Receptor dynamics regulates actin polymerization state through phosphorylation of cofilin in mast cells.

Aggregation of IgE bound to the high-affinity IgE receptor (FcεRI) by a multivalent antigen induces mast cell activation, while disaggregation of aggregated FcεRI by monomer hapten immediately terminates degranulation mediated by dephosphorylation of Syk and mediates a decrease in intracellular Ca2+ concentration ([Ca2+]i). The actin polymerization state is intimately involved in mast cell activation mediated by FcεRI aggregation. However, the relation between aggregation-disaggregation of FcεRI and actin rearrangement in mast cells is not well understood. The addition of a multivalent antigen rapidly depolymerized actin filaments, while the subsequent addition of monomer hapten rapidly recovered actin polymerization. Whereas cofilin, an actin-severing protein, was temporally dephosphorylated several minutes after a multivalent antigen stimulation and the addition of monomer hapten rapidly increased cofilin phosphorylation level within 30 s. The removal of extracellular Ca2+ instead of monomer hapten addition did not restore cofilin phosphorylation, suggesting that the significant decrease in [Ca2+]i by monovalent hapten was not a critical reason for the actin rearrangement. Additionally, monovalent hapten did not completely reduce [Ca2+]i in mast cells pretreated with jasplakinolide, an inhibitor of actin depolymerization. These results suggest that the multivalent antigen-induced actin depolymerization mediated by cofilin dephosphorylation, and the subsequent addition of monovalent hapten in the F-actin severing state efficiently elicited actin re-polymerization by cofilin phosphorylation.

Monomer hapten and hapten-specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms.

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.

Involvement of intracellular caveolin-1 distribution in the suppression of antigen-induced mast cell activation by cationic liposomes.

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross-linking of high-affinity immunoglobulin E receptor in a time- and dose-dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca2+ concentration ([Ca2+ ]i ) via the inhibition of store-operated Ca2+ entry. Further, we revealed that the mechanism underlying an impaired [Ca2+ ]i increase is the inhibition of the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Yet, how cationic liposomes inhibit the PI3K-Akt pathway is still unclear. Here, we focused on caveolin-1, a major component of caveolae, which is reported to be involved in the activation of the PI3K-Akt pathway in various cell lines. In this study, we showed that caveolin-1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin-1 to the plasma membrane and the colocalization of caveolin-1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.

Inhibition of degranulation in mast cells attached to a hydrogel through defective microtubule tracts.

Mast cells (MCs) are important effectors of the immediate allergic response. MCs are distributed throughout various tissues and organs, and adhere to extracellular matrix (ECM) with broad stiffness in the body. Here we compared cellular responses following antigen stimulation in MCs on glass-base dishes with and without a hydrogel. We found that an antigen-induced increase in intracellular Ca2+ concentration was suppressed slightly in cells on hydrogel-coated dishes compared with those on non-coated dishes, whereas their subsequent degranulation was largely inhibited in cells adherent to the hydrogel. Focusing on focal adhesions (FAs), vinculin was distributed in a dot-like manner at the bottom of resting cells on non-coated dishes but not on hydrogel-coated dishes. According to antigen stimulation, phosphorylation of focal adhesion kinase and additive vinculin accumulation to FAs were promoted in cells on non-coated dishes, but were diminished on hydrogel-coated dishes. Moreover, microtubule reorganization and acetylation (which have important roles in MC degranulation) were also suppressed in activated MCs adherent to the hydrogel. These findings suggest that adhesion to a hydrogel led to failure of composition of functional FAs and microtubule tracts, which resulted in suppression of MC degranulation following antigen stimulation.

Promotion of microtubule acetylation plays an important role in degranulation of antigen-activated mast cells.

OBJECTIVE: The aim of this study was to investigate whether microtubule acetylation is triggered by antigen stimulation and how it affects mast cell degranulation. METHODS: The RBL-2H3 cell line was used as a model for mast cells. Acetylation of α-tubulin was analyzed by Western blotting. Intracellular distribution of α-tubulin and acetylated α-tubulin was observed by immunostaining. Degranulation was monitored by measuring the activity of ß-hexosaminidase secreted into cell supernatants. Tukey-Kramer test was used to compare differences between groups. RESULTS: Microtubule acetylation proceeds globally in mast cell cytoplasm after antigen stimulation in addition to accelerated formation of microtubule-organizing centers. Pretreatment with 5Z-7-oxozeaenol (5 µmol/l), an inhibitor of TGF-ß-activated kinase 1, which is a key activator of α-tubulin acetyltransferase 1, did not affect the distribution and acetylation of microtubules in resting cells; however, it significantly suppressed antigen-evoked microtubule acetylation and their reorganization, and subsequent degranulation (95.0 ± 1.2% inhibition, n = 3, P < 0.01). CONCLUSIONS: These results provided new insight into the post-translational modifications of microtubule to regulate mast cell degranulation.

