To determine the ventricular looping pattern in relation to cardiac laterality, we studied rat embryos treated with retinoic acid (RA). A total of 243 Wistar rat embryos from an in vivo treated group (a single dose of 20-40 mg/kg all-trans RA administered to pregnant rats on day 6.5 to 9.5) and 29 control embryos were examined on day 13 of gestation. Twenty-nine embryos from the in-vitro treated group (treated by all-trans RA at 2 x 10(-7) M for 6 hr on day 9.0 or 9.5 during the entire embryo culture for 72 hr) and seven control embryos were examined on day 12 of gestation. Abnormalities in cardiac laterality and ventricular looping were found in the in-vivo groups treated on day 8.5 and 8.75 and in the in-vitro group on day 9.0. Among 25 animals with abnormal laterality, right isomerism was the most common feature (22 cases), while the type of ventricular looping varied. Cases with normal laterality had a low incidence of abnormal looping (1.4%). In rat embryos treated with all-trans RA, normal cardiac looping was expected when cardiac laterality was normal. But in cases with abnormal laterality, the type of abnormal ventricular looping was unexpected.
Animal ; Cell Division ; Female ; Heart/drug effects* ; Heart Defects, Congenital/pathology ; Heart Defects, Congenital/chemically induced* ; Heart Ventricle/pathology ; Heart Ventricle/abnormalities* ; Incidence ; Male ; Pregnancy ; Rats ; Rats, Wistar ; Tretinoin/pharmacology*

The anti-melanogenesis effect of glyceollins was examined by melanin synthesis, tyrosinase activity assay in zebrafish embryos and in B16F10 melanoma cells. When developing zebrafish embryos were treated with glyceollins, pigmentation of the embryos, melanin synthesis and tyrosinase activity were all decreased compared with control zebrafish embryos. In situ expression of a pigment cell-specific gene, Sox10, was dramatically decreased by glyceollin treatment in the neural tubes of the trunk region of the embryos. Stem cell factor (SCF)/c-kit signaling pathways as well as expression of microphthalmia-associated transcription factor (MITF) were determined by western blot analysis. Glyceollins inhibited melanin synthesis, as well as the expression and activity of tyrosinase induced by SCF, in a dose-dependent manner in B16F10 melanoma cells. Pretreatment of B16F10 cells with glyceollins dose-dependently inhibited SCF-induced c-kit and Akt phosphorylation. Glyceollins significantly impaired the expression and activity of MITF. An additional inhibitory function of glyceollins was to effectively downregulate intracellular cyclic AMP levels stimulated by SCF in B16F10 cells. Glyceollins have a depigmentation/whitening activity in vitro and in vivo, and that this effect may be due to the inhibition of SCF-induced c-kit and tyrosinase activity through the blockade of downstream signaling pathway.
Animals ; Embryo, Nonmammalian/drug effects ; Melanins/*biosynthesis ; Melanoma, Experimental/metabolism/pathology ; Mice ; Monophenol Monooxygenase/metabolism ; Phosphorylation/drug effects ; Pigmentation/drug effects ; Proto-Oncogene Proteins c-kit/*metabolism ; Pterocarpans/chemistry/*pharmacology ; SOXE Transcription Factors/metabolism ; Sesquiterpenes/chemistry/*pharmacology ; Signal Transduction/*drug effects ; Soybeans/*chemistry ; Stem Cell Factor/*pharmacology ; Zebrafish/embryology/metabolism

