Calcium signaling in B cells: Regulation of cytosolic Ca increase and its sensor molecules,STIM1 and STIM2

Calcium signals are crucial for diverse cellular functions including adhesion, differentiation, proliferation, effector functions and gene expression. After engagement of the B cell receptor, the intracellular calcium ion (Ca²⁺) concentration is increased promoting the activation of various signaling cascades. While elevated Ca²⁺ in the cytosol initially comes from the endoplasmic reticulum (ER), a continuous influx of extracellular Ca²⁺ is required to maintain the increased level of cytosolic Ca²⁺. Store-operated Ca²⁺ entry manages this process, which is regulated by an ER calcium sensor, stromal interaction molecule (STIM). STIM proteins sense changes in the levels of Ca²⁺ stored within the ER lumen and regulates the Ca²⁺-release activated Ca²⁺ channel in the plasma membrane. This review focuses on the signaling pathways leading to Ca²⁺ influx and the role of Ca²⁺ signals in B cell functions.     

Effect of chronic activation of 5-HT3 receptors on 5-HT3, 5-HT1A and 5-HT2A receptors functional activity and expression of key genes of the brain serotonin system

Among serotonin (5-HT) receptors, the 5-HT₃ receptor is the only ligand-gated ion-channel. Little is known about the interaction between the 5-HT₃ receptor and other 5-HT receptors and influence of 5-HT₃ chronic activation on other 5-HT receptors and the expression of key genes of 5-HT system. Chronic activation of 5-HT₃ receptor with intracerebroventricularly administrated selective agonist 1-(3-chlorophenyl)biguanide hydrochloride (m-CPBG) (14 days, 40 nmol, i.c.v.) produced significant desensitization of 5-HT₃ and 5-HT_1A receptors. The hypothermic responses produced by acute administration of selective agonist of 5-HT₃ receptor (m-CPBG, 40 nmol, i.c.v.) or selective agonist of 5-HT_1A receptor (8-hydroxy-2-(di-n-propylamino)tetralin) (8-OH-DPAT, 1 mg/kg, i.p.) was significantly lower in m-CPBG treated mice compared with the mice of control groups. Chronic m-CPBG administration failed to induce any significant change in the 5-HT_2A receptor functional activity and in the expression of the gene encoding 5-HT_2A receptor. Chronic activation of 5-HT₃ receptor produced no considerable effect on the expression on 5-HT₃, 5-HT_1A, and 5-HT transporter (5-HTT) and tryptophan hydroxylase-2 (TPH-2) genes – the key genes of brain 5-HT system, in the midbrain, frontal cortex and hippocampus. In conclusion, chronic activation of ionotropic 5-HT₃ receptor produced significant desensitization of 5-HT₃ and postsynaptic 5-HT_1A receptors but caused no considerable changes in the expression of key genes of the brain 5-HT system.     

Calcium fluxes cause nuclear shrinkage and the translocation of phospholipase C-δ1 into the nucleus

Phospholipase C-δ1 (PLCδ1) is the most fundamental form of the eukaryotic PLC and thought to play important roles in the regulation of cells. We previously reported that PLCδ1 shuttles between the cytoplasm and nucleus, and an influx of Ca²⁺ triggers the nuclear import of PLCδ1 via Ca²⁺-dependent interaction with importin β1, although the physiological meaning of this is unclear. Here we have examined the distribution of PLCδ1 using primary cultures of rat hippocampal neurons. Treatment of 7DIV neurons with ionomycin or thapsigargin caused the nuclear localization of PLCδ1 as has been observed in other cell lines. Similar results were obtained with neurons treated with glutamate, suggesting that the nuclear localization of PLCδ1 plays some roles in excitotoxicity associated with ischemic stress. Generally, cells undergoing ischemic or hypoxic cell death show nuclear shrinkage. We confirmed that a massive influx of Ca²⁺ caused similar results. Furthermore, overexpression of GFP-PLCδ1 facilitated ionomycin-induced nuclear shrinkage in embryonic fibroblasts derived from PLCδ1 gene-knockout mice (PLCδ1KO-MEF). By contrast, an E341A mutant that cannot bind with importin β1 and be imported into the nucleus by ionomycin and also lacks enzymatic activity did not cause nuclear shrinkage in PLCδ1KO-MEF. Nuclear translocation and the PLC activity of PLCδ1, therefore, may regulate the nuclear shape by controlling the nuclear scaffold during stress-induced cell death caused by high levels of Ca²⁺.     

Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function

Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer for which outcomes are poor when the disease is advanced. Although well-developed mouse models exist for embryonal and pleomorphic rhabdomyosarcomas, neither a spontaneous nor a transgenic mouse model of alveolar rhabdomyosarcoma has yet been reported. We report the first mouse model of alveolar rhabdomyosarcoma using a conditional Pax3:Fkhr knock-in allele whose activation in late embryogenesis and postnatally is targeted to terminally differentiating Myf6-expressing skeletal muscle. In these mice, alveolar rhabdomyosarcomas occur but at low frequency, and Fkhr haploinsufficiency does not appear to accelerate tumorigenesis. However, Pax3:Fkhr homozygosity with accompanying Ink4a/ARF or Trp53 pathway disruption, by means of conditional Trp53 or Ink4a/ARF loss of function, substantially increases the frequencies of tumor formation. These results of successful tumor generation postnatally from a target pool of differentiating myofibers are in sharp contrast to the birth defects and lack of tumors for mice with prenatal and postnatal satellite cell triggering of Pax3:Fkhr. Furthermore, these murine alveolar rhabdomyosarcomas have an immunohistochemical profile similar to human alveolar rhabdomyosarcoma, suggesting that this conditional mouse model will be relevant to study of the disease and will be useful for preclinical therapeutic testing.     

Vitamin D Regulation of Metal loproteinase Activity in Matrix Vesicles

Matrix vesicles (MVs) are enriched in matrix metalloproteinases (MMPs) capable of degrading proteoglycans. The aim of the present study was to identify which MMPs are present in MVs and determine whether these MMPs are regulated by l, 25-(OH)₂D₃ [1, 25] and 24, 25-(OH)₂D₃ [24, 25]. To do this, growth zone (GC) and resting zone (RC) chondrocytes were isolated from rat costochondral cartilage and placed into culture. At confluence, GCs were treated with 1, 25 and RCs with 24, 25 for 24 hours. MVs, plasma membranes (PMs), and conditioned media were then collected from the cultures. RT-PCR demonstrated the presence of mRNA for stromelysin-1 and 72 kDa gelatinase in both RCs and GCs. Casein zymography revealed activity at M_r 48 and 28 kDa in MV, but not PM or conditioned media; Western analysis confirmed that this activity was stromelysin-1. Gelatinolytic activity, at low levels, was also found in MVs, but not PMs or conditioned media. When enzyme activity was measured using a proteoglycan bead assay, it was found that both GCs and RCs produced MVs and PMs containing neutral metalloproteinase. Both cells also produced MVs and PMs containing plasminogen activator. The addition of 1, 25 to GCs caused a significant 4- to 5-fold increase in metalloproteinase activity in MVs, but not PMs. In contrast, MVs from cultures of RCs treated with 24, 25 contained decreased metalloproteinase activity; enzyme activity in PMs was unaffected by 24, 25. Plasminogen activator in MVs from RC was increased by treatment with 24, 25, while MV enzyme activity was decreased after treatment of GC cultures with 1, 25. This study shows that both RCs and GCs produce stromelysin-1 and 72 kDa gelatinase and that these enzymes are preferentially localized in MVs. Further, MMP and plasminogen activator activities in MVs and PMs are regulated by vitamin D metabolites.     

Adhesion molecules involved in hepoxilin A3-mediated neutrophil transepithelial migration

A common feature underlying active states of inflammation is the migration of neutrophils (PMNs) from the circulation and across a number of tissue barriers in response to chemoattractant stimuli. Although our group has recently established a discreet role for the PMN chemoattractant, hepoxilin A3 (HXA3) in the process of PMN recruitment, very little is known regarding the interaction of HXA3 with PMNs. To characterize further the event of HXA3-induced PMN transepithelial migration, we sought to determine the adhesion molecules required for migration across different epithelial surfaces (T84 intestinal and A549 airway cells) relative to two well-studied PMN chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4). Our findings reveal that the adhesion interaction profile of PMN transepithelial migration in response to HXA3 differs from the adhesion interaction profile exhibited by the structurally related eicosanoid LTB4. Furthermore, unique to PMN transepithelial migration induced by gradients of HXA3 was the critical dependency of all four major surface adhesion molecules examined (i.e. CD18, CD47, CD44 and CD55). Our results suggest that the particular chemoattractant gradient imposed, as well as the type of epithelial cell monolayer, each plays a role in determining the adhesion molecules involved in transepithelial migration. Given the complexities of these interactions, our findings are important to consider with respect to adhesion molecules that may be targeted for potential drug development.     

