de ecogenicidade variável. Trombose da veia esplênica e dobramento do baço. Os resultados obtidos nesse trabalho, indicaram que a retículo esplenite traumática causa sinais clínicos inespecíficos, severas alterações laboratoriais e principalmente que a ultrassonografia é um método eficiente para o diagnóstico dessa enfermidade uma vez que as lesões anatomopatológicas confirmaram os achados ultrassonográficos.(AU)
Subject(s)
Animals , Cattle , Peritonitis/veterinary , Peritonitis/diagnostic imaging , Reticulum/injuries , Reticulum/diagnostic imaging , Spleen/diagnostic imaging , Stomach Diseases/veterinary , Stomach Diseases/diagnostic imaging , Foreign-Body Reaction/veterinary , Ultrasonography/veterinaryABSTRACT
BACKGROUND: Chronic hyperglycemia has deleterious effects on pancreatic β-cell function and turnover. Recent studies support the view that cyclin-dependent kinase 5 (CDK5) plays a role in β-cell failure under hyperglycemic conditions. However, little is known about how CDK5 impair β-cell function. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. In this study, we examined the effect of myricetin on high glucose (HG)-induced β-cell apoptosis and explored the relationship between myricetin and CDK5. METHODS: To address this question, we subjected INS-1 cells and isolated rat islets to HG conditions (30 mM) in the presence or absence of myricetin. Docking studies were conducted to validate the interaction between myricetin and CDK5. Gene expression and protein levels of endoplasmic reticulum (ER) stress markers were measured by real-time reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: Activation of CDK5 in response to HG coupled with the induction of ER stress via the down regulation of sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) gene expression and reduced the nuclear accumulation of pancreatic duodenal homeobox 1 (PDX1) leads to β-cell apoptosis. Docking study predicts that myricetin inhibit CDK5 activation by direct binding in the ATP-binding pocket. Myricetin counteracted the decrease in the levels of PDX1 and SERCA2b by HG. Moreover, myricetin attenuated HG-induced apoptosis in INS-1 cells and rat islets and reduce the mitochondrial dysfunction by decreasing reactive oxygen species production and mitochondrial membrane potential (Δψm) loss. CONCLUSION: Myricetin protects the β-cells against HG-induced apoptosis by inhibiting ER stress, possibly through inactivation of CDK5 and consequent upregulation of PDX1 and SERCA2b.
Subject(s)
Animals , Rats , Apoptosis , Blotting, Western , Calcium-Transporting ATPases , Cyclin-Dependent Kinase 5 , Diabetes Mellitus, Type 2 , Down-Regulation , Endoplasmic Reticulum Stress , Endoplasmic Reticulum , Gene Expression , Genes, Homeobox , Glucose , Hyperglycemia , Insulin-Secreting Cells , Membrane Potential, Mitochondrial , Polymerase Chain Reaction , Reactive Oxygen Species , Reticulum , Reverse Transcription , Up-RegulationABSTRACT
Cyclic ADP-ribose (cADPR) releases Ca²⁺ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca²⁺ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca²⁺ ([Ca²⁺](i)) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca²⁺ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.
