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Background/Aim: Kuji amber is an interesting natural source for drug discovery because a new anti-allergic compound, named kujigamberol and several new compounds have been isolatated from it. It was important to evaluate the yield, biological activities and constituents of each methanol extract of Kuji, Iwaki, Choshi, Mizunami and Ube ambers in Japan in order to establish if additional new compounds could be identified in these ambers. Materials and Method: Biological activities of each extract were evaluated using growth-restoring activity of the mutant yeast strain involving Ca2+-signal transduction and inhibition activity of degranulation in rat basophilic leukemia (RBL)-2H3 cells. Constituents of each extract were analyzed by high performance liquid chromatography (HPLC). Results: All ambers except Ube amber have growth-restoring activity against the mutant yeast. Both Kuji and Iwaki ambers inhibited the degranulation of RBL-2H3 cells induced by the calcium ionophore A23187 in a dose dependent manner. The main biologically active compound in Kuji amber, kujigamberol, was also isolated from Iwaki amber and analyzed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Conclusion: Kuji and Iwaki ambers appeared to have the same origin. Choshi, Mizunami, and Ube ambers are valuable sources for biologically active compounds which are different from those of Kuji amber.

The ethanolic extract obtained from the stems of Glycosmis pentaphylla was found to suppress antigen-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells. Four new geranylated 2-quinolone alkaloids, named glycopentanolones A–D (1–4), and 12 known metabolites (5–16) were isolated from the ethanolic extract from the stems of G. pentaphylla using bioassay-guided fractionation. Their structures were elucidated by a combination of 1D and 2D NMR, and HRESI-MS. The inhibitory effects of the isolated constituents on β-hexosaminidase release from RBL-2H3 cells were examined, and compounds 1, 5, 8 and 11 exhibited potent inhibitory activity with IC50 values between 0.05 and 4.28 μM.

Ten new (1–10) and 26 known (11–36) compounds were isolated from Penicillium griseofulvum MCCC 3A00225, a deep sea-derived fungus. The structures of the new compounds were determined by detailed analysis of the NMR and HRESIMS spectroscopic data. The absolute configurations were established by X-ray crystallography, Marfey’s method, and the ICD method. All isolates were tested for in vitro anti-food allergic bioactivities in immunoglobulin (Ig) E-mediated rat basophilic leukemia (RBL)-2H3 cells. Compound 13 significantly decreased the degranulation release with an IC50 value of 60.3 μM, compared to that of 91.6 μM of the positive control, loratadine.

Degranulation of mast cells and basophils occurs after the cross-linking of FceRI receptor-bound IgE by multivalent allergens, resulting in the release of a range of de novo synthesized and preformed mediators of the allergic response. β-Hexosaminidase release is usually measured as a simple readout for degranulation. Furthermore, the rat basophilic leukemia (RBL)-2H3 cell line is commonly used for measuring degranulation, monitoring β-hexosaminidase release. Here, we describe surface-engineered and modified nanoparticles with specific ligands in order to study the signaling and cellular responses of the RBL-2H3 cell line.

The inhibitory activities of the antimycin-class antibiotics UK-2A, antimycin A, and splenocin B against the production of anti-inflammatory cytokine IL-4, which is related to IgE-mediated allergic responses in rat basophilic leukemia (RBL-2H3) cells, were evaluated. Although antimycin A and splenocin B showed cytotoxicity at concentrations at which IL-4 release from the cells was restricted, UK-2A was found to restrict IL-4 release without cytotoxicity. Three UK-2A analogues (4–6) were then synthesized and assessed. Compound 5 restricted IL-4 release dose-dependently without cytotoxicity, and its effect was more potent than that of UK-2A.

