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Medicinal cannabis has shown promise for the symptomatic treatment of Parkinson’s disease (PD), but patient exposure to whole plant mixtures may be undesirable due to concerns around safety, consistency, regulatory issues, and psychoactivity. Identification of a subset of components responsible for the potential therapeutic effects within cannabis represents a direct path forward for the generation of anti-PD drugs. Using an in silico database, literature reviews, and cell based assays, GB Sciences previously identified and patented a subset of five cannabinoids and five terpenes that could potentially recapitulate the anti-PD attributes of cannabis. While this work represents a critical step towards harnessing the anti-PD capabilities of cannabis, polypharmaceutical drugs of this complexity may not be feasible as therapeutics. In this paper, we utilize a reductionist approach to identify minimal essential mixtures (MEMs) of these components that are amenable to pharmacological formulation. In the first phase, cell-based models revealed that the cannabinoids had the most significant positive effects on neuroprotection and dopamine secretion. We then evaluated the ability of combinations of these cannabinoids to ameliorate a 6-hydroxydopmamine (OHDA)-induced change in locomotion in larval zebrafish, which has become a well-established PD disease model. Equimolar mixtures that each contained three cannabinoids were able to significantly reverse the OHDA mediated changes in locomotion and other advanced metrics of behavior. Additional screening of sixty-three variations of the original cannabinoid mixtures identified five highly efficacious mixtures that outperformed the original equimolar cannabinoid MEMs and represent the most attractive candidates for therapeutic development. This work highlights the strength of the reductionist approach for the development of ratio-controlled, cannabis mixture-based therapeutics for the treatment of Parkinson’s disease.

Nociceptive Transient Receptor Potential channels such as TRPV1 are targets for treating pain. Both antagonism and agonism of TRP channels can promote analgesia, through inactivation and chronic desensitization. Since plant-derived mixtures of cannabinoids and the Cannabis component myrcene have been suggested as pain therapeutics, we screened terpenes found in Cannabis for activity at TRPV1. We used inducible expression of TRPV1 to examine TRPV1-dependency of terpene-induced calcium flux responses. Terpenes contribute differentially to calcium fluxes via TRPV1 induced by Cannabis-mimetic cannabinoid/terpenoid mixtures. Myrcene dominates the TRPV1-mediated calcium responses seen with terpenoid mixtures. Myrcene-induced calcium influx is inhibited by the TRPV1 inhibitor capsazepine and Myrcene elicits TRPV1 currents in the whole-cell patch-clamp configuration. TRPV1 currents are highly sensitive to internal calcium. When Myrcene currents are evoked, they are distinct from capsaicin responses on the basis of Imax and their lack of shift to a pore-dilated state. Myrcene pre-application and residency at TRPV1 appears to negatively impact subsequent responses to TRPV1 ligands such as Cannabidiol, indicating allosteric modulation and possible competition by Myrcene. Molecular docking studies suggest a non-covalent interaction site for Myrcene in TRPV1 and identifies key residues that form partially overlapping Myrcene and Cannabidiol binding sites. We identify several non-Cannabis plant-derived sources of Myrcene and other compounds targeting nociceptive TRPs using a data mining approach focused on analgesics suggested by non-Western Traditional Medical Systems. These data establish TRPV1 as a target of Myrcene and suggest the therapeutic potential of analgesic formulations containing Myrcene.

Calcium entry is central to the functional processes in mast cells and basophils that contribute to the induction and maintenance of inflammatory responses. Mast cells and basophils express an array of calcium channels, which mediate responses to diverse stimuli triggered by small bioactive molecules, physicochemical stimuli and immunological inputs including antigens and direct immune cell interactions. These cells are also highly responsive to certain venoms (such as Hymenoptera envenomations), which cause histamine secretion, cytokine release and an array of pro-inflammatory functional responses. There are gaps in our understanding of the coupling of venom exposure to specific signaling pathways such as activation of calcium channels. In the present study, we performed a current survey of a model mast cell line selected for its pleiotropic responsiveness to multiple pro-inflammatory inputs. As a heterogenous stimulus, Hymenoptera venom activates multiple classes of conductance at the population level but tend to lead to the measurement of only one type of conductance per cell, despite the cell co-expressing multiple channel types. The data show that ICRAC, IARC, and TRPV-like currents are present in the model mast cell populations and respond to venom exposure. We further assessed individual venom components, specifically secretagogues and arachidonic acid, and identified the conductances associated with these stimuli in mast cells. Single-cell calcium assays and immunofluorescence analysis show that there is heterogeneity of channel expression across the cell population, but this heterogeneity does not explain the apparent selectivity for specific channels in response to exposure to venom as a composite stimulus.