Subject(s)
Action Potentials , Adenosine Diphosphate , Adenosine Triphosphatases , Calcium , Cyclic ADP-Ribose , Cytochalasin B , Endoplasmic Reticulum , Homeostasis , Muscle Cells , Myocytes, Cardiac , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases , Reticulum , Ryanodine Receptor Calcium Release Channel , TyrosineABSTRACT
Intracellular calcium (Ca²⁺) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H₂O₂) on intracellular Ca²⁺ accumulation in mouse pancreatic acinar cells. Perfusion of H₂O₂ at 300 µM resulted in additional elevation of intracellular Ca²⁺ levels and termination of oscillatory Ca²⁺ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca²⁺. Antioxidants, catalase or DTT, completely prevented H₂O₂-induced additional Ca²⁺ increase and termination of Ca²⁺ oscillation. In Ca²⁺-free medium, H₂O₂ still enhanced CCh-induced intracellular Ca²⁺ levels and thapsigargin (TG) mimicked H₂O₂-induced cytosolic Ca²⁺ increase. Furthermore, H₂O₂-induced elevation of intracellular Ca²⁺ levels was abolished under sarco/endoplasmic reticulum Ca²⁺ ATPase-inactivated condition by TG pretreatment with CCh. H₂O₂ at 300 µM failed to affect store-operated Ca²⁺ entry or Ca²⁺ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca²⁺ uniporter blocker, failed to attenuate H₂O₂-induced intracellular Ca²⁺ elevation. These results provide evidence that excessive generation of H₂O₂ in pathological conditions could accumulate intracellular Ca²⁺ by attenuating refilling of internal Ca²⁺ stores rather than by inhibiting Ca²⁺ extrusion to extracellular fluid or enhancing Ca²⁺ mobilization from extracellular medium in mouse pancreatic acinar cells.
Subject(s)
Animals , Mice , Acinar Cells , Antioxidants , Calcium , Carbachol , Catalase , Cell Membrane , Cytosol , Extracellular Fluid , Hydrogen Peroxide , Hydrogen , Ion Transport , Pancreatitis , Perfusion , Reactive Oxygen Species , Reticulum , Ruthenium Red , ThapsigarginABSTRACT
Seven Baladi goats of both sexes (9 to 24 months old) were used to describe the distribution pattern of ki-67, alpha smooth muscle actin (áSMA) and vimentin (VIM) in the reticulum and omasum. This study was carried out using the avidin-biotin immunoperoxidase method. Ki-67 immunostaining was restricted to the basal cells layer in the epithelia of both the reticulum and omasum, suggesting the importance of ki-67 in epithelial cells proliferation and keratin biosynthesis. Immunostaining for áSMA was detected in smooth muscle cells in reticular folds, omasal lamiae and muscularis in both the reticulum and omasum, indicating the critical role of áSMA in muscular motility. The widespread distribution of VIM immunostainings in epithelia, fibroblasts in lamina propria and submucosa, and endothelia of blood vessels supports the importance of VIM as an intermediate filament protein. Detection of VIM in glial cells of enteric plexuses indicates its supportive role in the nervous control of both reticulum and omasum. Overall, this immunohistochemical study revealed non-significant differences in the expression of ki-67, áSMA, and VIM between the reticulum and omasum. This study thus verifies the important roles of ki-67, áSMA and VIM in the structure and function of the reticulum and omasum of Baladi goats
No disponible
Subject(s)
Animals , Ki-67 Antigen/analysis , Vimentin/biosynthesis , Actins/biosynthesis , Reticulum/physiology , Omasum/physiology , Muscle, Smooth/physiology , Goats/physiology , Immunohistochemistry/methodsABSTRACT
PURPOSE: To investigate the biological effects of preservative-free artificial tear drops on cultured human corneal epithelial cells in vitro. METHODS: The effects of the preservative-free artificial tear drops (Kynex(R) 0.1%, Kynex II(R) 0.18% [Alcon, Seoul, Korea] and Hyaluni eye drops(R) 0.15%, 0.3% [Taejun, Seoul, Korea]) on the human corneal epithelial cells were evaluated. An methyl thiazolyl tetrazolium (MTT)-based colorimetric assay was performed to assess the cellular metabolic activity and a lactate dehydrogenase (LDH) leakage assay was used to determine cellular toxicity. The eye drop ingredients were analyzed for electrolyte composition, pH, and osmolarity. We performed a scratch assay and cellular morphology test using electronic microscopy. RESULTS: The metabolic activity of corneal epithelial cells was higher than controls at 24 hours after exposure and then decreased at 48 and 72 hours after exposure (p < 0.05). The LDH titers of the 4 eye drops were higher compared with controls (p < 0.05). Sodium hyaluronate 0.18% contained lower concentrations of Na+ or Cl- and showed lower osmolarity values compared with the other eye drops. The cellular migration based on the scratch assay was more delayed and cellular damage such as loss of microvilli, rough endothelial reticulum (RER), and mitochondria dilatation was greater than controls based on electron microscopy. CONCLUSIONS: Long-term exposure to preservative-free sodium hyaluronate eye drops may induce decreased metabolic activity and cellular damage. Thus, preservative-free artificial tears should be used carefully to prevent cellular toxicity.