Decreased intracellular granule movement and glucagon secretion in pancreatic α cells attached to superior cervical ganglion neurites.

Autonomic neurons innervate pancreatic islets of Langerhans and participate in the maintenance of blood glucose concentrations by controlling hormone levels through attachment with islet cells. We previously found that stimulated superior cervical ganglia (SCG) could induce Ca2+ oscillation in α cells via neuropeptide substance P using an in vitro co-culture model. In this study, we studied the effect of SCG neurite adhesion on intracellular secretory granule movement and glucagon secretion in α cells stimulated by low glucose concentration. Spinning disk microscopic analysis revealed that the mean velocity of intracellular granules was significantly lower in α cells attached to SCG neurites than that in those without neurites under low (2 mM), middle (10 mM), and high (20 mM) glucose concentrations. Stimulation by a low (2 mM) glucose concentration significantly increased glucagon secretion in α cells lacking neurites but not in those bound to neurites. These results suggest that adhesion to SCG neurites decreases low glucose-induced glucagon secretion in pancreatic α cells by attenuating intracellular granule movement activity.

Cationic liposomes suppress intracellular calcium ion concentration increase via inhibition of PI3 kinase pathway in mast cells.

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes (antisense DNA, plasmid DNA, siRNA, etc.) into target cells. Cationic liposomes are also known to affect cellular immunocompetences such as the mast cell function in allergic reactions. In particular, we previously showed that the cationic liposomes bound to the mast cell surface suppress the degranulation induced by cross-linking of high affinity IgE receptors in a time- and dose-dependent manner. This suppression is mediated by impairment of the sustained level of intracellular Ca2+ concentration ([Ca2+]i) via inhibition of store-operated Ca2+ entry (SOCE). Here we study the mechanism underlying an impaired [Ca2+]i increase by cationic liposomes in mast cells. We show that cationic liposomes inhibit the phosphorylation of Akt and PI3 kinases but not Syk and LAT. As a consequence, SOCE is suppressed but Ca2+ release from endoplasmic reticulum (ER) is not. Cationic liposomes inhibit the formation of STIM1 puncta, which is essential to SOCE by interacting with Orai1 following the Ca2+ concentration decrease in the ER. These data suggest that cationic liposomes suppress SOCE by inhibiting the phosphorylation of PI3 and Akt kinases in mast cells.

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions.

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion.

Effects of PIP2 on membrane fusion between mast cell SNARE liposomes mediated by synaptotagmin 2.

Recent studies have revealed that SNARE proteins are involved in exocytotic release in mast cells. Previously, we reported that mast cell SNARE proteins induce membrane fusion between liposomes. Moreover, we found that synaptotagmin 2, a candidate Ca2+ sensor for mast cell exocytosis, enhanced SNARE-mediated membrane fusion via Ca2+ and phosphatidylserine. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an acidic phospholipid like phosphatidylserine. In the present study, we investigated whether PIP2 is involved in the enhancement effect of synaptotagmin 2 on SNARE-mediated membrane fusion. PIP2 did not show any significant effect on SNARE-mediated membrane fusion by itself. In the presence of Ca2+, synaptotagmin 2 enhanced SNARE-mediated membrane fusion between liposomes containing PIP2. However, even in the presence of Ca2+, when we used 100% PC liposomes, synaptotagmin 2 did not show any significant effect on SNARE-mediated membrane fusion. These results indicated that PIP2 is involved in the enhancement effect of synaptotagmin 2 on membrane fusion between liposomes containing mast cell SNARE proteins.

Phosphorylation of syntaxin-3 at Thr 14 negatively regulates exocytosis in RBL-2H3 mast cells.

Recent studies have revealed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin-3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin-3. We found that Thr 14 of syntaxin-3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin-3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin-3 at Thr 14 (T14E) induced structural changes in syntaxin-3, and this mutation inhibited binding of syntaxin-3 to Munc18-2. These results suggest that phosphorylated syntaxin-3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin-3 and Munc18-2.

Impaired expression of the mitochondrial calcium uniporter suppresses mast cell degranulation.