The impact of several differentially functionalized SWCNTs, including pristine, unfunctionalized CNTs (U-CNTs) and differentially carboxy-functionalized CNTs (CNT-30, CNT-40 and CNT-50) on degranulation and histamine release of a model mast cell line (RBL-2H3 cells) was investigated. Greater declines in cell viability at 24h were noted when cells were exposed to U-CNTs (78.5 ± 3%) compared to CNT-30 (55.4 ± 4%), CNT-40 (58.4 ± 3%) and CNT-50 (55.8 ± 4%). U-CNT exposure caused a significantly greater decline in IgE-FceRI-mediated degranulation compared to acid functionalized CNT-30 at all concentrations tested. Importantly, removal of U-CNTs resulted in recovery degranulatory function while no recovery was found in cells exposed to CNT-30. U-CNT exposure resulted in a concomitant reduction in MAPK/ERK activation at 2 h declining to 53.0 ± 11% of control in the 250 mg L− 1 exposure group. However, similar to the degranulatory responses, MAPK/ERK activity could be rescued in RBL-2H3 cells exposed to U-CNTs but not in the AF-CNT exposures. Our results suggest that U-CNT result in reduced downstream activation of signaling kinases and suggests that the diminished degranulatory response observed after U-CNT exposure may result from a reduction in IgE-FceRI aggregation.

Summary Cell size homeostasis can be achieved by size checkpoints that couple cell size to cell-cycle progression or by alternative mechanisms such as constant extension. In mammalian cells, the existence of strict size checkpoints remains controversial due to the technical limitations in determining cell size directly and accurately. We developed a microfabricated channel system that linearizes mammalian cell growth and facilitates cell size measurements. By tracking cell length, while directly visualizing cell-cycle progression in rat basophilic leukemia cells and RAW 264.7 macrophages, we examined the mechanisms of size homeostasis and the existence of a size checkpoint at the G1/S transition. Our analysis revealed a two-tier size homeostasis mechanism where a G1 "sizer" or "adder" could operate, depending on the birth size of the cells.

Andrographis paniculata, âKing of bittersâ is a popularly known medicinal plant extensively used in many parts of the world for treatment of various diseases. Since recent past, anaphylactic/allergic type adverse events were reported upon A. paniculata usage, the study aimed to evaluate the anaphylactic and anaphylactoid potential of A. paniculata extract and andrographolide (a major phytoactive of A. paniculata). The anaphylactic potential was evaluated using active systemic anaphylaxis (ASA) assay in guinea pigs. Further, the release of allergic mediators was measured in immunoglobulin E (IgE) sensitized and non-IgE sensitized Rat Basophilic Leukemia (RBL-2H3) cell lines in-vitro. A. paniculata extract or andrographolide sensitized guinea pigs following the challenge antigen administration orally and intravenously did not demonstrate any clinical signs of anaphylaxis. IgE sensitized and non- IgE sensitized RBL-2H3 cells treated with A. paniculata extract did not induce release of allergic mediators. Whereas IgE sensitized and non- IgE sensitized RBL-2H3 cells treated with andrographolide demonstrated mild to moderate release of allergic mediators. A. paniculata extract has no anaphylactic and anaphylactoid potential in in-vivo and in-vitro studies. Whereas, andrographolide effects on allergic mediators in in-vitro studies needs to be scrutinized if they are of biologically important. Keywords: Andrographis paniculata extract, Andrographolide, Active systemic anaphylaxis, Anaphylactoid, &beta, &minus, Hexosaminidase, Leukotriene C4

A highly sensitive high performance liquid chromatography-electrochemical detection (HPLC-ECD) method being used to detect released serotonin in real time from rat basophilic leukemia 2H3 (RBL-2H3) cells which can be stimulated by potential allergens was established to evaluate the sensitization of potential allergens. Chromatographic separation was carried out on a Chromolith R Speed ROD RP-18e column (50 mm × 4.6 mm I.D., 2 μm). The linearity of serotonin in samples was good, with correlation coefficients greater than 0.9986 within the corresponding concentration range. The relative standard deviation (RSD) percentages were in the range of 0.72%–2.96% for inter-day precision and in the range of 2.02%–3.48% for intra-day precision. The relative errors (REs) were within ±3.21%. The recovery of serotonin was in the range of 100.98%–101.56%, and the RSDs of recovery were less than 2.28%. The method allows for simple sample preparation, short analysis time, and high sensitivity, specificity, and reliability. This method was successfully used to detect serotonin released by RBL-2H3 cells stimulated by different concentrations of schisandrin A and harpagoside. The results demonstrate that schisandrin A and harpagoside can trigger RBL-2H3 cells to release serotonin in a dose-dependent manner. The study contributes to the further use of evaluation of potential allergens.