The need for a multi-level biochemical approach to defeat cancer that will also support the host

Cited research papers support the main hypothesis that selected publications supply sufficient information for a combined multi-level treatment strategy against cancer that will also strengthen the host. The three major elements of the proposal are: (A) metastasis being separate from tumor growth requires specific antimetastatic treatments. For this, manipulation of the composition of phospholipids will alter cellular charge characteristics which are instrumental in adhesion. (B) Formate metabolism is at the center of many activities that are controlling tumor growth. The rational and consequences of this are as follows. Supply of formate depends mainly on serine, and consumption on conversion to CO(2) yielding needed NADPH. The remainder is used to complete IMP configuration with 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP). At homeostasis residual ZMP activates AMP-activated protein kinase (AMPK) to curb growth promoting phosphatidylinositol-3-kinase (PI3PK). Residual ZMP also activates the oxidation of choline to betaine supplying methyl groups needed for global methylation of DNA while increased oxidation of choline also alters cellular phospholipid composition (refer to metastasis). At low formate level, increased accumulated ZMP becomes pyrophosporylated to ZTP. AMPK activation shifts to PI3PK activity for insulin action restoring formate supplied by serine derived from glycolysis. Increased NADPH-generating glucose-6-phosphate dehydrogenase is diminishing NADP(+) required for dehydrogenation of formate. This is restoring the formate balance while lowering ZMP levels to that of homeostasis. Evidence suggests that transformed cells exceed up-regulation of formate thus suppressing all ZMP accumulations resulting in limited AMPK activation, cessation of choline oxidation to betaine and loss of global methylation of DNA. This scenario appears to be tied to tumor survival, a state that could be altered by metabolic interventions using mild agents as described in the research reports cited. (C) Because of a preponderance of pyrimidines in cancer supporting UTP requiring immune evasion, exogenous IMP may offset this imbalance and thus hinder tumor anti-immune activities while strengthen host immune functions. For studies to confirm the proposal, the overall expected result is that a combined administration of all these agents cited here will outperform any single agent considered so far for anticancer treatment.     

Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E2

Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E₂ (PGE₂) and prostaglandin F_2α (PGF_2α) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE_2, we found enhanced GJIC with 1 nM PGE₂. This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE₂ was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE₂ secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE₂ to the cells. Our findings show that PGE₂ may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE₂ secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells.     

Differential expression of multiple genes in association with MADH4/DPC4/SMAD4 inactivation in pancreatic cancer

The Gene Logic Inc. Gene Express(R) tools and Affymetrix GeneChip(R) arrays were utilized to discover genes differentially expressed in pancreatic cancers with MADH4/DPC4/SMAD4 gene inactivation. cDNA was prepared from thirteen pancreas cancer cell lines with known MADH4 status (5 with wild-type MADH4 and 8 with inactivated MADH4) and hybridized to the complete Affymetrix Human Genome U133 GeneChip(R) set (arrays U133 A,B) for simultaneous analysis of 45,000 gene fragments corresponding to 33,000 known genes. 25 known genes were identified as down-regulated at least three fold in the MADH4 mutant cancer cell lines. 9 were decreased in expression at least 5 fold, and 1 in particular (ID3) was decreased 23 fold. Only 2 of the 25 down-regulated genes (ID1 and ID3) have been previously reported as MADH4-dependent targets, and the remaining 23 genes represent potential novel direct or indirect MADH4 downstream targets. Immunolabeling for Id1 and Id3 did not show a relationship with known MADH4 status in pancreatic cancer tissues, suggesting additional regulation of these two genes than activation by MadH4. Further investigations to validate and to determine the significance of these candidate target genes in pancreatic carcinogenesis and progression are warranted.     

