In vitro screening of seaweed extract on the proliferation of mouse spleen and thymus cell

A total number of 31 types of seaweed were assessed with regard to their effects on the proliferation of mouse spleen and thymus cells in a culture, using an MTT reduction assay. Acetone:dichloromethane (1∶1) extracts of three seaweed plants:Derbesia marina, Sargassum sp., andHisikia fuziformis, exhibited significantly positive effects on the survival of mouse spleen and thymus cellsin vitro. The acetone: dichloromethane (1∶1) extracts ofSargassum sp., in particular, much more potent effects on thymus cell activation than did any of the other types of seaweed. However, the methanol extracts ofSargassum ringgoldianium andChondrus crispus exerted a stimulatory influence only on the proliferation of mouse spleen cells, whereas the methanol extracts ofGrateloupia lanceolata exhibited significant cell proliferation properties in both spleen and thymus cells.     

The effect of Na+-K+ ATPase inhibition by ouabain on histamine release from human cutaneous mast cells

Background: There are controversial reports on the effect of sodium-potassium adenosine triphosphatase (Na⁺-K⁺ ATPase) inhibition on mast cell mediator release. Some of them have indicated that ouabain (strophanthin G), a specific Na⁺-K⁺ ATPase inhibitor, inhibited the release, whereas the others have shown that ouabain had no effect or even had a stimulatory effect on the mediator secretion. Most of these studies have utilized animal-derived mast cells. The aim of this study was to determine the effect of Na⁺-K⁺ ATPase inhibition on human skin mast cells. Methods: Unpurified and purified mast cells were obtained from newborn foreskins and stimulated by calcium ionophore A23187 (1 μM) for 30 min following a 1 hr incubation with various concentrations (10⁻⁴ to 10⁻⁸ M) of ouabain. Histamine release was assayed by enzyme-linked immunosorbent assay (ELISA). Results: The results indicated that ouabain had no significant effect on the non-immunologic histamine release from human skin mast cells, in vitro. Conclusions: Na⁺-K⁺ ATPase inhibition by ouabain had no significant effect on the non-immunologic histamine release from human cutaneous mast cells and suggested differences between human and animal mast cells.     

Observations of Forsythia koreana methanol extract on mast cell-mediated allergic reactions in experimental models

To explore effects of Forsythia koreana methanol extract (FKME) on mast cell-mediated allergic and inflammatory properties, the effect of FKME was evaluated on compound 48/80-induced systemic anaphylaxis, ear swelling, and anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-induced passive cutaneous anaphylaxis (PCA). In addition, the effect of FKME was investigated on the histamine release from rat peritoneal mast cells (RPMCs) stimulated by compound 48/80, which promotes histamine release. The human mast cell line HMC-1 was stimulated by phorbol 12-myristate 13-acetate plus calcium ionophore A23187. Activated HMC-1 can produce several proinflammatory and chemotactic cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8. Cytokine levels in the culture supernatant were measured by an enzyme-linked immunosorbent assay. Cytotoxicity by FKME was determined by a 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. FKME inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling in mice. When 1 g/kg FKME was pretreated or posttreated with mice, compound 48/80-induced mice morality was 50 and 66.7%, respectively. One gram per kilogram of FKME pretreatment inhibited ear-swelling responses derived from compound 48/80 by 29.75%. A PCA reaction was inhibited by 17.9%. In an in vitro model, FKME (1 mg/ml) inhibited histamine release from the RPMCs by 13.8% and TNF-α, IL-6, and IL-8 production from HMC-1 cells by 71.16% (P < 0.001), 86.72% (P < 0.001), and 44.6%, respectively. However, FKME had no cytotoxic effects on cell viability. In conclusion, FKME inhibited not only systemic anaphylaxis and ear swelling induced by compound 48/80 but also inhibited a PCA reaction induced by anti-DNP IgE in vivo. Treatment with FKME showed significant inhibitory effects on histamine, TNF-α, IL-6, and IL-8 release from mast cells.     

Antiallergic mechanisms of beta-adrenergic stimulants in rats.

Antiallergic mechanisms of beta-adrenergic stimulants were investigated in rats. Isoproterenol administered intravenously inhibited IgE antibody-mediated homologous passive cutaneous anaphylaxis (PCA) and histamine-induced cutaneous reaction (HCR) elicited at the same time in the same rats significantly. The inhibition of PCA was more potent than that of HCR, suggesting that PCA is inhibited by at least 2 mechanisms. One is the inhibition of vascular permeability increase. In vivo histamine release in the rat peritoneal cavity caused by intravenous antigen was inhibited by the intravenous administration of isoproterenol or salbutamol dose-dependently. On the contrary, when the histamine release in the peritoneal cavity was caused by intraperitoneal antigen, isoproterenol or salbutamol administered simultaneously with antigen failed to inhibit the reaction. Furthermore, antigen-induced histamine release from sensitized peritoneal exudate cells in vitro was not inhibited by isoproterenol or salbutamol. These results indicate that the primary target of beta-adrenergic stimulants is the vascular endothelium, and that the direct inhibition of chemical mediator release from mast cells does not play an important role for the inhibition of PCA and in vivo histamine release in the peritoneal cavity in rats. Beta-adrenergic stimulants therefore may prevent intravenously administered antigen from activating sensitized mast cells through affecting endothelial cells.     