OBJECTIVES: This study was conducted in order to understand the cellular events associated with silica-induced pathogenesis of the rat lung. METHODS: Silicosis was induced by an intratracheal instillation of 50 mg of silica (SiO2, 0.15 - 10 micrometer) suspended in 500 microliter of a sterile saline solution in Sprague-Dawley rats weighing 200g. Silicotic nodules were excised from the rat lungs 4 weeks after silica instillation, then boiled for 4 days at 110 degrees in solution containing 2% SDS, 10 M urea and 40 mM DTT. The insoluble cellular encapsulates were electrophoresed on 4-12 % gradient SDS-PAGE, and the amino acid composition was analyzed. Affinity chromatographies of the homogenate supernatants of the control lung, silicotic nodule, and normal rat plasma were performed using rabbit IgG, anti-rat, cross-linked protein from the silicotic nodule. The amounts of N epsilon-(gamma-glutamyl) lysine cross-linked in the control lungs and silicotic nodules were determined using HPLC analysis. RESULTS: The remaining cross-linked protein was insoluble in the 10 M urea and 40 mM sulfhydryl reagents even under prolonged boiling conditions. The encapsulate revealed the retention of silica particles within the protein whose amino acid composition showed a high percentage of alanine, leucine and glycine. A 46 KDa protein was identified as a cross-linked protein in the silicotic nodule by affinity chromatography. The level of N epsilon-(gamma-glutamyl) lysine dipeptide in the nodule digest was prominently increased compared with that in the control lung. CONCLUSIONS: Transglutaminase (TGase)-catalyzed cross-linking appears to be involved in the silicotic nodule formation, and the 46 KDa protein may be cross-linked to itself and other extracellular matrix proteins during fibrosis and the formation of eventually insoluble nodule.
Alanine ; Animals ; Chromatography ; Chromatography, Affinity ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel ; Extracellular Matrix Proteins ; Fibrosis ; Glycine ; Immunoglobulin G ; Leucine ; Lung* ; Lysine ; Plasma ; Rats* ; Rats, Sprague-Dawley ; Silicon Dioxide ; Silicosis ; Sodium Chloride ; Sulfhydryl Reagents ; Urea

OBJECTIVES: This study was performed to examine the immunohistochemical distribution of TGase 1, 2, 3, coagulation factor XIII and N epsilon-(gamma-glutamyl) lysine cross-link in the silicotic nodules formed after an intratracheal instillation of the silica. METHODS: The immunohistochemical examinations used antibodies against TGase 1, 2, 3, coagulation factor XIII and N epsilon-(gamma-glutamyl) lysine isopeptide in the silicotic nodules induced after an intratracheal instillation of 50 mg of size fractionated, crystalline silica. RESULTS: A high level of TGase 3 was related to the severity of fibrosis in silicotic nodules and extracellular coagulation factor XIII was detected around the nodules. Expressions of both membrane-bound TGase 1 and TGase 2 were barely detected in the nodules although high expressions were detected in the intact lung. Formation of N epsilon-(gamma-glutamyl) lysine cross-links was increased in severe fibrotic nodules. CONCLUSIONS: TGase 3 might contribute to the eventual stone-like fibrosis via formation of N epsilon-(gamma-glutamyl) lysine cross-links. Futhermore, coagulation factor XIII plays a role in the formation of a provisional matrix which results in fibrogenesis during silicotic nodule formation.
Antibodies ; Blood Coagulation Factors* ; Crystallins ; Factor XIII* ; Fibrosis ; Immunohistochemistry* ; Lung ; Lysine* ; Plasma* ; Silicon Dioxide

Background/Aims: Angiogenesis is essential for the outgrowth and metastasis of tumors. The structure and characteristics of tumor vasculature differ from those of normal vessels. We compared the characteristics of differentially expressed genes in endothelial cells (ECs) isolated from gastric and normal cells. Methods: Previously, we had isolated pure tumor ECs (TECs) and normal ECs (NECs) from advanced gastric cancer (AGC) lesions and normal mucosal tissues, respectively. Using the oligomer chip platform of the Affymetrix GeneChip technology, genes that were expressed more than three-fold with a significance of p≤0.001 were measured. The intercellular adhesion molecule 1 (ICAM-1) was found to be overexpressed in the TECs compared to the normal gastric ECs. In this study, the upregulation of ICAM-1 was confirmed in cultured TECs by immunofluorescence. Results: The expression of ICAM-1 was upregulated in the ECs, as well as in the stromal and immune cells, in early human gastric preneoplastic and hepatic fibrotic tissues. Upregulation of ICAM-1 was observed in the TECs, immune cells, and cancer epithelial cells in AGC and hepatocellular carcinoma (HCC). These results suggest that increased ICAM-1 expression in the ECs of the tissue microenvironment progressively contributes to the recruitment of immune cells to promote inflammation, leading to fibrosis and tumorigenesis. Conclusions Therefore, upregulated ICAM-1 in the tissues in premalignant gastric diseases or hepatic fibrosis and their malignant cancers could be a promising target for disease prevention and treatment.