Mast cells play a critical role in allergic diseases. Therefore, development of new therapeutic agents that suppresses the activation of mast cells may help prevent or treat allergic diseases. Here, we investigated the anti-allergic effects of 4-chloro-cinnamaldehyde and 4-trifluoro-cinnamaldehyde in RBL-2H3 cells. β-Hexosaminidase assays revealed that degranulation of RBL-2H3 cells was decreased following treatment with 60μM 4-chloro-cinnamaldehyde or 4-trifluoro-cinnamaldehyde. Moreover, quantitative real-time reverse transcription polymerase chain reaction showed that the relative expression levels of tumor necrosis factor-α, interleukin-4, and cyclooxygenase-2 mRNAs were decreased in RBL-2H3 cells treated with 4-chloro-cinnamaldehyde and 4-trifluoro-cinnamaldehyde in a concentration-dependent manner. Additionally, 4-chloro-cinnamaldehyde blocked the phosphorylation of MKKs and MAPKs. These data clearly suggested that 4-chloro-cinnamaldehyde and 4-trifluoro-cinnamaldehyde had inhibitory effects on the inflammatory responses of mast cells and may have potential as novel therapeutic agents for the prevention or treatment of allergic diseases.

The research in our laboratory focuses on the isolation of saponins from cactus. In this study, we report five new triterpenoid saponins, dumortierinoside A methyl ester (1), pachanoside I1 (2), pachanoside D1 (3), gummososide A (4), and gummososide A methyl ester (5). Compounds 1–3 isolated from Isolatocereus dumortieri Backbg., and compounds 4 and 5 were isolated from Stenocereus alamosensis A. C. Gibson & K. E. Horak. Compound 2 possessed a new pachanane-type triterpene skeleton, pachanol I, in its aglycon. The aglycon of 3 was pachanol D, while those of 4 and 5 were both gummosogenin, which we have previously reported, but this is the first report of pachanol D and gummosogenin in their aglycon forms. Additionally, we evaluated the anti-type I allergy activity of the saponins with RBL-2H3 (Rat basophilic leukemia) cells by measuring the β-hexosaminidase release inhibitory activity. As a result of these studies, gummososide A methyl ester (5) was found to show activity (IC50 = 99.5 μM) and thurberoside A exhibited mild activity (IC50 = 166.9 μM).

In their article, “Graphene Oxide Nanosheets Stimulate Ruffling and Shedding of Mammalian Cell Plasma Membranes,” Sun et al.1 described how the early-stage interaction between graphene oxide (GO) flakes and rat basophilic leukemia (RBL) cells perturbed their plasma membrane without causing cytotoxicity. The formation of peripheral membrane structures detaching from the RBL cells (within 4 hr) was reported for two other mammalian cell types, MDS-MB-231 (human breast cancer) and NIH/3T3 (mouse fibroblast) cells.

We report on electron-spin resonance microscopy (ESRM) providing sub-micron resolution (~700nm) with a high spin concentration sample, i.e. lithium phthalocyanine (LiPc) crystal. For biomedical applications of our ESRM, we have imaged samples containing rat basophilic leukemia (RBL) cells as well as cancerous tissue samples with a resolution of several microns using a water soluble spin probe, Trityl_OX063_d24. Phantom samples with the nitroxide spin label, (15)N PDT, were also imaged to demonstrate that nitroxides, which are commonly used as spin labels, may also be used for ESRM applications. ESRM tissue imaging would therefore be valuable for diagnostic or therapeutic purposes. Also, ESRM can be used to study the motility or the metabolism of cells in various environments. With further modification and/or improvement of imaging probe and spectrometer instrumentation sub-micron biological images should be obtainable, thereby providing a useful tool for various biomedical applications.