The involvement of protein kinase C in exocytosis in mast cells.

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.     

Biochemical analysis of glucocorticoid-induced inhibition of IgE-mediated histamine release from mouse mast cells

Pretreatment of mouse mast cells with 10(-7) to 10(-6) M dexamethasone (DM) during overnight sensitization with mouse IgE antibody resulted in inhibition of antigen-induced histamine release and degranulation. The inhibition of both degranulation and histamine release increased linearly with the duration of the treatment; maximal inhibition was obtained after approximately 16 hr with DM. The addition of DM to sensitized mast cells immediately before antigen challenge did not affect the antigen-induced histamine release. DM interacted directly with mast cells by binding to DM-specific cytoplasmic receptors. The treatment of mast cells with DM did not affect the binding of IgE to mast cells or intracellular cAMP levels. Bridging of cell-bound IgE anti-DNP antibody on mouse mast cells either by multivalent DNP-HSA or by anti-IgE induced phospholipid methylation at the plasma membrane and Ca++ influx into the cells. Pretreatment of mast cells with DM inhibited the antigen-induced phospholipid methylation and Ca++ uptake but failed to affect histamine release by Ca++ ionophore A23187. The results suggest that DM treatment inhibits histamine release by the inhibition of the early stage of biochemical processes leading to opening Ca++ channels but does not affect the process distal to Ca++ influx or the binding of IgE molecules to IgE receptors.     

Depletion of mast cell ATP inhibits complement-dependent cytotoxic histamine release

Rabbit anti-rat mast cell antibody is capable of liberating histamine from rat peritoneal mast cells in the presence of complement. The cytotoxicity of this complement-mediated histamine release mechanism is attested by a substantial reduction of cell ATP, release of ⁵¹Cr and ⁸⁶Rb and lytic ultrastructural changes. Inhibition of complement-dependent cytotoxic histamine release can be achieved by depressing mast cell ATP with 2,4-dinitrophenol (1 mM), antimycin A (0.2 μM) or potassium cyanide (1 mM). Restoration of cell ATP is accompanied by reversal of the inhibition of the cytotoxic histamine release. Ultrastructural analysis and ⁵¹Cr release studies reveal that in mast cells depleted of ATP, cytolysis occurs but perigranule membranes remain intact, thus preventing histamine release.     

Inhibitory Effects of Apple Polyphenol on Induced Histamine Release from RBL-2H3 Cells and Rat Mast Cells

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC₅₀ values for histamine release were 30 μg/ml and 25 μg/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.     

Differentiation of the mechanisms inducing segmentation and asymetry of chromatids after treatment with 5-bromodeoxyuridine]

Rabbit anti-rat mast cell antibody is capable of liberating histamine from rat peritoneal mast cells in the presence of complement. The cytotoxicity of this complement-mediated histamine release mechanism is attested by a substantial reduction of cell ATP, release of ⁵¹Cr and ⁸⁶Rb and lytic ultrastructural changes. Inhibition of complement-dependent cytotoxic histamine release can be achieved by depressing mast cell ATP with 2,4-dinitrophenol (1 mM), antimycin A (0.2 μM) or potassium cyanide (1 mM). Restoration of cell ATP is accompanied by reversal of the inhibition of the cytotoxic histamine release. Ultrastructural analysis and ⁵¹Cr release studies reveal that in mast cells depleted of ATP, cytolysis occurs but perigranule membranes remain intact, thus preventing histamine release.     

Short-term sedimentation in Tagus estuary,Portugal: the influence of salt marsh plants

Short-term sediment deposition was studied at four salt marsh areas in the Tagus estuary. In areas covered with Sarcocornia perennis, Sarcocornia fruticosa, Halimione portulacoides and Spartina maritima and also in the non-vegetated areas, sedimentation was measured as the monthly accumulation of sediments on nylon filters anchored on the soil surface, from August 2000 to May 2001. Our experiments were used also to determine the influence of the different plant species in vertical accretion rates. Short-term sedimentation rates (from 2.8 to 272.3 g m⁻² d⁻¹) did show significant differences when the four salt marshes studied in the Tagus estuary were compared to each others. Salt marshes closer to the sediment sources had higher sedimentation rates. Our results suggest that the salt marsh type and surface cover may provide small-scale variations in sedimentation and also that sediment deposition values do change according to the position of the different plant species within the salt marsh. Sedimentation is an essential factor in salt marsh vertical accretion studies and our investigation may provide support to help forecast the adaptative response of the Tagus estuary wetlands to future sea level rise.     