Mast cell lipid bodies are key to initiation, maintenance and resolution of inflammatory responses in tissue. Mast cell lines, primary bone marrow-derived mast cells and peripheral blood basophils present a ‘steatotic’ phenotype in response to chronic insulin exposure, where cells become loaded with lipid bodies. Here we show this state is associated with reduced histamine release, but increased capacity to release bioactive lipids. We describe the overall lipid phenotype of mast cells in this insulin-induced steatotic state and the consequences for critical cellular lipid classes involved in stages of inflammation. We show significant insulin-induced shifts in specific lipid classes, especially arachidonic acid derivatives, MUFA and PUFA, the EPA/DHA ratio, and in cardiolipins, especially those conjugated to certain DHA and EPAs. Functionally, insulin exposure markedly alters the FcϵRI-induced release of Series 4 leukotriene LTC4, Series 2 prostaglandin PGD2, Resolvin-D1, Resolvin-D2 and Resolvin-1, reflecting the expanded precursor pools and impact on both the pro-inflammation and pro-resolution bioactive lipids that are released during mast cell activation. Chronic hyperinsulinemia is a feature of obesity and progression to Type 2 Diabetes, these data suggest that mast cell release of key lipid mediators is altered in patients with metabolic syndrome.

Cannabinoid compounds are potential analgesics. Users of medicinal Cannabis report efficacy for pain control, clinical studies show that cannabis can be effective and opioid sparing in chronic pain, and some constituent cannabinoids have been shown to target nociceptive ion channels. Here, we explore and compare a suite of cannabinoids for their impact upon the physiology of TRPV1. The cannabinoids tested evoke differential responses in terms of kinetics of activation and inactivation. Cannabinoid activation of TRPV1 displays significant dependence on internal and external calcium levels. Cannabinoid activation of TRPV1 does not appear to induce the highly permeant, pore-dilated channel state seen with Capsaicin, even at high current amplitudes. Finally, we analyzed cannabinoid responses at nociceptive channels other than TRPV1 (TRPV2, TRPM8, and TRPA1), and report that cannabinoids differentially activate these channels. On the basis of response activation and kinetics, state-selectivity and receptor selectivity, it may be possible to rationally design approaches to pain using single or multiple cannabinoids.

Ethnopharmacological relevance Traditional pharmacopeias have been developed by multiple cultures and evaluated for efficacy and safety through both historical/empirical iteration and more recently through controlled studies using Western scientific paradigms and an increasing emphasis on data science methodologies for network pharmacology. Traditional medicines represent likely sources of relatively inexpensive drugs for symptomatic management as well as potential libraries of new therapeutic approaches. Leveraging this potential requires hard evidence for efficacy that separates science from pseudoscience. Materials and methods We performed a review of non-Western medical systems and developed case studies that illustrate the epistemological and practical translative barriers that hamper their transition to integration with Western approaches. We developed a new data analytics approach, in silico convergence analysis, to deconvolve modes of action, and potentially predict desirable components of TM-derived formulations based on computational consensus analysis across cultures and medical systems. Results Abstraction, simplification and altered dose and delivery modalities were identified as factors that influence actual and perceived efficacy once a medicine is moved from a non-Western to Western setting. Case studies on these factors highlighted issues with translation between non-Western and Western epistemologies, including those where epistemological and medicinal systems drive markets that can be epicenters for zoonoses such as the novel Coronavirus. The proposed novel data science approach demonstrated the ability to identify and predict desirable medicinal components for a test indication, pain. Conclusions Relegation of traditional therapies to the relatively unregulated nutraceutical industry may lead healthcare providers and patients to underestimate the therapeutic potential of these medicines. We suggest three areas of emphasis for this field: First, vertical integration and embedding of traditional medicines into healthcare systems would subject them to appropriate regulation and evidence-based practice, as viable integrative implementation mode. Second, we offer a new Bradford-Hill-like framework for setting research priorities and evaluating efficacy, with the goal of rescuing potentially valuable therapies from the nutraceutical market and discrediting those that are pseudoscience. Third, data analytics pipelines offer new capacity to generate new types of TMS-inspired medicines that are rationally-designed based on integrated knowledge across cultures, and also provide an evaluative framework against which to test claims of fidelity and efficacy to TMS made for nutraceuticals., Graphical abstract Schematic grouping of traditional medicine approaches. Schematic representation of out-grouping of formalized phytomedicines and phytopharmacopeias from other traditional medicine approaches and pseudoscientific approaches. The potential of each group of approaches to be evaluated and distributed across axes of the potential or actual scientific proof of efficacy, plausibility of mechanism of action, and the potential for placebo effect (likely related to perceived complexity).Image 1