Subject(s)
Humans , Cornea , Dilatation , Epithelial Cells , Epithelium , Hyaluronic Acid , Hydrogen-Ion Concentration , L-Lactate Dehydrogenase , Microscopy , Microscopy, Electron , Microvilli , Mitochondria , Ophthalmic Solutions , Osmolar Concentration , Reticulum , Seoul , Sodium , TearsABSTRACT
The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.
Subject(s)
Humans , Bacteria , Bacterial Toxins , Calcium , Calcium Signaling , Calcium-Transporting ATPases , Cytokines , Epithelial Cells , Epithelium , Gram-Positive Bacteria , Inflammation , Inositol 1,4,5-Trisphosphate Receptors , Interleukin-8 , Peptidoglycan , Periodontal Diseases , Phospholipases , Reticulum , Thapsigargin , Toll-Like Receptor 2 , Toll-Like Receptors , Type C PhospholipasesABSTRACT
Six forestomachs of yaks (Bos grunniens) were studied with gross dissection and histological methods. It was found that the forestomach of yak consisted of the following three parts, rumen, reticulum and omasum, which were composed of the mucosa, submucosa, muscularis and serosa. In addition, the mucosal epithelium was covered with stratified squamous epithelium, with part of keratinized the shallow cells. Rumen, the mucosa of which formed ligulate papillae varying in size and shape, was no muscularis mucosa. Reticulum, consisted of a surface epithelium that invaginated to various extent into the lamina propria, formed various folds in shape, namely, grid-like small rooms. Furthermore, there are many secondary folds densely covered with keratinized papillae. The most striking feature of the omasum was to be formed the laminae omasi varying in length, with short and rough papillae distributing on both sides. Taken together, there was no glands within the mucosa and lamina propria of forestomach of yak, where diffuse lymphoid tissues can be observed clearly. It is, therefore, believed that the yak forestomach may have evolved those specific structural characteristics in response to the unique living environment and dietary habits impose on the Qinghai-Tibetan Plateau.
Seis preestómagos de yaks (Bos grunniens) fueron estudiados mediante disección macroscópica y métodos histológicos. Se encontró que el preestómago del yak constaba de tres partes: rumen, retículo y omaso, compuestas de mucosa, submucosa, muscular y serosa. Además, el epitelio de la mucosa se conformó con epitelio escamoso estratificado, con parte de células cornificadas superficiales. En el rumen, la mucosa formó papilas linguladas que variaron en tamaño y forma. El retículo, consistió en una superficie epitelial que se invaginó en distinta medida en la lámina propia, conformando varias formas de pliegues, es decir, cuadrículas como pequeños cubículos. Además, existían muchos pliegues secundarios densamente cubiertos con papilas cornificadas. La característica más llamativa del omaso, fue formar láminas que variaron en longitud, con papilas cortas y ásperas distribuidas en ambos lados. Tomados en conjunto, no hubo glándulas dentro de la mucosa y la lámina propia del preestómago del yak, donde los tejidos linfoides difusos se pueden observar claramente. Por lo tanto, creemos que esas características estructurales específicas del preestómago del yak pudieron haber evolucionado en respuesta a las condiciones de vida únicas y hábitos dietéticos que se presentan en la meseta de Qinghai-Tíbet.