We examined the effects of essential oil from Thujopsis dolobrata Sieb. et Zucc. var. hondai Makino (EOTD) (Cupressaceae) on atopic dermatitis (AD)-like skin lesions in NC/Nga mice. Treatment with EOTD twice daily for two weeks ameliorate AD-like skin lesions induced by DNCB (2,4 dinitrochlorobenzene), and clinical scores were reduced to 7.29, 7.07, and 4.5 points in the groups treated with 1.5%, 3.0%, and 6.0% extract (p

RBL-2H3細胞によるβ－ヘキソサミニダーゼ放出阻害活性が認められたヨモギ熱水抽出物と，同様の方法でヨモギ熱水抽出物をクロロゲン酸エステラーゼで処理したヨモギ酵素分解物を得た．ヨモギ酵素分解物のIC50値は0.447 mg/mlでヨモギIC50値と比較すると脱顆粒抑制活性が7倍強くなった．また，カフェ酸含量はヨモギ酵素分解物の4.65％であった．ヨモギ酵素分解物のHPLC分析で得た1画分にも脱顆粒抑制作用が認められた．HPLC画分を解析した結果，カフェ酸と同定されカフェ酸画分のIC50値23.3 g/mlは，ヨモギ酵素分解物のIC50値（0.447 mg/ml）とカフェ酸含有量（4.65％）から推算される値と一致した．このことより，ヨモギ酵素分解物の主要な脱顆粒抑制作用は，カフェ酸に由来することが示唆された．

In this study, we investigated the suppressive effects of ore minerals on the allergic cell damages and oxida- tive cell damages. The ore minerals significantly reduced the productions of tumor necrosis factor-alpha (TNF- ) and interleukin-4 (IL-4) in rat basophilic leukemia cells challenged with 2,4-dinitrophenol-bovine serum α albumin (DNP-BSA). Lipoxygenase activity was also reduced by the ore minerals. Moreover, the ore minerals showed weak protective effects on the oxidative damage induced by hydrogen peroxide in pig kidney cells and retinal ganglion cells. Photohemolysis of erythrocytes in the presence of rose-bengal as a sensitizer was also inhibited by ore minerals. These results suggest that the ore minerals may be useful as the protectant for allergic and oxidative cell damages.

The anti-allergic effects of commercial products from cooked burdock (burdock salad, fried up burdock and burdock boiled with vinegar) were investigated by analyzing their inhibitory effects on the enzymatic activity (cyclooxygenase-2, lipoxygenase, phospholipase A2 and hyaluronidase) and the suppression of degranulation from rat basophilic leukemia (RBL)-2H3 cells. The methanol extract of burdock boiled with vinegar did not inhibit the cyclooxygenase-2 activity in the experiments. Those of the burdock salad and fried up burdock exhibited inhibitory activities in all the experiments. The burdock salad extract strongly inhibited lipoxygenase, phospholipase A2 and hyaluronidase. Thus, commercial cooked burdock, in addition to raw burdock, has bioactivities corresponding to an anti-allergic effect.

In this study, the inhibitory activities of one hundred plant extracts were analyzed against the IgE-induced allergy reaction in the rat basophilic leukemia (RBL-2H3) cells. Release of IL-4 and beta-hexosaminidase from the IgE-sensitized RBL-2H3 cells treated with the plant extracts were measured. Additionally, the effects of the plant extracts on the cell viability were tested. From the analysis, 17 extracts including Rhamnus davurica Pall. (branch) significantly inhibited beta-hexosaminidase release at 20 µg/mL. And 41 plants such as Artemisia absinthium (leaf and stem) showed high suppressive activities on IL-4 release. All of the extracts proliferated the sensitized cells above 80% compared with control, which was not treated with plant extract samples but treated only with the buffer used for sample preparation. In conclusion, the result suggests that the plants with the inhibitory activities on allergy reaction at cell level are candidates for developing useful anti-allergy materials and need to be study more.

We have studied the role of phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) in activation of rat basophilic leukemia (RBL-2H3) cells.Antigen-mediated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase and beta-hexosaminidase secretion were measured using RBL-2H3 cells stably expressing PI4KIIalpha-yellow fluorescent protein (YFP) or its kinase-deficient mutant PI4KIIalpha (K151A)-YFP.Neither PI4KIIalpha-YFP nor PI4KIIalpha (K151A)-YFP were distributed on the plasma membranes but on the exocytotic vesicles. The RBL-2H3 cells stably expressing PI4KIIalpha-YFP showed significantly enhanced beta-hexosaminidase secretion but not an increase in [Ca(2+)](i) after antigen stimulation. The cells with PI4KIIalpha (K151A)-YFP showed no change in the [Ca(2+)](i) increase nor degranulation. The promotion of secretion by PI4KIIalpha-YFP was not observed using co-stimulation with Ca(2+) ionophore and the protein kinase C activator, phorbol myristate acetate.These results suggest that PI4KIIalpha plays a role in the exocytotic process downstream of Ca(2+) signaling in antigen-mediated mast cell activation.