Large membrane derangements in the form of non-detaching blebs or membrane protrusions occur in a variety of cell stress and physiological situations and do not always reflect apoptotic processes. They have been studied in model mast cells under conditions of cell stress, but their potential physiological relevance to mast cell function and formation in primary mast cells or basophils have not been addressed. In the current study, we examine the large, non-detaching, non-apoptotic, membrane structures that form in model and primary mast cells under conditions of stimulation that are relevant to allergy, atopy and Type IV delayed hypersensitivity reactions. We characterized the inflation kinetics, dependency of formation upon external free calcium and striking geometric consistency of formation for large plasma membrane blebs (LPMBs). We describe that immunologically stimulated LPMBs in mast cells are constrained to form in locations where dissociation of the membrane-associated cytoskeleton occurs. Mast cell LPMBs decorate with wheat germ agglutinin, suggesting that they contain plasma membrane (PM) lectins. Electrophysiological capacitance measurements support a model where LPMBs are not being formed from internal membranes newly fused into the PM, but rather arise from stretching of the existing membrane, or inflation and smoothing of a micro-ruffled PM. This study provides new insights into the physiological manifestations of LPMB in response to immunologically relevant stimuli and in the absence of cell stress, death or apoptotic pathways.

Kava is a soporific, anxiolytic and relaxant in widespread ritual and recreational use throughout the Pacific. Traditional uses of kava by indigenous Pacific Island peoples reflect a complex pharmacopeia, centered on GABA-ergic effects of the well-characterized kavalactones. However, peripheral effects of kava suggest active components other than the CNS-targeted kavalactones. We have previously shown that immunocytes exhibit calcium mobilization in response to traditionally prepared kava extracts, and that the kavalactones do not induce these calcium responses. Here, we characterize the complex calcium-mobilizing activity of traditionally prepared and partially HPLC-purified kava extracts, noting induction of both calcium entry and store release pathways. Kava components activate intracellular store depletion of thapsigargin-sensitive and -insensitive stores that are coupled to the calcium release activated (CRAC) current, and cause calcium entry through non-store-operated pathways. Together with the pepper-like potency reported by kava users, these studies lead us to hypothesize that kava extracts contain one or more ligands for the transient receptor potential (TRP) family of ion channels. Indeed, TRP-like conductances are observed in kava-treated cells under patch clamp. Thus TRP-mediated cellular effects may be responsible for some of the reported pharmacology of kava.

Kava ('Awa) is a traditional water-based beverage in Pacific island communities, prepared from the ground root and stems of Piper methysticum. Kava use is associated with an ichthyotic dermatitis and delayed type hypersensitivity reactions. In the current study we collated preparative methodologies from cultural practitioners and recreational kava users in various Pacific communities. We standardized culturally informed aqueous extraction methods and prepared extracts that were subjected to basic physicochemical analysis. Mast cells exposed to these extracts displayed robust intracellular free calcium responses, and concomitant release of proinflammatory mediators. In contrast, mast cells were refractory to single or combinatorial stimulation with kavalactones, including methysticin, dihydromethysticin and kavain. Moreover, we reproduced a traditional modification of the kava preparation methodology, pre-mixing with the mucilage of Hibiscus tiliaceus, and observed its potentiating effect on the activity of aqueous extracts in mast cells. Taken together, these data indicate that water extractable active ingredients may play a role in the physiological and pathophysiological effects of kava, and suggests that mast cell activation may be a mechanistic component of kava-related skin inflammations.