Subject(s)
Animals , Male , Female , Omasum/anatomy & histology , Reticulum/anatomy & histology , Rumen/anatomy & histology , Cattle/anatomy & histology , Stomach, Ruminant/anatomy & histology , TibetABSTRACT
PURPOSE: To investigate the chemotherapeutic effect of quercetin against cancer cells, signaling pathway of apoptosis was explored in human pancreatic cells. METHODS: Various anticancer drugs including adriamycin, cisplatin, 5-fluorouracil (5-FU) and gemcitabine were used. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nyltetra zolium bromide assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole nuclei staining and flow cytometry in PANC-1 cells treated with 50 microg/mL quercetin for 24 hours. Expression of endoplas mic reticulum (ER) stress mediators including, Grp78/Bip, p-PERK, PERK, ATF4, ATF6 and GADD153/CHOP proteins were measured by Western blot analysis. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. Quercetin induced the apoptosis of PANC-1, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G0/G1 fraction of cell cycle increase. But not adriamycin, cisplatin, gemcitabine, and 5-FU. PANC-1 cells were markedly sensitive to quercetin. RESULTS: Treatment with quercetin resulted in the increased accumulation of intracellular Ca2+ ion. Treatment with quercetin also increased the expression of Grp78/Bip and GADD153/CHOP protein and induced mitochondrial dysfunction. Quercetin exerted cytotoxicity against human pancreatic cancer cells via ER stress-mediated apoptotic signaling including reactive oxygen species production and mitochondrial dysfunction. CONCLUSION: These data suggest that quercetin may be an important modulator of chemosensitivity of cancer cells against anticancer chemotherapeutic agents.
Subject(s)
Humans , Apoptosis , Benzimidazoles , Blotting, Western , Carbocyanines , Cell Cycle , Cell Survival , Chromatin , Cisplatin , Deoxycytidine , DNA Fragmentation , Doxorubicin , Drug Therapy , Flow Cytometry , Fluorescence , Fluorouracil , Membrane Potential, Mitochondrial , Pancreatic Neoplasms , Quercetin , Reactive Oxygen Species , Reticulum , Rhodamine 123ABSTRACT
alpha-Mangostin is a xanthon derivative contained in the fruit hull of mangosteen (Garcinia mangostana L.), and the administration of alpha-Mangostin inhibited the growth of transplanted colon cancer, Her/CT26 cells which expressed Her-2/neu as tumor antigen. Although alpha-Mangostin was reported to have inhibitory activity against sarco/endoplasmic reticulum Ca2+ ATPase like thapsigargin, it showed different activity for autophagy regulation. In the current study, we found that alpha-Mangostin induced autophagy activation in mouse intestinal epithelial cells, as GFP-LC3 transgenic mice were orally administered with 20 mg/kg of alpha-Mangostin daily for three days. However, the activation of autophagy by alpha-Mangostin did not significantly increase OVA-specific T cell proliferation. As we assessed ER stress by using XBP-1 reporter system and phosphorylation of eIF2alpha, thapsigargin-induced ER stress was significantly reduced by alpha-Mangostin. However, coadministration of thapsigargin with alpha-Mangostin completely blocked the antitumor activity of alpha-Mangostin, suggesting ER stress with autophagy blockade accelerated tumor growth in mouse colon cancer model. Thus the antitumor activity of alpha-Mangostin can be ascribable to the autophagy activation rather than ER stress induction.
Subject(s)
Animals , Mice , Autophagy , Calcium-Transporting ATPases , Cell Proliferation , Colonic Neoplasms , Epithelial Cells , Fruit , Garcinia mangostana , Mice, Transgenic , Phosphorylation , Reticulum , Thapsigargin , Transplants , XanthonesABSTRACT
The receptor activator of NF-kappaB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-kappaB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) delta, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.