Calcium ion (Ca(2+)) uptake into the mitochondrial matrix influences ATP production, Ca(2+) homeostasis, and apoptosis regulation. Ca(2+) uptake across the ion-impermeable inner mitochondrial membrane is mediated by the mitochondrial Ca(2+) uniporter (MCU) complex. The MCU complex forms a pore structure composed of several proteins. MCU is a Ca(2+)-selective channel in the inner-mitochondrial membrane that allows electrophoretic Ca(2+) entry into the matrix. Mitochondrial Ca(2+) uptake 1 (MICU1) functions as a Ca(2+)-sensing regulator of the MCU complex. Previously, by microscopic analysis at the single-cell level, we found that during mast cell activation, mitochondria capture cytosolic Ca(2+) in two steps. Consequently, mitochondrial Ca(2+) uptake likely plays a role in cellular function through cytosolic Ca(2+) buffering. Here, we investigate the role of MCU and MICU1 in mitochondrial Ca(2+) uptake and mast cell degranulation using MCU- and MICU1-knockdown (KD) mast cells. Whereas MCU- and MICU1-KD mast cells show normal proliferation rates and mitochondrial membrane potential, they exhibit slow and reduced cytosolic and mitochondrial Ca(2+) elevation after antigen stimulation. Moreover, ß-hexosaminidase release induced by antigen was significantly suppressed in MCU-KD cells but not MICU1-KD cells. This suggests that both MCU and MICU1 are involved in mitochondrial Ca(2+) uptake in mast cells, while MCU plays a role in mast cell degranulation.

Novel mono substituted pyridoimidazoisoquinoliniums via a silver-catalyzed intramolecular cyclization and their applications in cellular imaging.

Cationic heterocycles, an important class of organic compounds soluble in polar solvents, have been gaining attention in the construction of fluorescent probes. This paper reports the quick synthesis of novel pyrido[1',2';2,3]imidazo[5,1-a]isoquinoliniums starting from 2-(2-ethynylphenyl)imidazo[1,2-a]pyridines at room temperature via intramolecular cyclization by employing a catalytic amount of silver trifluoromethanesulfonate in addition to lithium trifluoromethanesulfonate and silica gel as the counter anion source and additive, respectively. The designed pyridoimidazoisoquinoliniums consisted of an imidazo[1,2-a]pyridine fused isoquinolinium. The X-ray diffraction results revealed that pyrido[1',2';2,3]imidazo[5,1-a]isoquinolinium trifluoromethanesulfonate contained considerable planar parent skeletons and interacted by π-π stacking with neighbouring molecules. Furthermore, in a methanol solution the designed 6-phenyl derivative exhibited strong fluorescence in the 420-450 nm region in addition to strong mitochondrial specificity in a cell staining assay.

Substance P plays an important role in cell adhesion molecule 1-mediated nerve-pancreatic islet α cell interaction.

Autonomic neurons innervate pancreatic islets of Langerhans and maintain blood glucose homeostasis by regulating hormone levels. We previously showed that cell adhesion molecule 1 (CADM1) mediated the attachment and interaction between nerves and aggregated pancreatic islet α cells. In this study, we cocultured αTC6 cells, a murine α cell line, with mouse superior cervical ganglion (SCG) neurons. The oscillation of intracellular Ca(2+) concentration ([Ca(2+)]i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6(siRNA-CADM1) cells) in aggregates, respectively, within 1min after specific SCG nerve stimulation with scorpion venom. In αTC6(siRNA-CADM1) cells, the responding rate during 3min after SCG nerve stimulation significantly increased compared with that within 1min, whereas the increase in the responding rate was not significantly different in αTC6 cells. This indicated that the response of αTC6 cells according to nerve stimulation occurred more rapidly and effectively than that of αTC6(siRNA-CADM1) cells, suggesting CADM1 involvement in promoting the interaction between nerves and α cells and among α cells. In addition, because we found that neurokinin (NK)-1 receptors, which are neuropeptide substance P receptors, were expressed to a similar extent by both cells, we investigated the effect of substance P on nerve-α cell interaction. Pretreatment with CP99,994 (0.1µg/ml), an NK-1 receptor antagonist, reduced the responding rate of both cells, suggesting that substance P released from stimulated neurites was a mediator to activate αTC6 cells. In addition, α cells that were attached to neurites in a CADM1-mediated manner appeared to respond effectively to neurite activation via substance P/NK-1 receptors.

Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes.

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.

Effects of lipid composition in cationic liposomes on suppression of mast cell activation.

