Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues

BACKGROUND: The mucosal mast cell (MMC) granule-specific beta-chymase, mouse mast cell protease-1 (mMCP-1), is released systemically into the bloodstream early in nematode infection before parasite-specific IgE responses develop and TGF-beta1 induces constitutive release of mMCP-1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein-1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated. OBJECTIVE: To investigate the expression and to compare the mechanisms of constitutive release of the chymase, mMCP-1, and the chemokine, CCL2. METHODS: MMC homologues were generated by culturing bone marrow cells in the presence of TGF-beta1, IL-3, IL-9 and stem cell factor (SCF). The intracellular distribution of mMCP-1 and CCL2 was examined by confocal microscopy. The involvement of the Golgi complex and of protein synthesis in the constitutive release of mMCP-1 and CCL2 was investigated using the Golgi-disrupting agent brefeldin A and cycloheximide to block protein synthesis. Secreted analytes were quantified by ELISA. RESULTS: mMCP-1 colocalized with Golgi matrix protein 130 but was most abundant in the granules, whereas CCL2 was not found in the granules but appeared to be located uniquely in the Golgi complex. Extracellular release of mMCP-1 was significantly inhibited ( approximately 40%) by cycloheximide and by the Golgi-disrupting agent brefeldin A, indicating both continuous protein synthesis and transportation via the Golgi complex are required for optimal mMCP-1 secretion. A similar but more marked inhibitory effect with both compounds was demonstrated on the constitutive secretion of CCL2. CONCLUSION: The culture conditions that promote mMCP-1 expression and release by MMC homologues also promote the expression and release of CCL2. Constitutive release involves de novo protein synthesis and requires a functional Golgi complex, suggesting that similar mechanisms of extracellular secretion operate for both mediators.     

Effect of neurotropin on seizure activity in picrotoxin kindling

Department of Normal Physiology, N. I. Pirogov Odessa Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR G. N. Kryzhanovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 3, pp. 236–239, March, 1992.     

Heat shock preconditioning modulates proliferation and apoptosis after superficial injury in isolated guinea pig gastric mucosa via an eicosanoid and protein synthesis-dependent mechanism

AIM: In restitution after superficial injury of the gastric mucosa, the epithelial continuity is restored by cellular migration. We have shown that heat shock preconditioning inhibits restitution after superficial injury. This study investigates the effect of heat shock preconditioning on tissue proliferation and apoptosis. EXPERIMENTAL DESIGN: Paired guinea pig gastric mucosae were mounted and perfused in Ussing chambers (37 degrees C). After heat shock preconditioning (42 degrees C) (30 min) and normothermic recovery (37 degrees C) (150 min) or normothermic perfusion, a superficial injury was induced by luminal exposure to 1.25 mol/L NaCl (5 min) followed by a 3 h restitution. During perfusion, the mucosa was exposed to 30 micromol/L arachidonic acid (AA) to enhance heat shock response, to 50 micromol/L quercetin (Q) to inhibit the metabolism of arachidonic acid via lipoxygenases, to 50 micromol/L indomethacin (In) to inhibit the metabolism of arachidonic acid via cyclo-oxygenases, or to 150 micromol/L cycloheximide (CHX) to inhibit de novo protein synthesis. After the experiment the mucosa was prepared for immunohistochemical analysis of the expression of Mib-1 proliferation antigen and pro-apoptotic protein Bax. RESULTS: Heat shock decreased Mib-1/Bax ratio and this effect was maintained after superficial injury and exposure to Q, to AA+CHX or to In+CHX. Exposure to CHX, to AA, to In+Q, to In+AA, In+AA+Q or to In+AA+CHX, however, blocked the effect of heat shock preconditioning. The decreasing effect of heat shock preconditioning on Mib-1/Bax ratio could be reversed by exposure to AA+Q or to In. CONCLUSION: The heat-preconditioning-induced effects on the mucosa are reversible and sensitive to exogenous pharmacological modulation. Heat shock preconditioning inhibits proliferation of superficially injured isolated gastric mucosa by a mechanism involving eicosanoid pathways and de novo protein synthesis.     

黄直丝链霉菌生产的新的细胞周期抑制剂(-)-脱水亚胺环己酮和l-亚胺环己酮

采用tsFT210细胞的流式细胞术活性筛选模型,通过活性跟踪分离,从黄直丝链霉菌18522发酵物中分离得到两个具有细胞周期抑制活性的化合物,并根据波谱数据和理化性质分别鉴定为(-)-脱水亚胺环己酮(1)和l-亚胺环己酮(2).活性测试结果表明,1和2将tsFT210细胞的细胞周期抑制在G0/G1期,最低有效浓度分别为23.8 μmol/L(1)和10.8 nmol/L(2).化合物1为新天然产物,也是新的亚胺环己酮类细胞周期抑制剂.     