Subject(s)
Animals , Mice , Blood Proteins , Boron Compounds , Calcium-Transporting ATPases , Cell Membrane , Dentin , Estrenes , Inositol , Inositol 1,4,5-Trisphosphate , Inositol 1,4,5-Trisphosphate Receptors , Macrophages , NF-kappa B , Osteoclasts , Phosphoproteins , Proteins , Pyrrolidinones , Receptor Activator of Nuclear Factor-kappa B , Reticulum , Signal Transduction , Tumor Necrosis Factor-alpha , Type C PhospholipasesABSTRACT
Teeth develop via a reciprocal induction between the ectomesenchyme originating from the neural crest and the ectodermal epithelium. During complete formation of the tooth morphology and structure, many cells proliferate, differentiate, and can be replaced with other structures. Apoptosis is a type of genetically-controlled cell death and a biological process arising at the cellular level during development. To determine if apoptosis is an effective mechanism for eliminating cells during tooth development, this process was examined in the rat mandible including the developing molar teeth using the transferase-mediated dUTP-biotin nick labeling (TUNEL) method. The tooth germ of the mandibular first molar in the postnatal rat showed a variety of morphological appearances from the bell stage to the crown stage. Strong TUNEL-positive reactivity was observed in the ameloblasts and cells of the stellate reticulum. Odontoblasts near the prospective cusp area also showed a TUNEL positive reaction and several cells in the dental papilla, which are the forming pulp, were also stained intensively in this assay. Our results thus show that apoptosis may take place not only in epithelial-derived dental organs but also in the mesenchyme-derived dental papilla. Hence, apoptosis may be an essential biological process in tooth development.
Subject(s)
Animals , Rats , Ameloblasts , Apoptosis , Biological Phenomena , Cell Death , Crowns , Dental Papilla , Ectoderm , Epithelium , Hypogonadism , In Situ Nick-End Labeling , Mandible , Mitochondrial Diseases , Molar , Neural Crest , Odontoblasts , Ophthalmoplegia , Reticulum , Tooth , Tooth GermABSTRACT
Regulators of G-protein signaling (RGS) proteins are regulators of Ca2+ signaling that accelerate the GTPase activity of the G-protein alpha-subunit. RGS1, RGS2, RGS4, and RGS16 are expressed in the pancreas, and RGS2 regulates G-protein coupled receptor (GPCR)-induced Ca2+ oscillations. However, the role of RGS4 in Ca2+ signaling in pancreatic acinar cells is unknown. In this study, we investigated the mechanism of GPCR-induced Ca2+ signaling in pancreatic acinar cells derived from RGS4-/- mice. RGS4-/- acinar cells showed an enhanced stimulus intensity response to a muscarinic receptor agonist in pancreatic acinar cells. Moreover, deletion of RGS4 increased the frequency of Ca2+ oscillations. RGS4-/- cells also showed increased expression of sarco/endoplasmic reticulum Ca2+ ATPase type 2. However, there were no significant alterations, such as Ca2+ signaling in treated high dose of agonist and its related amylase secretion activity, in acinar cells from RGS4-/- mice. These results indicate that RGS4 protein regulates Ca2+ signaling in mouse pancreatic acinar cells.
Subject(s)
Animals , Mice , Acinar Cells , Amylases , Calcium-Transporting ATPases , GTP Phosphohydrolases , GTP-Binding Proteins , Pancreas , Proteins , Receptors, Muscarinic , Reticulum , RGS ProteinsABSTRACT
Inositol 1,4,5-trisphosphate receptors (InsP3Rs) modulate Ca2+ release from intracellular Ca2+ store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block InsP3Rs, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of Ca2+ release from intracellular Ca2+ stores and Ca2+ entry through store-operated Ca2+ (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the Ca2+ entry, but significantly inhibited carbamylcholine (CCh)-induced Ca2+ release. In contrast, 2-APB did not block CCh-induced Ca2+ release, but remarkably blocked SOC-mediated Ca2+ entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP3-induced Ca2+ release, but 2-APB at lower concentration, which effectively blocked Ca2+ entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of InsP3-induced Ca2+ release and direct stimulation of Ca2+ release. Based on the results, we concluded that caffeine is useful as an inhibitor of InsP3R, and 2-APB at lower concentration is considered a blocker of Ca2+ entry through SOC channels in the pancreatic acinar cell.