BACKGROUND: T-SPOT.TB is a sensitive test that detects interferon-gamma producing T-cells in tuberculosis patients following stimulation with tuberculosis-specific antigens. Our study was aimed to investigate the possible causes of false negative results of the test by analyzing the patients with positive acid-fast bacilli (AFB) culture and negative T-SPOT.TB results. METHODS: We investigated 138 patients with positive AFB culture results reported between January 2009 and April 2010. Medical records of these patients were reviewed for the results of T-SPOT.TB test, AFB culture, PCR for Mycobacterium tuberculosis (TB-PCR), chest X-ray, drug treatment, etc. Diagnosis of tuberculosis was confirmed by positive TB-PCR or identification of Mycobacterium tuberculosis (MTB). Sensitivity of T-SPOT.TB test was calculated and the possible causes of AFB culture positive and T-SPOT.TB negative results were analyzed. RESULTS: T-SPOT.TB test was performed in 63 of the 138 patients with AFB culture positive results. Fifty-six (88.9%) were positive and 7 patients (11.1%) were negative on T-SPOT.TB test. Of these 7 negative cases, 4 were confirmed as nontuberculous mycobacteria (NTM), 2 were suspected as NTM and diagnosis could not be confirmed in 1. Six of these 7 patients were over 70 yr old and 6 patients had lymphocytopenia. T-SPOT.TB negative results were not observed in any of the 44 patients confirmed to have active tuberculosis (sensitivity 100%). CONCLUSIONS: Our results suggest that T-SPOT.TB test is very sensitive for diagnosing active tuberculosis. NTM may be the main cause of AFB culture positive and T-SPOT.TB negative results, but MTB infection in immunocompromised patients also has to be considered.
Adult ; Aged ; Aged, 80 and over ; Bacillus/*isolation & purification ; Culture Media ; Female ; Humans ; Lymphocyte Count ; Lymphopenia/diagnosis/microbiology ; Male ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Tuberculosis/*diagnosis/microbiology/radiography

Morus alba L. is well-known for its medicinal and economic value, particularly in Asian countries. Among the isolated compounds from this plant, steppogenin is exhibited as a flavonoid with promising pharmacological properties. This study focused on isolating bioactive compounds, notably steppogenin, from the ethyl acetate extract of M. alba. Additionally, a high-performance liquid chromatography-diode array detector (HPLC-DAD) method for the simultaneous quantification of steppogenin and isolated compounds was developed and validated. The calibration curve showed excellent linearity, with a correlation coefficient (R 2 ) value greater than 0.9957. The limit of detection (LOD) ranged from 0.006 to 0.018 μg/mL, whereas the limit of quantification (LOQ) ranged from 0.020 to 0.061 μg/mL. In precision tests conducted intra-day and inter-day, the accuracy was between 97.32% and 106.39%, with relative standard deviations (RSD) less than 2.27% and 1.65%, respectively. The presence of steppogenin and other flavonoids was confirmed by the study, contributing to the understanding of the chemical composition of M. alba. This validated analytical method offers a reliable means of quantifying steppogenin and aiding future research into its therapeutic potential.

A 36-year-old primivida, at 29 weeks of pregnancy with no smoking history, was admitted to the hospital complaining of cough for 3 days. Chest X-rays revealed atelectasis of the right upper lobe, and a transbronchial lung biopsy confirmed primary lung adenocarcinoma. After consulting with obstetricians and neonatologists, we wanted to deliver the child and treat the mother with chemoradiotherapy. But as she was adamantly opposed to treatment until fetal lung maturation was complete, we planned to delay the birth until 34 weeks, deliver the baby by caesarian section, and then treat the mother. However, maternal hypoxia and fetal distress resulted in an emergency delivery at 30 weeks. After delivery, we treated the mother's brain metastases with radiation therapy and systemic cisplatin-pemetrexed, but she deteriorated and expired 95 days after the diagnosis. Lung cancer during pregnancy is a rare disease and raises many medical and ethical issues in deciding the best course of therapy. We describe our clinical approach and review the potentially challenging features of managing a pregnant patient with lung cancer.
Adenocarcinoma ; Adult ; Anoxia ; Biopsy ; Brain ; Chemoradiotherapy ; Child ; Cough ; Emergencies ; Fetal Distress ; Humans ; Lung ; Lung Neoplasms ; Mothers ; Neoplasm Metastasis ; Parturition ; Pregnancy ; Pulmonary Atelectasis ; Rare Diseases ; Smoke ; Smoking ; Thorax