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.

幼若イネ中のポリフェノールを精製し構造解析とRBL-2H3に対する脱顆粒阻害作用を検討した．（1）幼若イネより固相抽出カラムを用いた精製によりポリフェノールを18.8％含む画分を得た．このイネ固相抽出精製物はHPLCにより5つの画分に分離された．（2）HPLCで分離された5つの画分に含まれる物質はフラボンを基本骨格にした類似の化合物であり，そのうち4つの成分はC-グリコシド結合を有する化合物と判明した．残りの1つは2種の混合物であったが，ともにC-グリコシドに加えてアグリコンのフラボンの水酸基のひとつに有機酸が結合した新規化合物である可能性が示唆された．（3）これら幼若イネのフラボンC-グリコシドは脱顆粒阻害作用を有していた．

The anti-allergic and anti-oxidative activities of curcumin-related compounds (glycosides, reductants and bis-demethoxy analogs) were investigated to elucidate the underlying active mechanisms and structural features of curcumin in exerting these activities. The anti-allergic activities were assessed by measurement of histamine release from rat basophilic leukemia cells, RBL-2H3. Curcumin and tetrahydrocurcumin (THC) caused a marked decrease in histamine release. Glycosides of curcumin, bis-demethoxycurcumin and THC also inhibited the release of histamine, though less potently than curcumin did. The anti-oxidative activities were assessed by measurement of cell-free or cellular radical scavenging. All compounds but diglycosides or bis-demethoxycurcumin analogs distinctly exerted anti-oxidative effects. The relationship between both of these activities revealed that all compounds with potent radical scavenging activities caused a definite decrease in histamine release, but some compounds with non-potent radical scavenging activities also inhibited the histamine release. These results suggest that the hydroxy groups of curcumin play a significant role in exerting both the anti-oxidative and anti-allergic activities, and that most of the compounds develop the anti-allergic activities through mechanisms related to anti-oxidative activities, but some through mechanisms unrelated to anti-oxidation activity.

被験農薬として10種類のピレスロイド系農薬及び25種類の有機リン系農薬について, 即時型アレルギー作用の指標となるin vitroにおけるRBL-2H3細胞の化学伝達物質である顆粒内酵素β-ヘキソサミニダーゼの遊離に及ぼす影響を検討した. その結果, β-ヘキソサミニダーゼの遊離を亢進させる農薬として, ピレスロイド系農薬のペルメトリン, ピレトリンと有機リン系農薬のEPN, プロチオホス, テルブホスが見いだされた. これらの農薬は比較的高濃度 (200μmol/L) で遊離亢進作用を示したが, 細胞の生存率にはほとんど影響を与えなかったことから, 本亢進作用はIgE抗体を介した化学伝達物質遊離反応系の亢進によることが示唆された.

Compound 4k N-[5-(4-fluoro)phenoxythien-2-yl]methanesulfonamide is representative of a new class of potent inhibitors of 5-lipoxygenase (5-LO). These versatile compounds exhibit dose-dependent inhibition of 5-LO with IC50s ranging from 20–100 nM in the rat basophilic leukemia (RBL-1) cell homogenate assay and submicromolar IC50s in both the RBL-1 and human peripheral blood leukocyte (PBL) whole cell assays. Compound 4k also showed significant anti-inflammatory activity in the adjuvant arthritic rat at an oral dose of 3 mg/kg.

Three known sesquiterpenoids of the lactarane and secolactarane type, deoxylactarorufin A (1), blennin A (2) and blennin C (3), have been obtained from cultures of Lentinellus cochleatus (Basidiomycetes) together with the new metabolites (Z)-2-chloro-3-(4-me-thoxyphenyl)-2-propen-l-ol (4) and lentinellone (5), a protoilludane derivative. The structures were determined by spectroscopic investigations. 1, 2 and 3 are potent inhibitors of leukotriene biosynthesis in rat basophilic leukemia (RBL-1) cells and human peripheral blood leukocytes (PBL).