Subject(s)
Animals , Mice , Acinar Cells , Boron Compounds , Caffeine , Calcium , Carbachol , Inositol 1,4,5-Trisphosphate Receptors , Membranes , ReticulumABSTRACT
The effects of unilateral sciatic neurectomy on gastrocnemius muscle were studied in adult male rats. The morphological changes of both gastrocnemius muscles were observed by light and electron microscopy. Western blot analysis was performed to study the protein expression. Following the denervation, the affected muscle weights decreased significantly than normal. And the denervation led to a significant reduction in the area and diameter of muscle fibers on light microscopy. The affected muscle fibers showed the decreasing in size and the irregularity of myofibrilar arrangement on electron microscopy. On transverse view, the area of affected muscle fibers were smaller than normal. Their myofibrils were smaller and very irregular in size. The thin and thick myofilaments were not regular and partially lost. Mitochondria between myofibrils and subsarcolemmal area in affected muscle fibers were damaged and partially lost. The sacoplasmic reticulum and T-tubules were mostly lost and irregular. Some satellite cells were observed adjacent the muscle fiber, but they were inactive morphologically. On longitudinal view, most of myofibrils in affected muscle fibers were lost generally or partially although the their most sarcomeres were regularly arranged. Z line and myofilaments were lost partially and were partially irregularly arranged. The loss of thin myofilaments was more than that of thick myofilaments. Much debris of cell organelles were scattered among myofibrils. The expression of MyoD and myogenin were decrease significantly and the expression of p-mTOR and p-FOXO1 were decreased, too. On the other hand, MuRF1 was increased significantly. Taken together, the main effect of gastrocnemius muscle by sciatic neurectomy is the atrophy as a result of the loss of myofilaments and cell organelles through the decrease of protein synthesis and the increase of protein degradation.
Subject(s)
Adult , Animals , Humans , Male , Rats , Atrophy , Blotting, Western , Denervation , Hand , Light , Microscopy , Microscopy, Electron , Mitochondria , Muscle, Skeletal , Muscles , Myofibrils , Myogenin , Organelles , Proteolysis , Reticulum , Sarcomeres , Sciatic Nerve , Weights and MeasuresABSTRACT
Tooth is formed by the reciprocal interactions between the ectoderm and ectomesenchyme derived from neural crest. It has not been clear that neuronal factors involved in the morphogenesis and differentiation of tooth. To identify the roles of neuronal factors during the tooth development, the expression patterns and localization of Uchl1 were investigated in the developing mouse tooth germ by in situ hybridization and immunohistochemistry. Uchl1 transcripts were weakly expressed in the oral epithelium and dental lamina at bud stage. However, expression of Uchl1 was not found in the oral epithelium from cap stage and observed in the inner enamel epithelium, stellate reticulum and dental papilla. From the bell stage, Uchl1 was expressed in the inner enamel epithelium and ameloblasts. Uchl1, was appeared to be localized in the inner enamel epithelium and differentiating ameloblasts of molar and incisors at neonates. Uchl1 was localized strongly in the fully differentiated ameloblasts and adjacent papillary layer whereas localized weakly in the odontoblasts of the molar at postnatal day 5. From these results, Uchl1 was expressed and localized in the differentiating dental epithelium and ameloblasts during tooth development. The results suggest that neuronal protein, Uchl1 may play roles in the histo- and cyto-differentiation of non-neuronal dental epithelium.