A 36-year-old primivida, at 29 weeks of pregnancy with no smoking history, was admitted to the hospital complaining of cough for 3 days. Chest X-rays revealed atelectasis of the right upper lobe, and a transbronchial lung biopsy confirmed primary lung adenocarcinoma. After consulting with obstetricians and neonatologists, we wanted to deliver the child and treat the mother with chemoradiotherapy. But as she was adamantly opposed to treatment until fetal lung maturation was complete, we planned to delay the birth until 34 weeks, deliver the baby by caesarian section, and then treat the mother. However, maternal hypoxia and fetal distress resulted in an emergency delivery at 30 weeks. After delivery, we treated the mother's brain metastases with radiation therapy and systemic cisplatin-pemetrexed, but she deteriorated and expired 95 days after the diagnosis. Lung cancer during pregnancy is a rare disease and raises many medical and ethical issues in deciding the best course of therapy. We describe our clinical approach and review the potentially challenging features of managing a pregnant patient with lung cancer.
Adenocarcinoma ; Adult ; Anoxia ; Biopsy ; Brain ; Chemoradiotherapy ; Child ; Cough ; Emergencies ; Fetal Distress ; Humans ; Lung ; Lung Neoplasms ; Mothers ; Neoplasm Metastasis ; Parturition ; Pregnancy ; Pulmonary Atelectasis ; Rare Diseases ; Smoke ; Smoking ; Thorax

Proton beam is useful to target tumor tissue sparing normal cells by allowing precise dose only into tumor cells. However, the cellular and molecular mechanisms by which proton beam induces tumor cell death are still undefined. We irradiated three different tumor cells (LLC, HepG2, and Molt-4) with low energy proton beam (35 MeV) with spread out Bragg peak (SOBP) in vitro, and investigated cell death by MTT or CCK-8 assay at 24 h after irradiation. LLC and HepG2 cells were sensitive to proton beam at over 10 Gy to induce apoptosis whereas Molt-4 showed rather low sensitivity. Relative biological effectiveness (RBE) values for the death rate relative to gamma-ray were ranged from 1.1 to 2.3 in LLC and HepG2 but from 0.3 to 0.7 in Molt-4 at 11 d after irradiation by colony formation assay. The typical apoptotic nuclear DNA morphological pattern was observed by staining with 4'-6-diamidino-2-phenylindole (DAPI). Tiny fragmented DNA was observed in HepG2 but not in Molt-4 by the treatment of proton in apoptotic DNA fragment assay. By FACS analysis after stained with FITC-Annexin-V, early as well as median apoptotic fractions were clearly increased by proton treatment. Proton beam-irradiated tumor cells induced a cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and procaspases-3 and -9. Activity of caspases was highly enhanced after proton beam irradiation. Reactive oxygen species (ROS) were significantly increased and N-acetyl cysteine pretreatment restored the apoptotic cell death induced by proton beam. Furthermore, p38 and JNK but not ERK were activated by proton and dominant negative mutants of p38 and JNK revived proton-induced apoptosis, suggesting that p38 and JNK pathway may be activated through ROS to activate apoptosis. In conclusion, our data clearly showed that single treatment of low energy proton beam with SOBP increased ROS and induced cell death of solid tumor cells (LLC and HepG2) in an apoptotic cell death program by the induction of caspases activities.
Apoptosis/*radiation effects ; Caspases/*metabolism ; Cell Line, Tumor ; DNA Fragmentation/radiation effects ; Dose-Response Relationship, Radiation ; Enzyme Activation/radiation effects ; Flow Cytometry ; Gamma Rays ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism ; Neoplasms/*enzymology/*pathology ; *Protons ; Reactive Oxygen Species/*metabolism ; p38 Mitogen-Activated Protein Kinases/metabolism