Two types of three-dimensional (3-D) optical circuits based on waveguide films are proposed and experimentally demonstrated. Type 1 is "Stacked waveguide films with 45deg mirrors". Waveguide films with 70-mum-thick undercladding layers and 32-mum-wide cores with air cladding are fabricated by the built-in mask method using photo-definable materials. The films are stacked by contacting their undercladding layers. Optical Z-connections are formed by aligning the 45deg mirrors in upper and lower films. The mirror-to-mirror distance is ~170 mum. When a probe beam of 650 nm or 1.3 mum in wavelength is introduced into input in the first film, it is transmitted to the second film through optical Z-connection and is observed at output. Loss at the optical Z-connection for 1.3-mum wavelength is ~14 dB, which might be due to leakage of probe beams reflected from the mirror with large diverging angles. Type 2 is "Waveguide films with vertical waveguides". After coating a photo-refractive layer of 500-mum thickness on the back of a cladding layer of a waveguide film, a 405-nm wavelength write beam is introduced into input. Then, a vertical waveguide of 3-D self-organized lightwave network (3-D SOLNET) is grown in the photo-refractive layer above a 45deg mirror. A probe beam guided in the vertical waveguide is observed with a beam size close to that in the core of the waveguide film. Loss reduction at the optical Z-connection is expected by combining optical circuits of Types 1 and 2 to insert the 3-D SOLNET between waveguide films as well as by decreasing cladding layer thicknesses

The inositol (1,4,5) trisphosphate 3-kinase (ITP3K) phosphorylates Ins (1,4,5) P3 to produce Ins (1,3,4,5) P4. The ITP3K substrate, InsP3, and its product, InsP4, both have the potential to regulate mast cell function. Here, we explore the effects of dominant inhibition of ITP3K upon secretory responses and Ras GTPase activation following antigenic cross-linking of the mast cell immunoreceptor, FcvarepsilonRI. Inhibition of ITP3K potentiates both calcium release from intracellular stores and calcium-dependent secretory responses in mast cells. Moreover, mast cells with dominantly inhibited ITP3K display constitutive activation of Ras and certain Ras effector pathways. We propose three mechanisms by which ITP3K inhibition could influence Ras activation. The protection of InsP3 that results from ITP3K inhibition may lead to enhanced activation of calcium-sensitive Ras-GAPs or -GRFs. Similarly, the deficit in InsP4 may change the behavior of the InsP4 receptor, the GAP1(IP4BP). Our data are inconsistent with calcium-sensitive Ras-GAP activation being the primary consequence of ITP3K inhibition in mast cells. Rather, we observe potentiation of Ras responses in mast cells transfected with dominant negative GAP1(IP4BP). Moreover, shRNA-mediated knockdown of GAP1(IP4BP) potentiates FcvarepsilonRI-mediated Ras activation, indicating that this InsP4-binding GAP protein may be used by the FcvarepsilonRI immunoreceptor to regulate Ras.

Cellular lipotoxicity manifests as the steatotic accumulation of lipid droplets or lipid bodies, and/or induction of phospholipidosis. Lipotoxicity can be induced by hyperinsulinemia/nutrient overload, cationic amphiphilic drugs (CAD), and innate immunological stimuli, all of which are stimuli relevant to mast cell physiology. Hyper-accumulation of mast cell lipid bodies in response to hyperinsulinemia has been documented but lipotoxicity in response to CAD or innate immunologic stimuli has not been analysed comparatively. Moreover, gaps in our understanding of this steatosis remain, specifically as to whether hyperinsulinemia-driven steatosis in these cells attains lipotoxic levels or is accompanied by phospholipidosis. To compare endocrine, pharmacological and innate immunological stimuli for their ability to induce steatosis and phospholipidosis in a rat basophilic leukemia mast cell model (RBL2H3), differential fluorescence microscopy staining and quantitation of phospholipidosis and steatosis in the RBL2H3 cell line was examined in response to applied endocrine, pharmacological and innate immunological stimuli. The three classes of stimuli differentially induced phospholipidosis and steatosis. PPARγ up-regulation was not uniformly associated with the expansion of the lipid body population. Fluorescence imaging of lipid-enriched structures generated in response to lipotoxic cationic amphiphilic drugs, chronic insulin exposure and TLR2/4 ligands revealed differential staining patterns when visualized using lipophilic dyes. It is concluded that lipotoxicity-inducing pathways in this model mast cell system are diverse, and include steatotic responses to an endocrine stimulus, as well as phospholipidosis responses to cationic lipophilic drugs not previously described in this cell type.

