Intracellular pH regulation of osteoblasts is of a great importance in the process of bone formation and resorption, and has been suggested to be mediated via intracellular Ca2+ and cAMP messenger systems. To elucidate the mechanism of modulation of intracellular pH by parathyroid hormone and PMA(Phorbo1-12-myristate-13-acetate), effects of these agonists on the individual transporter system, Na+-H+ antiporter and Cl−-HCO3-(−OH−) exchanger, were investigated. Intracellular pH and Ca2+ were measured by using the fluorescent dye BCECF and fura-2, respectively, in UMR-106 cell monolayer grown on glass coverslip. Addition of tumor promotor, PMA(luM) caused 0.14 unit pH rise of resting intracellular pH(pHi) and 38% increase of the initial rate of pHi recovery after cytosolic acid load. Perfusion of Cl−-free solution resulted in rapid cytosolic alkalinization of which the rate was increased 26% by preincubation of PMA. Ca2+ ionophore, ionomycin (1uM) decreased resting pHi by 0.17 unit, but had no effect on the initial rate of pHi recovery after cytosolic acid load. However, the addition of ionomycin augmented the initial rate of pHi increase after Cl−-depletion outside the cells by 34% over the control. Stimulation of cells with parathyroid hormone(10-8M) caused an initial acidification (0.27 unit) followed by cytosolic alkalinization, with inhibiting effect on the initial rate of pHi recovery after acid load (42%). But parathyroid hormone did not have any significant effect on the rate of pHi increase after Cl−-depletion. PMA caused a sustained increase of intracellular Ca2+, of which the peak depended on the concentration of Ca2+ in extracellular medium. Ionomycin caused a transient increase of Ca2+ but PTH had no significant increase of intracellular Ca2+ in the concentration range of 10-6M to 10-12M tested. 10-8M PTH increased cAMP levels by about 10-fold and 10-10M PTH did by 1.6-fold. PMA, which increased cytosolic Ca2+ concentration, also had an stimulatory effect on cAMP production in the concentration range of 10-5M to 10-6M by 2-fold. These findings suggest that in UMR-106 cells Ca2+ and cAMP can influence pHi by altering the activity of pHi regulatory transporter system, and parathyroid hormones modulate pHi by inhibiting Na+-H+ antiporter via intracellular increase of cAMP, which is probably accounts for the inhibitory effect of parathyroid hormone on the proliferation of osteoblasts.
Cytosol ; Fura-2 ; Glass ; Hydrogen-Ion Concentration ; Ion Transport ; Ionomycin ; Osteoblasts ; Osteogenesis ; Parathyroid Hormone ; Perfusion