Subject(s)
Animals , Humans , Infant, Newborn , Mice , Ameloblasts , Dental Enamel , Dental Papilla , Ectoderm , Epithelium , Immunohistochemistry , In Situ Hybridization , Incisor , Molar , Morphogenesis , Neural Crest , Neurons , Odontoblasts , Reticulum , Tooth Germ , ToothABSTRACT
A T-box transcription factor gene, Tbx1 is a principal candidate of the most frequent chromosomal deletion syndrome found in human, DiGeorge/velocardiofacial syndrome which is a complex developmental disorder associated with cardiac outflow tract abnormalities, mid facial dysmorphology, velopharyngeal insufficiency and submucosal cleft palate. We performed in situ hybridization against mouse embryo from E13.5 (bud stage) to E18.5 (late bell stage) in order to analyze the expression patterns of Tbx1 in the developing mouse first molar, a derivative of the first pharyngeal arch. Tbx1 transcripts were found in the dental lamina and its surrounding mesenchyme at E13.5 and in the dental organ except enamel knot at E14.5 (cap stage). Tbx1 was strongly expressed in the cervical loop and stratum intermedium but was weak in the dental papilla and dental follicle at E15.5 (early bell stage). At E18.5, Tbx1 was strongly expressed not only in the dental organ (bell stage) except stellate reticulum but also dental papilla and dental follicle adjacent to the inner dental epithelium. In conclusion, Tbx1 transcripts were specifically expressed both in the dental epithelium and surrounding mesenchyme of developing tooth from initiation to bell stage, which were the most similar with those of Sox9 but little different from those of Pitx2 and ectodin. These results strongly suggested that Tbx1 may play a role as a transcription factor regulating proliferation and differentiation of both dental epithelium and mesenchyme through the tooth development.
Subject(s)
Animals , Humans , Mice , Branchial Region , Cleft Palate , Dental Enamel , Dental Papilla , Dental Sac , Embryonic Structures , Epithelium , In Situ Hybridization , Mesoderm , Molar , Reticulum , Tooth , Transcription Factors , Velopharyngeal InsufficiencyABSTRACT
Dentinogenic ghost cell tumor (DGCT) is an uncommon odontogenic tumor. It is characterized by islands of odontogenic epithelial cells that contain numerous ghost cells and dysplastic dentin. Occasionally, DGCT combines with other odontogenic tumors, such as ameloblastoma. We report here on a 21-year-old female who complained of a tender solid mass in the left maxilla for the 7 month previous to her admission. MRI revealed a relatively well demarcated mass in the left maxilla with heterogenous signal intensity, measuring 3.2 x 2.8 cm, and this mass had invaded the left palate. Microscopically, the tumor was composed of nests of odontogenic epithelium that contained ghost cells and calcification with dysplastic dentin, which is all consistent with DGCT. Localized area showed odontogenic epithelial follicles that had peripheral palisading and satellite reticulum without ghost cells and dentin, and this is consistent with ame- loblastoma. The immunohistochemistry revealed cytokeratins, EMA, S100 and Bcl-2 positivity in areas of the DGCT and ameloblastoma. In the ameloblastoma, Bcl-2 positivity was noted in the palisading basal cells. We concluded that the tumor was an ameloblastoma associated with DGCT.
Subject(s)
Female , Humans , Young Adult , Ameloblastoma , Dentin , Epithelial Cells , Epithelium , Immunohistochemistry , Islands , Keratins , Magnetic Resonance Imaging , Maxilla , Odontogenic Tumors , Palate , ReticulumABSTRACT
BACKGROUND: Reticulum fibers represent a special type of thin collagen fiber that measures from 0.2 to 1.0 micrometer in diameter. A reticulum stain is currently used for diagnosing liver cirrhosis, chemodectoma, differentials of lymphoid tissue tumors and vascular tumors. In particular, it has been used for diagnosing sarcoidosis in the field of dermatology. OBJECTIVE: This study was conducted to ascertain whether reticulum fibers appeared when diseases showing granulomatous reaction were stained with a reticulum stain. METHODS: Patients who had been clinically or histopathologically diagnosed as having a granulomatous disease were used in this study. Granulomatous diseases included: sarcoidosis, leprosy, skin tuberculosis, lupus miliaris disseminatus faciei, granuloma annulare, paraffinoma, silicon granuloma or foreign body granuloma. A patient without a graunlomatous disease was used as the control. Hematoxylin-eosin staining and reticulum staining using Gomori's silver impregnation method were performed in all cases. RESULTS: A reticulum stain revealed a network of reticulum fibers surrounding and permeating the granulomas of various forms in patients with a granulomatous disease, although it was less abundant in sarcoidosis. CONCLUSION: We concluded that reticulum fibers appear in all granulomatous diseases.