Cutaneous mast cell responses to physical (thermal, mechanical, or osmotic) stimuli underlie the pathology of physical urticarias. In vitro experiments suggest that mast cells respond directly to these stimuli, implying that a signaling mechanism couples functional responses to physical inputs in mast cells. We asked whether transient receptor potential (vanilloid) (TRPV) cation channels were present and functionally coupled to signaling pathways in mast cells, since expression of this channel subfamily confers sensitivity to thermal, osmotic, and pressure inputs. Transcripts for a range of TRPVs were detected in mast cells, and we report the expression, surface localization, and oligomerization of TRPV2 protein subunits in these cells. We describe the functional coupling of TRPV2 protein to calcium fluxes and proinflammatory degranulation events in mast cells. In addition, we describe a novel protein kinase A (PKA)–dependent signaling module, containing PKA and a putative A kinase adapter protein, Acyl CoA binding domain protein (ACBD)3, that interacts with TRPV2 in mast cells. We propose that regulated phosphorylation by PKA may be a common pathway for TRPV modulation.

TRPV ion channels transduce a range of temperature stimuli. We proposed that analysis of the protein- protein interactions made by TRPV2 might give insight into the key issues surrounding this channel. These issues include the potential functional significance of TRPV2 in non-sensory tissues, the molecules involved in transducing its activation signal(s) and the mechanism by which its trafficking to the cell surface is regulated. Here we describe the interaction of TRPV2 channel with the RGA gene product. RGA is a four-transmembrane domain, intracellularly localized protein. RGA associates with TRPV2 in a rat mast cell line that is a native context for both proteins. The interaction between TRPV2 and RGA is transient and occurs intracellularly. RGA does not accompany TRPV2 to the cell surface. Formation of the TRPV2/ RGA complex is dependent upon a cellular glycosylation event, suggesting that RGA may play a chaperone or targeting role for TRPV2 during the maturation of the ion channel protein. These data record a novel protein-protein interaction for TRPV2 and provide a foundation for future study of the potential regulatory contribution of RGA to TRPV2 function. J. Cell. Biochem. 91: 808-820, 2004. 2004 Wiley-Liss, Inc.

Cannabinoid modulation of immune responses is a pathological consequence of marijuana abuse and a potential outcome of therapeutic application of the drug. Moreover, endogenous cannabinoids are physiological immune regulators. In the present report, we describe alterations in gene transcription that occur after cannabinoid exposure in a mast cell line, RBL2H3. Cannabinoid exposure causes marked changes in the transcript levels for numerous genes, acting both independently of and in concert with immunoreceptor stimulation via FcεRI. In two mast cell lines, we observed mRNA and protein expression corresponding to both CB1 and CB2 cannabinoid receptor isoforms, contrary to the prevailing view that CB1 is restricted to the CNS. We show that coexpression of the two isoforms is not functionally redundant in mast cells. Analysis of signaling pathways downstream of cannabinoid application reveals that activation of extracellular signal-regulated kinase, AKT, and a selected subset of AKT targets is accomplished by CB2 ligands and nonselective CB1/CB2 agonists in mast cells. CB1 inhibition does not affect AKT or extracellular signal-regulated kinase activation by cannabinoids, indicating that CB2 is the predominant regulatory receptor for these kinases in this cell context. CB1 receptors are, however, functional in these mast cells, since they can contribute to suppression of secretory responses.

Low Pressure Plasma Spraying (LPPS) is a promising coating method for Functionally Graded Material (FGM) expected to be able to reduce the thermal stress in high temperature environments such as a gas turbine. In this paper, we report the effect of the composition profile and coating density of LPPS sprayed FGM, consisting of ZrO2–8 wt%Y2O3 (YSZ) top coating, YSZ–Ni–20 wt%Cr (NiCr) FGM coating, NiCr under coating and copper substrate, on the thermal shock resistance evaluated by a modified temperature difference test. The density of YSZ and NiCr coating was successfully controlled by the chamber pressure and initial particle size in the range from 5.43 to 5.79 g/cm3 and from 7.89 to 8.09 g/cm3, respectively. For an YSZ composition profile from NiCr under coating to YSZ top coating (in FGM), the highest thermal shock resistance was obtained when the fraction of YSZ increased with gentle slope just over NiCr coating and acute slope just under YSZ coating. Also, the higher density coatings tended to perform the higher thermal shock resistance. Initial cracks formed in the YSZ top coating propagated into YSZ parts in FGM coating through the grain boundary of YSZ and/or the interface between flattened NiCr and YSZ particles. After the cracks connected, the coupled cracks caused the coating spallation.

