The role of intracellular Ca2+ in the degranulation of skinned mast cells

The intracellular pH of rat peritoneal mast cells was slightly acidic and compound 48/80 induced a decrease in the cytoplasmic pH of these cells. By means of chemical skinning, it was revealed that perfusion with Ca2+ or inositol 1,4,5-trisphosphate (IP3) induced degranulation dose-dependently in mast cells at concentrations higher than 10 microM and 0.1 microM, respectively. Na+ was essential for the release of histamine from mast cells. An assay based on the binding of 45Ca to mast cell fragments revealed that the intracellular Ca store of the mast cell is located in the endoplasmic reticulum. IP3 liberated Ca from the endoplasmic reticulum.     

Antiallergic effects of astemizole on immediate type hypersensitivity reactions.

Astemizole (0.5-5 mg/kg, p.o.) dose-dependently inhibited heterologous and homologous PCA reactions in rats at ID50 values of 1.48 mg/kg and 2.37 mg/kg, respectively. The inhibitory effect of astemizole on heterologous PCA was most remarkable when this compound was given p.o. 2 h prior to antigen challenge. Astemizole (0.1-5 mg/kg, p.o.) dose-dependently inhibited experimentally-induced asthma in guinea pigs at an ID50 of 0.86 mg/kg. Ex vivo, astemizole (0.5-5 mg/kg, p.o.) inhibited antigen-induced histamine release from lung pieces of sensitized guinea pigs. In in vitro experiments, the drug dose-dependently inhibited antigen-induced histamine and SRS-A releases from guinea pig lung pieces at concentrations of 0.05-10 microM. Furthermore, astemizole (0.1-10 microM) inhibited the histamine release induced by compound 48/80 and antigen-antibody reaction from rat peritoneal mast cells, and at 0.1-500 nM inhibited both leukotriene C4- and platelet-activating factor (PAF)-induced contraction of isolated guinea pig trachea at submicromolar concentrations. Astemizole not only inhibited 45Ca uptake into rat mast cells but also prevented the Ca2+ release from the intracellular Ca store induced by compound 48/80, although this compound did not affect the histamine release from permeabilized mast cells induced by Ca2+. Our results suggest that one of the antiallergic mechanisms of astemizole may be an inhibition of signal transduction from the mast cell membrane to the intracellular systems.     

Bronchial responsiveness and acute bronchodilator response in chronic obstructive pulmonary disease and diffuse panbronchiolitis.

BACKGROUND--Diffuse panbronchiolitis (DPB) is characterised clinically by chronic airflow limitation and respiratory tract infection, and pathologically by chronic bronchiolar inflammation. To elucidate the functional differences between chronic obstructive pulmonary disease (COPD) and DPB the bronchial responsiveness to methacholine was compared in 64 patients with COPD and 32 patients with DPB, and the bronchodilator response was compared in 72 patients with COPD and 49 with DPB. METHODS--Bronchial responsiveness to methacholine was determined by the dosimeter method and expressed as PD20FEV1, and bronchodilator response was measured as the change in percentage predicted response with 5 mg nebulised salbutamol. RESULTS--Baseline FEV1 was similar in the two groups of patients. Patients with COPD were more responsive to methacholine than were those with DPB (geometric mean PD20FEV1 8.87 v 48.0 cumulative units). Reversibility of air flow obstruction, expressed as the difference between the percentage predicted postbronchodilator FEV1 and prebronchodilator FEV1, was significantly larger in patients with COPD than in those with DPB (7.87 (6.52)% v 4.16 (4.43)%). CONCLUSIONS--The observation that patients with DPB differ substantially in bronchial responsiveness from those with COPD is thought to reflect the difference in the mechanisms of these two diseases--that is, airway disease in DPB and more parenchymal disease in the group of patients with COPD. The nature of bronchiolar inflammation in COPD and DPB is also different, possibly explaining the difference in bronchial responsiveness. More fixed airflow limitation as a result of structural bronchiolar lesions in DPB will explain the smaller reversibility of airflow obstruction.     

Role of the cytoskeleton in Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

In order to study the role of the cytoskeleton in histamine release from mast cells, the effects of cytochalasin D, cholchicine and vinblastine on Ca²⁺ release from the intracellular Ca store induced by compound 48/80 were investigated by means of a video-intensified microscopy system. When the quin 2-loaded mast cells were stimulated by 0.35 g/ml of compound 48/80, a rapid increase in intracellular Ca²⁺ was observed. At concentrations higher than 10^–6 M, both colchicine and vinblastine pretreatments significantly inhibited the increase in intracellular Ca²⁺ concentrations caused by compound 48/80, although cytochalasin D had no effect. When permeabilized mast cells were exposed to potassium-antimonate solution, microtubules became attached to the endoplasmic reticulum, where many dots of Ca-antimonate were observed; in some areas, the microtubules interconnected the endoplasmic reticulum and granules in the mast cells. From the results of the present study, it was assumed that microtubules play some important role in the processes leading to Ca²⁺ release from the intracellular Ca store.     