The isolation and some properties of guinea pig mesenteric mast cells

A technique is described for obtaining isolated mast cells from guinea-pig mesentery by an enzymatic digestion process using hyaluronidase and collagenase. One to 4 × 10⁶ mast cells were obtained from the mesentery of each animal. Isolated mast cells from guinea-pigs of about 400 g were approximately spherical with a mean diameter of 6.1 μm and a mean histamine content of 8.8 pg. Studies on isolated mast cells from sensitised animals showed that the cells were still capable of an anaphylactic release of histamine when challenged with the appropriate antigen. Isolated mast cells did not sensitise when incubated with antibody dissolved in physiological saline but sometimes became weakly sensitised when incubated with the same antibody in isotonic-buffered glucose. Mast cells were found to survive in culture but they were no longer capable of an antigen-induced histamine release.     

Inhibitory effect of polyphenol-enriched apple extracts on mast cell degranulation in vitro targeting the binding between IgE and FcepsilonRI

Extracts from immature fruit of the apple (Rosaceae, Malus sp.), which contain procyanidins (polymers of catechins) as the major ingredients, are known to inhibit histamine release from mast cells. We analyzed in this study the mechanism for the anti-allergic activity of two polyphenol-enriched apple extracts. These extracts, termed "crude apple polyphenol (CAP)" and "apple condensed tannin (ACT)", reduced the degranulation of mast cells caused by cross-linking of the high-affinity receptor for IgE (FcepsilonRI) with IgE and the antigen in a dose-dependent manner. Furthermore, western blotting revealed that phosphorylation of the intracellular signal-transduction molecules caused by cross-linking of FcepsilonRI was markedly decreased by the addition of CAP or ACT. We then analyzed the effects of CAP and ACT on the binding of the IgE antibody to FcepsilonRI on mast cells, which is the first key step in the allergic reaction mediated by mast cells, and found that this binding was markedly inhibited by both CAP and ACT. These results indicate that the inhibition of binding between FcepsilonRI and IgE by either CAP or ACT was the probable cause of the suppression of mast cell activation. This is the first report demonstrating the molecular mechanism for the anti-allergic effect of procyanidin-enriched extracts from apples.     

Preliminary study of taxonomy ofAzotobacter andAzomonas by using rocket line immunoelectrophoresis

Rocket line immunoelectrophoresis was used to study the taxonomy ofAzotobacter andAzomonas assessed by reaction with antiserum to the AVO₂ strain ofAzotobacter. Forty-five cultures, comprising seventeen species in five genera, showed that antigen “β”, like high-titer somatic agglutination, was restricted to all 11 strains ofAzotobacter vinelandii and to one strain which has been namedAzotobacter macrocytogenes (10EM). A thermoresistant antigen (“γ”) was found to be shared by all strains and species ofAzotobacter andAzomonas investigated.     

Habitat segregation in a salt marsh among adult sticklebacks (Gasterosteidae)

Synopsis The spatial distribution, seasonal abundance and diel activity of four sticklebacks,Gasterosteus aculeatus, G. wheatlandi, Pungitius pungitius, andApeltes quadracus. coexisting in a St. Lawrence salt marsh were examined to see how these closely related species share their habitat. While all four species breed in the Riviè des Vases, a tidal creek, only three species are found in the adjacent salt marsh pools,A. quadracus being absent. Results are interpreted in terms of avoidance of interspecific competition for space during the relatively short breeding season at this latitude.     

Antibacterial activity of some salt marsh halophytes and mangrove plants against methicillin resistant Staphylococcus aureus

The antibacterial activity of aqueous and methanol extracts of leaves/shoots of five salt marsh halophytes and six mangroves was studied against methicillin resistant, clinical isolates of Staphylococcus aureus. There was a clear comparability between the salt marsh halophytes and mangroves in their antibacterial action. The mangrove plants possessed higher antibacterial potency than the salt marsh halophytes. The highest activity was recorded with the methanol extract of Excoecaria agallocha followed by the methanol extracts of Aegiceras corniculatum, Lumnitzera racemosa and Ceriops decandra. The minimum inhibitory concentration (MIC) values ranged from 0.125 to 4 mg/mL and 1 to 16 mg/mL for methanol and aqueous extracts, respectively. Further separation of active principle from the potent mangrove plant will be useful for the control of drug resistant strains of S. aureus.     