Previously, we demonstrated that aggregates of the high affinity receptor for IgE (FcϵRI), formed by the binding of chemically cross-linked oligomers of IgE, continue to signal early and late cellular responses long after the formation of new aggregates is blocked. In the present work, we explore quantitatively the relationship between aggregation of the receptors and one of the earliest biochemical changes this initiates. We compare the time course of aggregate formation, inferred from studies of the binding of dimers of IgE, and the time course of phosphorylation of tyrosines on receptor subunits when the receptors are aggregated. A simple model does not fit the data. It appears that aggregates formed late in the response are less effective signaling units than those formed initially. We propose new explanations for the persistence of the response and the unusual kinetics.

Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-α production in rat basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced analog, 5′,6′-dimethyl-1,1′:4′1″-terphenyl-2′,3′,4,4″-tetraol (2) with a comparable inhibitory activity (IC50 = 0.02 nM) to that of 1. The synthesis involved double Suzuki–Miyaura coupling as a key step, and required only five steps from commercially available 3,4-dimethylphenol. For identification of the target molecule, fluorescent and biotinylated derivatives of 2 were prepared through a ‘click’ coupling process.

In order to study the mechanism(s) through which certain biologically active lipids, such as the hydroxyeicosanoids (HETEs), exert their effects, it is necessary to distinguish between binding of these lipids to cells and their cellular metabolism. A novel and simple method is described for the simultaneous determination of [3H]15-hydroxyeicosanoid (15-HETE) binding to cells and cellular [3H]15-HETE metabolism. The method involves initial separation of radiolabeled cells by filtration, filter extraction of cellular lipids by methanol, and thin-layer chromatography (or high performance liquid chromatography) determination of both nonesterified 15-HETE bound to cells and 15-HETE incorporation into cellular phospholipids. The method was applied to both PT-18 mast/basophil cells and rat basophilic leukemia (RBL-1) cells and should be applicable to other cells as well as other metabolizable hydroxy fatty acids or lipids.

The first synthesis of the 4,5 diphosphonate analog (4,5 IPn2; compound 2) of D,L-4,5 myo-inositol 4,5 diphosphate 1 (4,5 IP2) is described. Key steps in the synthesis include the introduction of the diphosphonate into dimesylate 5 using sodium diethyl phosphite in DME to give 6. The 3,6 trans hydroxyl groups were introduced into 6 using the Backvall trans-diacetoxylation reaction. The use of the C2 symmetry in the synthesis was utilized to introduce the final two setereocenters by osmylation of 14 to give compound 2 in an overall yield of 4.5% in only nine steps starting form readily available phthalic acid. Alternatively, the key intermediated 6 can be constructed by introducing the diphosphonate into compound 8 followed by conversion of 6. This results in an overall yield of 7% for the synthesis of 4,5 IPn2 (2) from butadiene and dimethyl fumarate. Compound 2 did not mobilize calcium in rat basophilic leukemia cells but is stable in vivo and can potentially be used for the production of antibodies.

A series of styrylpyrazoles, styrylisoxazoles, and styrylisothiazoles were prepared and found to be dual inhibitors of 5-lipoxygenase and cyclooxygenase in rat basophilic leukemia cells. Compounds from this series also were found to inhibit the in vivo production of LTB4 when dosed orally in rats. Among these compounds, di-tert-butylphenols 19 and 33 exhibit oral activity in various models of inflammation and, most importantly, are devoid of ulcerogenic potential.

A series of 1,2,4-oxadiazoles and 1,2,4-thiadiazoles containing a 2,6-di-tert-butylphenol substituent were prepared and evaluated as dual inhibitors of 5-lipoxygenase and cyclooxygenase in rat basophilic leukemia (RBL-1) cells. Several of these compounds show oral efficacy in the rat carrageenan footpad edema (CFE) and mycobacterium footpad edema (MFE) antiinflammatory models, without concomitant gastric ulceration. Structure-activity relationships are discussed. The best compounds (ID40 values in MFE of 3-8 mg/kg po) contain guanidine-derived substituents on the heterocyclic ring.