Regulation of the epithelial sodium channel by phosphatidylinositides: experiments, implications, and speculations

Recent studies suggest that the activity of epithelial sodium channels (ENaC) is increased by phosphatidylinositides, especially phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃). Stimulation of phospholipase C by either adenosine triphosphate (ATP)-activation of purinergic P2Y receptors or epidermal growth factor (EGF)-activation of EGF receptors reduces membrane PI(4,5)P₂, and consequently decreases ENaC activity. Since ATP and EGF may be trapped in cysts formed by the distal tubule, it is possible that ENaC inhibition induced by ATP and EGF facilitates cyst formation in polycystic kidney diseases (PKD). However, some results suggest that ENaC activity is increased in PKD. In contrast to P2Y and EGF receptors, stimulation of insulin-like growth factor-1 (IGF-1) receptor by aldosterone or insulin produces PI(3,4,5)P₃, and consequently increases ENaC activity. The acute effect of aldosterone on ENaC activity through PI(3,4,5)P₃ possibly accounts for the initial feedback for blood volume recovery after hypovolemic hypotension. PI(4,5)P₂ and PI(3,4,5)P₃, respectively, interacts with the N terminus of β-ENaC and the C terminus of γ-ENaC. However, whether ENaC selectively binds to PI(4,5)P₂ and PI(3,4,5)P₃ over other anionic phospholipids remains unclear.     

A G alpha-dependent pathway that antagonizes multiple chemoattractant responses that regulate directional cell movement

Chemotactic cells, including neutrophils and Dictyostelium discoideum, orient and move directionally in very shallow chemical gradients. As cells polarize, distinct structural and signaling components become spatially constrained to the leading edge or rear of the cell. It has been suggested that complex feedback loops that function downstream of receptor signaling integrate activating and inhibiting pathways to establish cell polarity within such gradients. Much effort has focused on defining activating pathways, whereas inhibitory networks have remained largely unexplored. We have identified a novel signaling function in Dictyostelium involving a Galpha subunit (Galpha9) that antagonizes broad chemotactic response. Mechanistically, Galpha9 functions rapidly following receptor stimulation to negatively regulate PI3K/PTEN, adenylyl cyclase, and guanylyl cyclase pathways. The coordinated activation of these pathways is required to establish the asymmetric mobilization of actin and myosin that typifies polarity and ultimately directs chemotaxis. Most dramatically, cells lacking Galpha9 have extended PI(3,4,5)P(3), cAMP, and cGMP responses and are hyperpolarized. In contrast, cells expressing constitutively activated Galpha9 exhibit a reciprocal phenotype. Their second message pathways are attenuated, and they have lost the ability to suppress lateral pseudopod formation. Potentially, functionally similar Galpha-mediated inhibitory signaling may exist in other eukaryotic cells to regulate chemoattractant response.     

Modulation of histamine H3 receptor function by monovalent ions

Monovalent ions differently affect ligand binding to G protein-coupled receptors (GPCRs) by as yet poorly defined mechanisms. In particular, NaCl often decreases the affinity of agonists but increases it for antagonists. We examined the effect of various monovalent ions on human histamine H₃ receptor (hH₃R), co-expressed with mammalian G proteins (Gα_i1, Gα_i2, Gα_i3 or Gα_o1, and β₁γ₂ dimers, respectively) in Sf9 insect cell membranes, with respect to agonist binding and G protein activation. NaCl (100 mM) had no effect on affinity of the agonist [³H]N^α-methylhistamine ([³H]NAMH). In steady-state GTPase assays, the endogenous agonist histamine had a lower potency and the inverse agonist thioperamide had a higher potency, when NaCl (100 mM) was present. Monovalent ions reduced H₃R-regulated signalling in the order of efficacy Li⁺ ∼ Na⁺ ∼ K⁺ < Cl⁻ < Br⁻ < I⁻. NaCl had a stronger effect on basal hH3R-signalling when Gα_i3 was co-expressed. Asp80^2.50, a putative interaction site for Na⁺, was mutated to Asn80^2.50 (D2.50N-hH₃R). Strikingly, the mutation was unable to activate Gα_i3 at all. The effects can be explained by a model, where (i) monovalent ions as well as a charge-neutralizing mutation of Asp80^2.50 generally reduce the interaction of hH₃R with G proteins, (ii) monovalent anions increase the affinity of G proteins for GDP and thus, indirectly affect their interaction with hH₃R and, (iii) Asp80^2.50 is a key residue for hH₃R/Gα_i3-protein activation. The latter result suggests that hH₃R/G protein-coupling interfaces may differ even between closely related subunits.     