Bleomycin, the generic name for a group of sulfur- containing polypeptide antibiotics derived from Streptomyces verticillus, has been used as a single agent and in combination for treatment of various neoplasms and viral diseases. Recently, intradermal bleomycin injections have been shown to bring about significant improvements in keloid and hypertrophic scar. However, the mechanism by which this drug acts on keloid is not entirely clear. In this study, the effects of bleomycin on growth rate, apoptosis, production of transforming growth factor(TGF-beta) and expression of its receptor, secretion of fibronectin were evaluated in keloid and normal human dermal fibroblasts. Human keloid and normal fibroblasts were primarily cultured from earlobe keloids of three female patients and treated with various concentration of bleomycin. Cell toxicity was assessed by MTT assay, growth rate and apoptosis was assessed by FACS, production of fibronectin by immunoprecipitation and western blot, TGF-beta secretion by ELISA, and expression of TGF-beta receptor by western blot, respectively. The obtained results are follows : Bleomycin induced cell toxicity dose-dependently in keloid fibroblasts in the range of 0.0012-0.075mg/ml, and the MTT90 and MTT50 values of bleomycin were 0.0081mg/ml and 0.0352mg/ml, respectively. Even in lower concentration (MTT90) of bleomycin, the cell growth was significantly suppressed in both normal and keloid fibroblasts, with the latter more suppressed than the former. Keloid fibroblasts secreted more TGF-beta than normal fibroblasts, but there was no significant difference of TGF-beta secretion between the group with bleomycin treatment and the untreated control group. There was no significant effect of bleomycin on the suppression of the expression of TGF-beta receptor and the production of fibronection in fibroblast. Keloid fibroblasts responded to bleomycin more sensitively than normal fibroblasts, and the percentage of apoptosis was higher in keloid fibroblasts than in normal fibroblast at the MTT50 concentration of bleomycin. Bleomycin had growth-inhibitory effect with inducing the apoptosis directly in lower concentration(MTT90). Therefore, clinical application with lower concentration than 1.0 mg/ml is adviced. Further studies, including clinical demonstration, will be required to better elucidate the anti-keloid effect of bleomycin depending on the dosage and the additive effect of different anti-keloid action by other agents. In summary, bleomycin may be a drug of promise in the treatment of keloid and hypertrophic scar.
Anti-Bacterial Agents ; Apoptosis* ; Bleomycin* ; Blotting, Western ; Cicatrix, Hypertrophic ; Enzyme-Linked Immunosorbent Assay ; Female ; Fibroblasts* ; Fibronectins ; Humans ; Immunoprecipitation ; Keloid* ; Receptors, Transforming Growth Factor beta ; Streptomyces ; Transforming Growth Factor beta ; Virus Diseases

Interleukin (IL)-8 and vascular endothelial growth factor (VEGF) are important factors that induce the migration and proliferation of endothelial cells, increase the vascular permeability, and the modulate chemotaxis of monocytes. These molecules have been found in human atherosclerotic plaques. However, it is not clear whether the circulating levels of IL-8 and VEGF correlate with the extents of carotid stenosis. In this study, we investigated the relationship between circulating levels of IL-8 as well as VEGF and the extents of carotid stenosis. Sera from 41 patients with carotid stenosis were assessed for concentrations of IL-8 and VEGF by enzyme-linked immunosorbent assay. The degree of stenosis of extracranial carotid artery was calibrated by carotid B- mode ultrasonography. The serum concentration of IL-8 (r=-0.04733, p>0.05) was not correlated with the degree of stenosis. However, the serum concentration of VEGF (r=0.4974, p<0.01) was significantly correlated with the degree of carotid stenosis. These findings suggest that increased serum level of VEGF might be a marker for higher degree of stenosis of extracranial carotid artery.
Adult ; Aged ; Carotid Artery Diseases/blood ; Carotid Stenosis/*blood ; Disease Progression ; Endothelial Growth Factors/*blood ; Female ; Human ; Interleukin-8/*blood ; Lymphokines/*blood ; Male ; Middle Age

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix. However, the molecular mechanisms underlying this pathologic deposition of extracellular matrix still remain to be elucidated. In this study, the effects of anti-keloid drugs (triamcinolone(R), 5-FU(R), bleomycin(R), verapamil(R)) on the expression of fibronectin and TGF-beta and its receptor in keloid fibroblasts were evaluated in vitro. Human keloid fibroblasts(KFs) and normal human dermal fibroblasts(NHDFs) were isolated from earlobe keloids. Immunoprecipitation and Western blot of fibronectin, ELISA of TGF-beta secretion and Western blot of TGF-beta receptor were performed using the primary cultured fibroblasts treated with various drugs. TGF-beta secretion was increased in keloid fibroblasts compared to NHDFs. TGF-beta promoted fibronectin synthesis in keloid fibroblasts. These results substantiate the hypothesis that the elevated levels of TGF-beta play a potential role in keloid pathogenesis. 5-FU suppressed profoundly TGF-beta secretion. Triamcinolone and verapamil inhibited significantly fibronectin synthesis and secretion, and also suppressed TGF-beta receptor expression in keloid fibroblasts. In conclusion, it is postulated that the combination of low dose of 5-FU and triamcinolone or verapamil may be useful in keloid treatment by the additive effect of different anti-keloid action.
Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Extracellular Matrix ; Fibroblasts* ; Fibronectins* ; Fluorouracil ; Humans ; Immunoprecipitation ; Keloid* ; Receptors, Transforming Growth Factor beta ; Transforming Growth Factor beta ; Triamcinolone ; Verapamil ; Wounds and Injuries