While atomic force microscopy (AFM) has become a promising tool for visualizing membrane morphology of cells, many studies have reported the presence of artifacts such as cliffs on the edges of cells. These artifacts shield important structural features such as lamellopodia, filopodia, microvilli and membrane ridges, which represent characteristic status in signaling processes such as spreading and activation. These cliff-like edges arise from a premature contact of the probe side contact with the cell prior to the probe top apex-cell contact. Carbon nanotube (CNT) modified AFM probes were utilized to address this drawback. Using rat basophilic leukemia (RBL) cells, this work revealed that CNT probes diminish cliff-like artifacts and enabled visualization of entire membrane morphology and structural features in three dimensions. The high aspect ratio of CNT probes provides a very effective remedy to the cliff-like artifacts as well as tip convolution of conventional probes, which shall enhance the validity and application of AFM in cellular biology research.

This paper describes the total synthesis of thelephantin G, thus revising the proposed structure 1 to 2. The key steps involved a double Suzuki−Miyaura coupling and an esterification reaction. By a similar strategy, ganbajunins D and E (3 and 4) were also prepared. Compound 2 strongly inhibited TNF (tumor necrosis factor)-α production in rat basophilic leukemia (RBL-2H3) cells: IC50 = 3.5 nM, while a mixture of 1 and its regioisomer 15 showed no such activity.

Comparison is made between several synthetic stereo and positional isomers of d -myo-inositol 1,4,5-trisphosphate ( d -myo-1,4,5-IP3) with respect to their ability to mobilize calcium from the internal stores of saponin-permeabilized rat basophilic leukemia cells. d - and l -myo-Inositol 1,4,5-trisphosphates, d - and l -myo-inositol 2,4,5-trisphosphates, d - and l -chiro-inositol 1,3,4-trisphosphates, d,l -trans-1,2-cyclohexane-diol bisphosphate, d,l -myo-inositol 4,5-bisphosphate, l -glycerol 1,2-bisphosphate, glycerol 1,3-bisphosphate and d,l -(1R,3R,4R)-1-phosphoryloxymethyl-trans-3,4-cyclohexanediol bisphosphate were tested. The analogs, each of which contains a vicinal trans-1,2-diol-bisphosphosphate motif, displayed potencies that were distributed over a 104-fold range of concentration and fell into 4 distinct classes of activity.

In this paper we describe our work that uses acoustics and dielectrophoresis to move particles/cells in microsystems. These particle manipulation technologies are used to create a high throughput system for spatiotemporal analyses of signaling within biological cells. The fabrication scheme and the focusing characteristics of the system are presented. The system allows the focusing characteristics to be tuned and optimized by changing the frequency of actuation for both the dielectrophoretic and piezoelectric electrodes. In addition to system characterization, we present initial data positioning rat basophilic leukemia (RBL) cells along the diffusion boundary of two different flow streams.

Ion channels play key roles in cell physiology and underlie a broad spectrum of disorders. To this day, the gold standard for studying ion channels is the patch clamp technique. Patch clamping involves careful positioning of a fine-tipped glass micropipette onto the surface of the cell to form a high-resistance (1 Gohms) seal ("gigaseal"), a procedure that is laborious, vibration-sensitive, and not easily amenable to automation. In addition, the solution inside the pipette cannot be easily exchanged. Recently reported patch clamp chips offer the potential of increased throughput, but to date the overall per-cell performance of most designs has been very low when compared to pipettes, and/or the fabrication process is prohibitively expensive. Here we demonstrate a replica-molded elastomeric patch clamp chip incorporating nanofabricated constrictions, which delivers high-stability gigaseals, with success rates comparable to those of pipettes, using rat basophilic leukemia (RBL) cells. The high stability enables exchanges of both the extracellular and intracellular solution during whole-cell recordings. In a sample of 103 experiments, 66 cells (64%) were successfully immobilized at the patch aperture; 38 cells (58% of immobilized cells, 37% of all cells) were successfully gigasealed; and 25 cells (65% of gigasealed cells, 34% of immobilized cells, 24% of all cells) were successfully perforated for whole-cell access. In the last group of 27 experiments, 79% of the cells could be immobilized, of which 68% could be gigasealed and 46% perforated for whole-cell access, indicating that dexterity is important.