There is well-established variability in the numbers of lipid bodies (LB) in macrophages, eosinophils, and neutrophils. Similarly to the steatosis observed in adipocytes and hepatocytes during hyperinsulinemia and nutrient overload, immune cell LB hyper-accumulate in response to bacterial and parasitic infection and inflammatory presentations. Recently we described that hyperinsulinemia, both in vitro and in vivo, drives steatosis and phenotypic changes in primary and transformed mast cells and basophils. LB reach high numbers in these steatotic cytosols, and here we propose that they could dramatically impact the transcytoplasmic signaling pathways. We compared calcium release and influx responses at the population and single cell level in normal and steatotic model mast cells. At the population level, all aspects of FcεRI-dependent calcium mobilization, as well as activation of calcium-dependent downstream signalling targets such as NFATC1 phosphorylation are suppressed. At the single cell level, we demonstrate that LB are both sources and sinks of calcium following FcεRI cross-linking. Unbiased analysis of the impact of the presence of LB on the rate of trans-cytoplasmic calcium signals suggest that LB enrichment accelerates calcium propagation, which may reflect a Bernoulli effect. LB abundance thus impacts this fundamental signalling pathway and its downstream targets.

The zebrafish is the first vertebrate organism used for large-scale genetic screens seeking genes critical to development. These screens have been quite successful, with more than 1800 recessive mutations discovered that speak to morphogenesis of the vertebrate embryo. The cloning of the mutant genes depends on a dense genetic map. The 2000 markers we present here, using microsatellite (CA) repeats, provides 1.2-cM average resolution. One centimorgan in zebrafish is about 0. 74 megabase, so, for many mutations, these markers are close enough to begin positional cloning by YAC walks.

Carbon nanotubes (CNT) are environmental challenges to the respiratory and gastrointestinal mucosa, and to the dermal immune system. Mast cells (MC) are pro-inflammatory immunocytes that reside at these interfaces with the environment. Mast cells are sources of pro-inflammatory mediators (histamine, serotonin, matrix-active proteases, eicosanoids, prostanoids, cytokines and chemokines), which are released in a calcium-dependent manner following immunological challenge or physico-chemical stimulation. Since C-60 fullerenes, which share geometry with CNT, are suppressive of mast cell-driven inflammatory responses, we explored the effects of unmodified SWCNT aggregates on mast cell signaling pathways, phenotype and pro-inflammatory function. We noted SWCNT suppression of antigen-induced signalling pathways and pro-inflammatory degranulation responses. Mast cells recognize unmodified SWCNT by remodeling the plasma membrane, disaggregating the cortical actin cytoskeleton and relocalizing clathrin. Clathrin was also identified as a component of an affinity-purified 'interactome' isolated from MC using an SWCNT affinity matrix for mast cell lysates. Together, these data are consistent with the ability of SWCNT to suppress mast cell pro-inflammatory function via a novel recognition mechanism.

Functionally gradient materials (FGMs) display continuously or discontinuously varying compositions and/or microstructures over definable geometrical distances. The gradients can be continuous on a microscopic level, or they can be laminates comprised of gradients of metals, ceramics, polymers, or variations of porosity/density. Several processing techniques have been explored for the fabrication of FGMs for structural applications, e.g., powder metallurgy, thermal spraying, in situ synthesis, self-propagating high-temperature synthesis, reactive infiltration, etc. Physical and chemical vapor deposition (CVD) techniques are also being explored to process FGM films with nanometer level gradients in composition. This article addresses the issues related to thermal-spray processing of FGMs and will only peripherally compare the advantages and limitations of thermal spray versus other processing techniques as reported in the literature.In thermal spraying, feedstock material (in the form of powder, rod, or wire) is introduced into a combustion or plasma flame. The particles melt in transit and impinge on the substrate where they flatten, undergo rapid solidification, and form a deposit through successive impingement. Thermal spraying has been traditionally employed to produce a variety of protective coatings of ceramics, metals, and polymers on a range of substrates. More recently, the process has been used for spray-forming structural components.Arc spray, combustion, and plasma are the major techniques comprising thermal spray. These classifications are based on the type of heat source and the method by which feedstock is injected. Arc-spray processes use electrically conductive wire as feedstock, while combustion methods use powder or wire.