Anti-HIV-1 efficacy of extracts from medicinal plants

The anti-HIV-1 activities of butanol, hexane, chloroform and water extracts from four widely used folk medicinal plants (Sophora flavescens, Tulipa edulis, Herba ephedra, and Pachyma hoelen Rumph) were evaluated in this study. The hexane extract of Pachyma hoelen Rumph, PH-4, showed effective inhibition against HIV-1. The 50% effective concentration (EC₅₀) of PH-4 was 37.3 μg/ml in the p24 antigen assay and 36.8% in the HIV-1 recombinant RT activity test (at 200 μg/ml). In addition, the PH-4 showed the protective effect on the infected MT-4 cells, with a 58.2% rate of protection. The 50% cytotoxic concentration (CC₅₀) of PH-4 was 100.6 μg/ml. These results suggest that PH-4 from Pachyma hoelen Rumph might be the candidate for the chemotherapy agent against HIV-1 infection with further study.     

Change of hemoagglutinin and hemolysin titers in hemolymph of gastropod molluscs in response to immunization with sheep erythrocytes

This work deals with analysis of changes of the levels of hemoagglutinins (HA) and hemolysins (HL) in hemolymph of three gastropod species,Planorbius corneus, Lymnea stagna-lis, andAchatina fulica, in response to immunization with sheep erythrocytes (ShE). The levels of HA and HL were determined before immunization and 3, 5, 10, and 15 days after the antigen administration. The HA titer was estimated from hemoagglutination reaction, the HL titer was measured spectrophotometrically from release of hemoglobin from ShE. It has been found that after immunization the titer of HA falls by 72–88% for the first 3–5 days, but returns to the initial values or even exceeds them statistically significantly by the10–15th days. The time of the decrease of the HA titers correlates inversely with the body mass of the animals, while the amplitude of this decrease is directly proportional to the body mass. The HL titer either changes parallel to the HA titer (inL. stagnalis) or increases statistically significantly by 60–65% during the entire period of observation (in two other species studied). It is suggested that inP. coneus andA. fulica, HA and HL are different compounds.     

Fritillaria ussuriensis Extract Inhibits the Production of Inflammatory Cytokine and MAPKs in Mast Cells

Fritillaria ussuriensis (FU, derived from the bulbs of various species of the genus Fritillaria, including Fritillaria thunbergii Miq.) is used in herbal medicine to treat conditions such as eczema, skin burns, and frostbite. In this study, we investigated the mechanism of the anti-allergy effect of FU. FU extract (80 mg/kg), orally administered to Sprague-Dawley (SD) rats, significantly inhibited the passive cutaneous anaphylaxis (PCA) reaction. It inhibited the compound 48/80-induced release of histamine from rat peritoneal mast cells in a concentration-dependent manner. Significant inhibitory effects of the FU extract on IL-6, IL-8, and TNF-α (1, 10, and 100 μg/mL) were observed in HMC-1 cells. Treatment with FU attenuated PMA plus A23187-induced phosphorylation of all three MAPKs, especially at concentrations of 10 and 100 μg/mL. Further, it (80 mg/kg) led to significant inhibition of mast-cell accumulation in ear tissue at the chronic phase. These results indicate that it inhibits allergic reactions.     

Application of suppression subtractive hybridization (SSH) to cloning differentially expressed cDNA inDunaliella salina (chlorophyta) under hyperosmotic shock

Two subtracted cDNA libraries ofDunaliella salina (Volvocales, Chlorophyceae) under different hyperosmotic shock were constructed using the suppression subtractive hybridization (SSH) method. The mRNA isolated from algae grown without stress was used as a “driver”, and the mRNAs isolated from algae 16 h (short-term treatment) or 7 d (long-term treatment) after salt stress were used as “testers”. The differentially expressed cDNA fragments inD. salina under salt stress were identified by screening these 2 libraries. Two cDNA fragments,D27 andD114, were identified from clones pL27 and pL114 after the long-term treatment. Three cDNA fragments,D21, D39, andD88, were identified from clones pSh21, pSh39, and pSh88 after the short-term treatment. The homology analysis revealed that D27 was highly similar (91%) to the subunit V of PS I reaction center inChlamydomonas reinhardtii. D21 was similar to fructose-1,6-diphosphate aldolase (78.4%). After searching GenBank with the sequences ofD39, D88, andD114, no similar sequences were found. Northern analysis revealed that the expression levels of all 5 cDNAs were increased significantly after salt stress. This means that SSH can be used in cloning differentially expressed cDNAs inD. salina under salt stress. The expression ofD27, D21, andD88 wasde novo induced by salt stress, and the expression ofD114 andD39 was increased from a relatively lower level; this indicates that all 5 cDNAs might exert an influence on the alga under hyperosmotic shock.