The effect of surface topography and chemistry on cellular response is of fundamental importance, especially where living systems encounter device surfaces as in medical implants, tissue engineering, and cell-based sensors. To understand these biological processes on surfaces, there is a widespread interest in tailored surface-active materials produced by a combination of surface chemistry coupled to advanced patterning processes. We utilize self-assembled monolayers (SAMs) as molecular templates with submicrometer-scale spatial resolution to engage and cluster IgE receptors on rat basophilic leukemia (RBL) mast cells. Bioactive templates consisted of gold arrays on silicon with patterns from 1 mum down to 45 nm. These gold arrays served as molecular tethering sites, enabling covalent binding of functionalized self-assembled monolayers of alkanethiols. The free ends of the monolayers were functionalized with 2,4-dinitrophenyl(DNP)-caproate-based ligands which interact specifically with anti-DNP IgE bound to its high affinity cell surface receptor, FcepsilonRI on RBL mast cells. Present results on structures 1 mum down to 600 nm in size indicate that these ligand-immobilized patterned arrays can function as a powerful tool for visualization and systematic characterization of cell membrane involvement in IgE receptor-mediated immune cell signaling.

The search for functional foods in recent years has led to a heightened interest on Chinese herbs and plants as sources of medicine. In this study using in vitro assays, we report the anti-allergic activities of fractions extracted with ethanol and hot water from Saussurea inrolucrata, a plant from Tian Shan, China. A crude polyphenol fraction, which had been obtained from the extracts of the plant by reverse-phase column chromatography, strongly inhibited the release of β -hexosaminidase from rat basophilic leukemia (RBL-2H3) cells stimulated by antigen stimulation. The fraction also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in antigen-stimulated RBL-2H3 cells. While this plant has been used for many years as a medicinal tea in China, its extracts’ anti-allergenic properties as well as the nature of its anti-allergens have not yet been reported to date.

We have studied here the effects of ITIM-bearing FcgammaRIIB2 on the FcvarepsilonRI-dependent activation of rat basophilic leukemia (RBL-2H3) cells by atomic force microscopy (AFM). AFM images showed that ruffling and degranulation of RBL-2H3 cells were significantly reduced but the cell spreading was not by the co-clustering of FcgammaRIIB2 and FcvarepsilonRI. From the results it was shown that the co-clustering of ITIM-bearing negative co-receptors in a single RBL cell hardly induce the degranulation though the [Ca(2+)](i) increased transiently in the cell. This suggested that secretory responses and the membrane ruffling which were induced by the crosslinkng of FcgammaRII should be negatively controlled by the co-clustering of FcgammaRII.

Nonexcitable cells do not express voltage-activated Na+ channels. Instead, selective Na+ influx is accomplished through GTP-activated Na+ channels, the best characterized of which are found in renal epithelia. We have described recently a GTP-dependent Na+ current in rat basophilic leukemia (RBL) cells that differs from previous reported Na+ channels in several ways including selectivity, pharmacology and mechanism of activation. In this report, we have investigated the biophysical properties of the RBL cell Na+ current using the whole cell patch-clamp technique. Following activation by 250-500 microM GTP gamma S, hyperpolarizing steps to a fixed potential (-100 mV) from a holding potential of 0 mV evoked transient inward Na+ currents that declined during the pulse. If the holding potential was made more positive (range 0 to +100 mV), then the amplitude of the transient inward current evoked by the hyperpolarization increased steeply, demonstrating that the conductance of the channels was voltage-dependent. Using a paired pulse protocol (500 msec pulses to -100 mV from a holding potential of 0 mV), it was found that the peak amplitude of the current during the second pulse became larger as the interpulse potential became more positive. In addition, increasing the time at which the cells were held at positive potentials also resulted in larger currents, indicating a time-dependent conductance change. With symmetrical Na+ solutions, outward currents were recorded at positive potentials and these demonstrated both a time- and voltage-dependent increase in conductance. The results show that a nonvoltage activated Na+ channel in an electrically nonexcitable cell undergoes prominent voltage-dependent transitions. Possible mechanisms underlying this voltage dependency are discussed.