Endocrine regulation of inflammatory potential in mast cells identifies insulin as a driving force for lipid body biogenesis and eicosanoid secretion. Lipid bodies are most studied in adipocytes, where the lipogenic action of insulin initiates their formation. Here, we test the hypothesis that insulin may regulate lipid body content in mast cells and hence, modify their proinflammatory potential. Our data show that insulin causes lipid body accumulation in RBL2H3 and BMMCs. Lipid body accumulation in mast cells is associated with enhanced levels of leukotriene-synthesizing enzymes (LTC4S and 5-LO). Increased basal and antigen-stimulated release of LTC4 is observed in insulin-treated mast cells. Concomitantly, the insulin-containing lipogenic stimulus induces a phenotypic change in mast cells, where this enhancement in leukotriene levels is accompanied by a marked down-regulation in secretory granule content and release in response to stimulus. Mast cells exposed to insulin exhibit altered scatter and fluorescence properties, accumulating in a SSCloFSChi population that exhibits decreased BS staining and degranulation responses and is enriched in NR-positive lipid bodies and eicosanoid synthesis enzymes. Lipid body accumulation in mast cells is mechanistically distinct from the process in adipocytes; for example, it is independent of PPARγ up-regulation and does not involve significant accumulation of conjugated glycerides. Thus, chronic exposure to metabolic stimuli, such as insulin, may be a determinant of the proinflammatory potential of the mast cell.

Concentrations of blood group A-specified alpha(1-->3)-N-acetylgalactosaminyltransferase (A enzyme) were measured in human plasma of blood groups A1, A2, and A3 by using chemically synthesized H disaccharides and H type 1 and type 2 trisaccharides attached to hydrophobic aglycones as acceptors. When the trisaccharides were used as acceptors, enzyme activities were reduced in samples from A2 and A3. However, the H disaccharides were shown to be good acceptors even for enzymes from A2 and A3, and no significant difference in enzyme concentration was detected in any of the plasma tested.

Experimental equipment was developed to investigate commutation phenomena in gasoline and to test commutator materials. Experimental results show that the duration of commutation arc in gasoline is shortened by 30-50% than in air. Two reasons are determined by analyzing the results. One is that the residual current of commutation in gasoline is smaller than in air. The other is that arc voltage in gasoline becomes higher than in air. >

A child's head-sized tumor on the upper back developed in a 43-year-old man. In spite of an extensive operation for the tumor, he died of multiple metastasis 15 months after the operation. Histologically, tumor cells proliferated throughout the dermal and subcutaneous regions, and a boxed-in appearance was noted with silver staining. Because electron microscopic observations strongly suggested a smooth muscle origin, we diagnosed this case as cutaneous and subcutaneous leiomyosarcoma.

A new-type camera, HDD/DVD hybrid camcorder, was proposed, whose advantages are; (1) long-time recording on built-in HDD, (2) disc interoperability with DVD players, and (3) stand-alone dubbing function (times2 speed). The new camera, actually developed cost-effectively, is already in the market. This paper presents technically how the HDD/DVD hybrid camcorder was designed effectively.

A Blu-ray disc (BD) camera was developed with high definition (HD) video technologies; (1) 5 mega pixel CMOS imager, (2) H.264 video codec, and (3) BD compatible recording. The BD camera enables one-hour HD video recording.

[1] The Research Group for Semi-controlled Earthquake-generation Experiments in South African deep gold mines (SeeSA) has continuously monitored strain changes with a resolution of 24 bit 25 Hz at the Bambanani mine near Welkom. An Ishii borehole strainmeter was installed at a depth of 2.4 km near the potential M ∼ 3 earthquake source area. Instantaneous strain steps of ∼10−4 strains associated with two M2 events were observed within a length of seismic fault. These steps were followed by significant post-seismic creep-like drift, but not preceded by forerunners. Analysis of the continuous 25 Hz data reveals many smaller steps with much longer durations (100 ms ∼ 100 s) than seen in normal earthquakes (−1 < M < 2) with source durations of 1 ms∼50 ms. Some of the especially slow steps were preceded by accelerations in strain, the maximum being as large as one-third of the step.

Cannabinoids are broadly immunosuppressive, and anti-inflammatory properties have been reported for certain marijuana constituents and endogenously produced cannabinoids. The CB2 cannabinoid receptor is an established constituent of immune system cells, and we have recently established that the CB1 cannabinoid receptor is expressed in mast cells. In the present study, we sought to define a role for CB1 in mast cells and to identify the signalling pathways that may mediate the suppressive effects of CB1 ligation on mast cell activation. Our results show that CB1 and CB2 mediate diametrically opposed effects on cAMP levels in mast cells. The observed long-term stimulation of cAMP levels by the Gαi/o-coupled CB1 is paradoxical, and our results indicate that it may be attributed to CB1-mediated transcriptional regulation of specific adenylate cyclase isoenzymes that exhibit superactivatable kinetics. Taken together, these results reveal the complexity in signalling of natively co-expressed cannabinoid receptors and suggest that some anti-inflammatory effects of CB1 ligands may be attributable to sustained cAMP elevation that, in turn, causes suppression of mast cell degranulation.