Germ cell transplantation: a review and progress report on ICSI from spermatozoa generated in xenogeneic testes

Results from the transplantation of donor male germ cells into xenogeneic recipient seminiferous tubules indicate that donor spermatogonia are capable of differentiating to form spermatozoa morphologically characteristic of the donor species. Germ cell transplantation procedures combined with developments in freezing, culturing or enriching germ cell populations have applications of paramount importance in medicine, basic sciences and animal reproduction. Additionally, these techniques can serve as an alternative approach for gonadal protection and fertility preservation in patients with cancer. This article is a chronological critical review of the technological advances that followed the initial successful transplantation of mouse germ cells into recipient mice. Furthermore, the factors responsible for the immunological privilege properties of the testis and the parameters influencing the potential of mammalian germ cells to undergo mitosis and meiosis within a xenogeneic testis are described. Finally, the role of human germ cell transplantation procedures in the therapeutic management of non-obstructive azoospermia is discussed.     

Comparison of the inhibitory effects of glucocorticoids on the expression of eotaxin in airway epithelial cell line BEAS-2B]

OBJECTIVE: Inhaled corticosteroids play a pivotal role in the treatment of asthma. To observe the mechanisms of glucocorticoids, we focused our study on the comparison of several glucocorticoids' effects on eotaxin expression in the airway epithelial cells. METHODS: Airway epithelial cell line BEAS-2B was cultured in vitro. Cells were preincubated with or without glucocorticoids (becromethasone dipropionate; BDP, budesonide; BUD, fluticasone propionate; FP) and stimulated with TNFalpha and/or IL-4. Protein levels of eotaxin in the supernatants of the cultured cells were determined by ELISA. RESULTS AND CONCLUSIONS: TNFalpha and IL-4 increased the levels of eotaxin in BEAS-2B cells. Combination of these cytokines synergistically upregulated the eotaxin expression as reported previously. Each glucocorticoid significantly inhibited the expression of eotaxin protein induced with TNFalpha and IL-4 and the compared efficacy was in order of FP>BUD>BDP. FP seemed most potent and the inhibitory effect was also observed with relatively low concentration such as 10 (-10)M. Taken together, the comparison of the potency of each glucocorticoid using airway epithelial cells may reflect the efficacy of these drugs in asthmatics.     

Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans

Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.     

Role of microfilaments in the exocytosis of rat peritoneal mast cells

When rat peritoneal mast cells were treated with the potent histamine releaser compound 48/80 in the presence of tetramethylrhodamine-labeled G-actin, the fluorescent G-actin particles were bound to the surface of extruded granules and to the cell surface. When rhodamine-phalloidin was incorporated into permeabilized rat mast cells in a Ca2+-free medium, rhodamine fluorescence was observed on the cell surface accompanied by serpentine ridges which appeared in the resting state. After perfusion with a cytosol-like solution containing Ca2+, rhodamine fluorescence appeared on the cell surface as a distinct network formation. In some cases, circular fluorescences which appeared to surround the extruded pores were observed in the cell membrane. These findings indicate the existence of actin filaments in the cell membrane and/or subplasmalemmal network. In whole-mount preparations, the granules were surrounded very densely with microfilaments of various widths. After exposure to compound 48/80, granules protruding through the cell membrane were wrapped in many filaments. The extruded granules located in the periphery of the cells were connected by many filamentous structures and disruptions in the middle of these connections were occasionally observed. In some cases, circular configurations of microfilaments were observed at the bottom of the extruded granules and in others dense gatherings of microfilaments were seen just beneath the granules, as if the latter were being pushed up and out of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Highly sensitive quantitative telomerase assay of diagnostic testicular biopsy material predicts the presence of haploid spermatogenic cells in therapeutic testicular biopsy in men with Sertoli cell-only syndrome

The role of a telomerase assay in the recognition of Sertoli cell-only syndrome with testicular foci of haploid cells was evaluated. Men with Sertoli cell-only syndrome (n = 23) were given a new diagnostic testicular biopsy. Part of the biopsy was stained and the remainder was processed for the quantitative telomerase assay. After 3-13 months, a therapeutic testicular biopsy was performed. This material was minced and then examined using confocal laser scanning microscopy and fluorescent in-situ hybridization. Histology of diagnostic testicular biopsy material confirmed the diagnosis of Sertoli cell-only syndrome in all the participants. All seven men with a telomerase assay value in their diagnostic testicular biopsy of >42 total product generated (TPG) U/microg protein had haploid cells (i.e. spermatozoa and/or spermatids) in their therapeutic testicular biopsy. Among participants with telomerase assay values <42 TPG U/microg protein, only one man had haploid cells in his therapeutic testicular biopsy. Thus, telomerase assay values >42 TPG U/microg protein in the diagnostic biopsy identified 87.5% of the Sertoli cell-only syndrome men with haploid cells in their therapeutic testicular biopsy. Significantly higher values of the telomerase assay were found in men with testicular foci of haploid cells than in men without these foci. The use of a quantitative telomerase assay biopsy appears to be important for identifying those men with Sertoli cell-only syndrome who have foci of haploid cells and can be candidates for assisted reproduction techniques.