Effects of calcium-sensing receptors on apoptosis in rat hippocampus during hypoxia/reoxygenation through the ERK1/2 pathway

Objectives: To explore the effects of calcium-sensing receptors (CaSR) on apoptosis in rat hippocampus during hypoxia/reoxygenation (H/R). Methods: After rat hippocampus was isolated, the cultures were subjected to H/R, and meanwhile gadolinium chloride (GdCl₃, agonist of CaSR) and NPS 2390 (antagonists of CaSR) were added to reperfusion solution. The number of hippocampal neuron, cell viability and apoptosis rate were determined by inverted microscope, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometer (FCM), respectively. Besides, caspase-3, Bax, cytochrome C (Cyt-c), extracellular signal-regulated protein kinase (ERK) 1/2, pERK1/2, P38 and pP38 were analyzed by Western blotting. Results: The hippocampal neuron number and cell viability were significantly decreased during H/R, and were further significantly reduced when co-treatment with CaSR agonist GdCl₃. But the effects of GdCl₃ were attenuated by NPS-2390. Whereas, apoptosis rate, the expression level of caspase-3, Bax and Cyt-c were all significantly increased under H/R condition, and was further significantly increased by GdCl₃, but were reversed by NPS-2390 (P < 0.05). Moreover, there were no significant differences in expression of ERK1/2, P38 and pP38 among different groups. However, the expression of pERK1/2 was significantly increased during H/R, but was significantly reduced by NPS 2390 (P < 0.05). Conclusion: The results suggest that CaSR might play significant roles in the induction of hippocampus apoptosis in rat during H/R through phosphorylation of ERK1/2.     

Differential distribution of γ-aminobutyric acidA, receptor subunit (α1, α2, α3, α5 and β2+3) immunoreactivity in the medial prefrontal cortex of the rat

A detailed mapping of the γ-aminobutyric acid (GABA)_A receptor subunits (α₁, α₂, α₃ and β₂₊₃) in the infralimbic/ventral prelimbic region (IL/vPL) of the rat frontal cortex was carried out using subunit-specific antibodies. The α₁ and β₂₊₃ subunit antibodies immunostained all layers of the IL/vPL region. Layers II and III displayed immunostaining of cell bodies whereas I, V and VI showed predominantly neuropil staining. The size of the α₁-positive cell bodies corresponded to that of small interneurons (range, 20–55 μm²; mean ± SEM, 37 ± 5.5 μm²) as well as pyramidal cells or large interneurons (range, 87–135 μm²; mean ± SEM, 103.4 ± 9.7 μm²). However, β₂₊₃ antibody immunostained only small cell bodies. Immunoreactivity for α₂ was restricted to layers I and II, whereas α₃ and α₅ subunit expression was seen only in layer VI. The antibody to the α₂ subunit immunostained small cell bodies (range, 29–63 μm²; mean ± SEM, 32 ± 4.5 μm²) in layer II, resembling interneurons. Conversely, both α₃ and α₅ antibodies immunostained large cell bodies (range, 94–151 gmm²; mean ± SEM, 115.7 ± 13.4 μm²), consistent with pyramidal cell labelling in layer VI.     

A k2A-positive Klebsiella pneumoniae causes liver and brain abscess in a Saint Kitt's man

Klebsiella pneumoniae isolated in community-acquired pneumonia is increasingly found in primary pyogenic liver abscesses. The presence of magA in K. pneumoniae has been implicated in hypermucoviscosity and virulence of liver abscess isolates. The K2 serotype has also been strongly associated with hypervirulence. We report the isolation of non-magA, K2 K. pneumoniae strain from a liver abscess of a Saint Kitt''s man who survived the invasive syndrome.     

An engineered Plasmodium falciparum C-terminal 19-kilodalton merozoite surface protein 1 vaccine candidate induces high levels of interferon-gamma production associated with cellular immune responses to specific peptide sequences in Gambian adults naturally exposed to malaria

The 19-kDa C-terminal region of merozoite surface protein 1 (MSP1(19)), a major blood stage malaria vaccine candidate, is the target of cellular and humoral immune responses in humans naturally infected with Plasmodium falciparum. We have previously described engineered variants of this protein, designed to be better vaccine candidates, but the human immune response to these proteins has not been characterized fully. Here we have investigated the antigenicity of one such variant compared to wild-type MSP1(19)-derived protein and peptides. Gambian adults produced both high T helper type 1 (Th1) [interferon (IFN)-γ] and Th0/Th2 [interleukin (IL)-13 and sCD30] responses to the wild-type MSP1(19) and the modified protein as wells as to peptides derived from both forms. Response to the modified MSP1(19) (with three amino acid substitutions: Glu27Tyr, Leu31Arg and Glu43Leu) relative to the wild-type, included higher IFN-γ production. Interestingly, some peptides evoked different patterns of cytokine responses. Modified peptides induced higher IL-13 production than the wild-type, while the conserved peptides P16 and P19 induced the highest IFN-γ and IL-13 and/or sCD30 release, respectively. We identified P16 as the immunodominant peptide that was recognized by cells from 63% of the study population, and not restricted to any particular human leucocyte antigen D-related (HLA-DR) type. These findings provide new and very useful information for future vaccine development and formulation as well as potential Th1/Th2 immunmodulation using either wild-type or modified protein in combination with their peptides.     