Water transport in highly-permeable membranes is facilitated by some specialized pathways, which are called aquaporins (AQP). AQP1 (AQP-CHIP) is the first recognized aquaporin identified from red cells and renal proximal tubules. Up until now 4 other aquaporin homologs have been reported. Each aquaporin has its unique tissue distribution and regulatory mechanisms. To elucidate molecular mechanisms for their transcription regulation and tissue-specific expression isolation of aquaporin genes is required. To clone promoters of the AQP family mouse genomic library was screened by the 1st exon-specific probe of AQP4, and 5 different plaques were positively hybridized. Phage DNAs were purified and characterized by restriction mapping and sequencing. One of them is the mouse AQP-CD gene. The gene was consisted of 4 exons and the exon-intron boundaries of mouse AQP-CD gene were identified at identical positions in other related genes. The 5'-flanking region of AQP-CD gene contains one classic TATA box, a GATA consensus sequence, an E-box and a cyclic AMP-responsive element. The cloning of the mouse AQP-CD gene, of which product is expressed in the collecting duct and is responsible for antidiuresis by vasopressin, will contribute to understand the molecular mechanisms of tissue-specific expression and regulation of AQP-CD gene under various conditions.
Animals ; Aquaporin 2 ; Aquaporins ; Bacteriophages ; Clone Cells* ; Cloning, Organism* ; Consensus Sequence ; DNA ; Exons ; Genomic Library ; Humans ; Membranes ; Mice* ; Restriction Mapping ; TATA Box ; Tissue Distribution ; Vasopressins ; Water

OBJECTIVE: Henoch-Schonlein purpura (HSP) is a systemic vasculitis, characterized by small-vessel leukocytoclastic vasculitis with the deposition of immune complexes containing IgA. It is the most common acute vasculitic disorder affecting children but is relatively uncommon in adults. We investigated the clinical features and factors affecting the prognosis of adult HSP in Korea. METHODS: From 1996 to 2011 seventy patients over 15 years of age with HSP were retrospectively analyzed. RESULTS: Thirty eight patients (54.3%) were female and the age at disease onset ranged from 15 to 75 years (35.0+/-15.8 years). Purpuric skin rash was observed in all patients and arthralgia was present in 34 patients (48.6%). GI symptoms and kidney involvements were observed in 28 patients (40.0%) and 34 patients (48.6%), respectively. Complete remission was achieved in 46 patients (65.7%). The remission group showed a lower incidence of hematochezia (p=0.044), hematuria (p=0.008), and proteinuria (p=0.011) at diagnosis than the no remission group. About 10% of adult HSP patient progressed to chronic kidney disease (CKD), which showed higher a incidence of nephrotic range proteinuria. Only nephrotic range proteinuria at diagnosis was a significant risk factor for CKD (OR=16.7, p=0.008, 95% CI=2.1~133.1). CONCLUSION: Hematochezia, hematuria and proteinuria at the diagnosis of HSP are important prognostic factors in predicting remission. In addition, HSP patients with nephrotic range proteinuria at diagnosis have an increased risk of renal failure.
Adult ; Antigen-Antibody Complex ; Arthralgia ; Child ; Exanthema ; Female ; Gastrointestinal Hemorrhage ; Hematuria ; Humans ; Immunoglobulin A ; Incidence ; Kidney ; Korea ; Prognosis ; Proteinuria ; Purpura, Schoenlein-Henoch ; Renal Insufficiency ; Renal Insufficiency, Chronic ; Retrospective Studies ; Risk Factors ; Systemic Vasculitis ; Vasculitis ; Vasculitis, Leukocytoclastic, Cutaneous