We previously showed that cationic liposomes composed of cholesteryl-3ß-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) inhibited mast cell degranulation mediated by the cross-linking of high-affinity IgE receptors (FcεRI). In this study, we prepared three kinds of cationic liposomes composed of OH-Chol and DOPE in different ratios (0.28, 0.60, and 0.86 of OH-Chol in mol ratio, named as L-liposome, M-liposome, and H-liposome, respectively) and investigated their effects on mast cell activation. We found that mast cell degranulation evoked with antigen was inhibited by pretreatment with cationic liposomes in the composite ratio-dependent manner of OH-Chol and that the H-liposome showed the highest inhibitory effect on degranulation among three kinds of liposomes. Store-operated Ca2+ entry, phosphorylation of PI3K and Akt, and IL-4 secretion after antigen stimulation were reduced in dose-dependent manner of each liposome, but there were no differences between H-liposome and M-liposome. Meanwhile, microtubule acetylation, which is involved in the secretory granule transport, was significantly suppressed by H-liposome compared with M-liposome. These data suggested that the lipid composition in cationic liposomes themselves largely influenced the inhibition of mast cell activation as well as the efficiency of gene transfection.

Effects of Cot expression on the nuclear translocation of NF-kappaB in RBL-2H3 cells.

Cot is a serine/threonine protein kinase and is classified as a mitogen-activated protein (MAP) kinase kinase kinase. Overexpression of this protein has been shown to activate the extracellular signal-regulated kinase, the c-Jun N-terminal kinase, and the p38 MAP kinase pathways and to stimulate NF-AT and NF-kappaB-dependent transcription. Here we have shown that Cot kinase activity is intimately involved in the high affinity receptor for IgE (FcvarepsilonRI)-mediated nuclear translocation of NF-kappaB1 independent of NF-kappaB-inducing kinase (NIK) in rat basophilic leukemia (RBL-2H3) cells. A transfected green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1) resided in the cytoplasm in RBL-2H3 cells and it remained in the cytoplasm even when Cot tagged with red fluorescent protein (Cot-RFP) was co-expressed. Western blotting analysis showed that IkappaB kinases (IKKs) were expressed in RBL-2H3 cells but NIK was not. GFP-NF-kappaB1 translocated from the cytoplasm to the nucleus after the aggregation of FcvarepsilonRI in Cot-transfected cells but not in kinase-deficient Cot-transfected cells. This finding gives a new insight into the role of Cot in the FcvarepsilonRI-mediated NF-kappaB activation in mast cells.

Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix.

Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.

Gene transfection efficiency into dendritic cells is influenced by the size of cationic liposomes/DNA complexes.

Cationic liposomes have attracted recent attention as DNA vaccine carriers that can target dendritic cells (DCs). In general, cationic liposome/DNA complexes (lipoplexes) are taken up by various cells via clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, or phagocytosis, with the mode of endocytosis determining further intracellular trafficking pathways. Moreover, the physicochemical properties of cationic lipoplexes, including lipid composition, shape, size, and charge, influence transfection efficiency, affecting uptake and subsequent intracellular pathways. To develop cationic liposomes as potential DNA vaccine carriers, the objective of this study was to study the effect of lipoplex size on DNA transfection efficiency in DCs. We explored the size-dependent endocytosis pathway and the intracellular trafficking of cationic lipoplexes using bone marrow derived dendritic cells (BMDCs). Our results indicated that small-sized lipoplexes (approximately 270nm diameter) were taken up by BMDCs via caveolae-mediated endocytosis, which led to a non-degradative pathway, whereas larger-sized lipoplexes (approximately 500nm diameter) were taken up by BMDCs via clathrin-mediated endocytosis and micropinocytosis, which led to a lysosomal degradation pathway. These findings suggest that, by regulating the size of lipoplexes, it may be possible to develop cationic liposomes as DNA vaccine therapies for targeting DCs.

Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 µm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 µm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity.

Effects of propolis on cell growth and gene expression in HL-60 cells.

Brazilian propolis obtained from honeybee hives was extracted with water or ethanol. Cell growth-inhibitory activities of these propolis extracts were found in HL-60 human myeloid leukemia cells. The extracts-induced apoptosis in the cells, which was characterized by morphological and nucleosomal DNA fragmentation analysis. The apoptosis was mainly attributed to the induction of granulocytic differentiation, which was evaluated by nitro blue tetrazolium (NBT) reducing assays and cytofluorometric analysis for the expression of cell surface marker CD11b. DNA microarray analysis was performed to examine the gene expression profiles in the propolis-treated HL-60 cells accompanied with granulocytic differentiation, which were compared with those in all-trans retinoic acid-treated cells. Several genes were up- or down-regulated. Two genes encoding S100 calcium binding protein A9 and ferritin, heavy polypeptide 1 were up-regulated, which were also confirmed by semi-quantitative reverse transcriptase-PCR (RT-PCR). Propolis-induced growth inhibition in HL-60 cells was, at least in part, due to differentiation with gene expression profiles, which are similar to those induced by all-trans retinoic acid.