Characterization of thromboxane A2 receptor and TRPV1 mRNA expression in cultured sensory neurons

Thromboxane A₂ (TxA₂) is an arachidonic acid metabolite that stimulates platelet aggregation and vasoconstriction when released from platelets and other cell types during tissue trauma. More recent research has demonstrated that TxA₂ can also stimulate vagal and spinal sensory nerves. The purpose of this study was twofold. One, we compared the expression of the TxA₂ receptor (TxA2R) in neurons from two sensory ganglia: the nodose ganglion (NG) containing cell bodies of vagal afferent nerves and the thoracic dorsal root ganglion (DRG) containing cell bodies of spinal afferent nerves. Two, we determined if TxA2R co-localizes with mRNA for the nociceptive marker, TRPV1, which is the receptor for the noxious substance capsaicin. We found a greater percentage of neurons in the NG that are positive for TxA2R expression than in the DRG. We also found that there was no correlation of expression of TxA2R with TRPV1. These data suggest that while TxA2R is expressed in both vagal and spinal neurons, TxA₂ may elicit stronger vagal or parasympathetic reflexes in the rabbit when released during tissue trauma depending on the location of release. Our data also indicate that TxA₂ is likely to stimulate both nociceptive and non-nociceptive neurons thereby broadening the types of neurons and reflexes that it may excite.     

Fates of Fe3O4 and Fe3O4@SiO2 nanoparticles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques

Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as the magnetic resonance imaging (MRI) contrast agent in biomedical studies and clinical applications, with special interest recently in in vivo stem cell tracking. However, a full understanding of the fate of SPIOs in cells has not been achieved yet, which is particularly important for stem cells since any change of the microenvironment may disturb their propagation and differentiation behaviors. Herein, synchrotron radiation-based X-ray fluorescence (XRF) in combination with X-ray absorption spectroscopy (XAS) were used to in situ reveal the fate of Fe₃O₄ and Fe₃O₄@SiO₂ NPs in human mesenchymal stem cells (hMSCs), in which the dynamic changes of their distribution and chemical speciation were precisely determined. The XAS analysis evidences that Fe₃O₄ NPs cultured with hMSCs are quite stable and almost keep their initial chemical form up to 14 days, which is contradictory to the previous report that Fe₃O₄ NPs were unstable in cell labeling assessed by using a simplified lysosomal model system. Coating with a SiO₂ shell, Fe₃O₄@SiO₂ NPs present higher stability in hMSCs without detectable changes of their chemical form. In addition, XRF analysis demonstrates that Fe₃O₄@SiO₂ NPs can label hMSCs in a high efficiency manner and are solely distributed in cytoplasm during cell proliferation, making it an ideal probe for in vivo stem cell tracking. These findings with the help of synchrotron radiation-based XAS and XRF improve our understanding of the fate of SPIOs administered to hMSCs and will help the future design of SPIOs for safe and efficient stem cells tracking.     

C. elegans longevity pathways converge to decrease mitochondrial membrane potential

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (ΔΨ_m) across the inner membrane. In this work, we show that a lower ΔΨ_m is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower ΔΨ_m than wild type animals. The lower ΔΨ_m of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease ΔΨ_m. Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates ΔΨ_m. We conclude that longevity pathways converge on the mitochondria and lead to a decreased ΔΨ_m. Our results are consistent with the ‘uncoupling to survive’ hypothesis, which states that dissipation of the ΔΨ_m will extend lifespan.     

Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain

Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as “lipid rafts” obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP₂ is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP₂ in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP₂ in SPM-derived DRM, we measured the amount of PIP₂ in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cδ1 as a PIP₂ binding probe. About 10% of the PIP₂ in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP₂ in DRM prepared from SPM.