Expression of inducible nitric oxide synthase in cultured smooth muscle cells from rat mesenteric lymphatic vessels

OBJECTIVE: The objective was to devise a method for establishing cultures of rat mesenteric lymphatic vessel smooth muscle cells (LSMC) and to investigate if inducible nitric oxide synthase (iNOS) expression could be activated in LSMC treated with bacterial lipopolysaccharide (LPS). METHODS: LSMC were successfully grown from explanted rat lymphatic microvessels and maintained by subculture. Treatment of LSMC for 24 h with LPS (1-100 microg/mL) activated iNOS protein induction, associated with (1) assay of increased nitrite concentrations in the medium representing cellular nitric oxide synthesis, and (2) demonstration of iNOS in cell extracts by Western blotting. RESULTS: The protein synthesis inhibitor cycloheximide (10 microM) blocked both LPS-induced nitrite formation and iNOS protein expression in LSMC. 1400 W (1 microM), a selective iNOS inhibitor, prevented LPS-induced nitrite formation but not iNOS expression. As well as induction of iNOS by LPS, "constitutive" iNOS was present in some cultures, producing nitrite in amounts that were also subsequently reduced after cell treatment with 1400 W. CONCLUSION: Rat mesenteric LSMC produce nitrite and express iNOS in response to bacterial LPS. Cultured LSMC may provide a useful model for studying mechanisms of iNOS induction in relation to possible influences of iNOS upon lymphatic vessel function.     

Targeting V-ATPase in primary human monocytes by archazolid potently represses the classical secretion of cytokines due to accumulation at the endoplasmic reticulum

The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10–100 nM) increases the vesicular pH in these cells. Archazolid (10 nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000 nM. Of interest, secretion of interleukin-1β was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.     

The role of protein synthesis in adrenocorticotrophin action: Effects of cycloheximide and puromycin on the steroidogenic response of isolated adrenocortical cells

The kinetics of the corticosteroidogenic response to adrenocorticotrophin (ACTH) have been investigated using collagenase dispersed adrenocortical cells. Following the addition of ACTH at a concentration that was maximal for steroidogenesis, there was a time-lag of about 3 min before increased steroidogenesis became apparent. This lag was extended (about two-fold) in the presence of a half-maximal concentration of ACTH. Preincubation of cells with submaximal concentrations of both cycloheximide and puromycin extended the time-lag observed following ACTH addition. Increasing doses of cycloheximide or puromycin concomitantly inhibited protein synthesis and steroidogenesis. Moreover cycloheximide, at a dose that halved protein synthesis, also inhibited steroidogenesis by 54–61% for a range of ACTH concentrations (1 × 10⁻⁴ to 1 × 10⁻² I.U./ml).It is concluded that the delay before ACTH-stimulated steroidogenesis is not attributable solely to the time taken for ribosomes to read of from the mRNA strand, the code for protein regulator(s). The results are discussed in terms of steroidogenic mechanisms whereby ACTH either induces de novo protein synthesis or activates a pre-existing, but labile, protein. In this latter scheme the steroidogenic rate observed under various conditions would be directly dependent upon the intracellular level of such an activated protein regulator. The half-life of labile protein, implicated as regulating steroidogenesis, was estimated at 2–4 min in this adrenal cell suspension system.     

Corticosteroids as long-term regulators of the insulin effectiveness in mouse 3T3 adipocytes

Summary Since corticosteroid treatment is often accompanied by insulin resistance, we explored the role of corticosteroids in the regulation of the insulin effectiveness in cultured 3T3 (mouse) adipocytes. Exposure of the fat cells to dexamethasone or corticosterone (0–5 days) induced a time-, concentration-, and protein synthesis-dependent and reversible decrease in insulin binding and in basal and insulin-stimulated 2-deoxyglucose uptake. The decrease in binding (50%) was primarily due to a decrease in receptor affinity i. e. to an increase in the rate of dissociation of insulin from its receptors, and was independent from the effects of pH and temperature on the affinity. The reduction in the 2-deoxyglucose uptake (30–50%) was due to a decrease in the hexose transport capacity rather than to a decrease in the phosphorylation component of the 2-deoxyglucose uptake process. Lineweaver-Burk analysis revealed the dexamethasone induced a decrease in the apparent V_max of the transport system i. e. in the number or activity of the hexose transporters. The effect of dexamethasone seemed to be superimposed on that of long-term insulin treatment, suggesting a different mechanism. It is concluded that corticosteroids act as long-term regulators of the insulin effectiveness by influencing the rate at which insulin dissociates from its receptors and by altering the number or activity of the hexose transporters by a common mechanism, which differs from that of the long-term regulatory effect of insulin.     

放线菌酮和TNFα协同诱导Lovo细胞凋亡

目的探索放线菌酮和ＴＮＦα协同诱导人大肠癌Ｌｏｖｏ细胞凋亡的发生规律．方法用细胞体外培养方法,以放线菌酮和ＴＮＦα均小剂量作用于人大肠癌Ｌｏｖｏ细胞,经Ｈｏｅｃｈｓｔ３３２５８荧光染色,光镜观察形态改变,并用流式细胞术检测ＤＮＡ断裂情况．结果单用ＴＮＦα５×１０５Ｕ／Ｌ或放线菌酮４ｍｇ／Ｌ,均未引起Ｌｏｖｏ细胞凋亡效应．而两者协同,随作用１２ｈ～４８ｈ,Ｌｏｖｏ细胞渐出现明显的凋亡形态学改变和ＤＮＡ断裂．结论小剂量的放线菌酮和ＴＮＦα协同作用于人大肠癌Ｌｏｖｏ细胞,出现明显的凋亡效应．