Recently many small D.C. motors have been used for automotive electrical equipment. In particular, motors for fuel pumps play an important role of supplying the engine with gasoline. Therefore, high reliability and long life are required for the motors. The feature of these motors is that gasoline flows inside the motor and commutation is done in gasoline. This paper is a fundamental investigation of the commutation phenomena in gasoline. We have already reported in a previous paper on fundamental characteristics of the commutation arc such as arc duration, arc voltage and residual current. In this paper, the experimental results of arc duration, residual current and arc voltage are reported for both commutator materials of Cu and Ag/Ni. And the wear of a brush made of metallic-carbon is measured, respectively on Cu and Ag/Ni commutators. When the silver-nickel commutator is used, brush wear is slighter in gasoline than in air. When the copper commutator is used, brush wear in gasoline is similar to that in air. The influence of gasoline on commutation arc voltage is also described.

A 73-year-old man presented with acute renal failure due to bilateral hydronephroses. Urography and CT scan revealed stenosis and medial deviation of the bilateral ureters surrounded by a soft tissue mass at the level of the 4-5th lumber vertebrae. Under a diagnosis of retroperitoneal fibrosis, bilateral nephrostomies were temporarily placed and prednisolone administration was initiated at a dose of 20 mg/day. Ureteral passage markedly improved within 2 weeks in association with a decrease in size of the retroperitoneal soft tissue mass. The steroid therapy was continued for 4 months with decreasing dosing schedule. The patient has been doing well without any signs of recurrence for 3 months after the cessation of the steroid therapy.

The CYP17 gene (CYP17) codes for the cytochrome P450c17alpha enzyme, which mediates two key steps in the sex steroid synthesis. There is a polymorphism (a T-to-C substitution) in the 5'-untranslated region, which may influence the transcription level of CYP17 mRNA. There is a continuing controversy as to whether the variant allele is associated with a subset of breast cancer or polycystic ovary syndrome. In prostate cancer research, there are contradictory data concerning the CYP17 risk allele. We explored the association between CYP17 polymorphism and a risk of prostate cancer or benign prostatic hyperplasia (BPH) in a Japanese population. This study included 252 prostate cancer patients, 202 BPH patients, and 131 male controls. A 451-bp fragment encompassing the polymorphic site was amplified by PCR, treated with restriction enzyme MspA1, and electrophoresed on an agarose gel. The MspA1-undigested allele with the published sequence and the MspA1-digested variant allele were designated as A1 and A2, respectively. There was a significant difference (P0.05) in the genotypes between prostate cancer patients and male controls, and between BPH patients and male controls. Men with the A1/A1 CYP17 genotype had an increased risk of prostate cancer [odds ratio (OR), 2.57; 95% confidence interval (CI) = 1.39-4.78] and BPH (OR, 2.44; 95% CI = 1.26-4.72) compared with those with the A2/A2 genotype. Men with the A1/A2 genotype had an intermediate increased risk of prostate cancer (OR, 1.45; 95% CI = 0.84-2.54) and BPH (OR, 1.60; 95% CI = 0.89-2.87) compared with those with the A2/A2 genotype. The trend of an increasing risk of prostate cancer and BPH with an increasing number of the A1 allele was statistically significant (prostate cancer versus male control, P = 0.003; OR, 1.57; 95% CI = 1.16-2.12; BPH versus male control, P = 0.008; OR, 1.55; 95% CI = 1.12-2.13). There was no significant association between the CYP17 genotype and the tumor status (grade and stage) of prostate cancer. Our results suggest that the A1 allele of the CYP17 polymorphism is associated with an increased risk of prostate cancer and BPH, with a gene dosage effect. However, the CYP17 genotype does not seem to influence the disease status in prostate cancer.

The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor. VirA also responds to specific monosaccharides, which enhance vir expression. These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential. To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity. The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars. Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes. In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region. We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein. Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars.

Recent studies have suggested that vitamin D is an important determinant of prostate cancer risk and inherited polymorphisms in the 3'-untranslated region (3'UTR) of the vitamin D receptor (VDR) gene are associated with the risk and progression of prostate cancer. This study was conducted to explore the association of VDR gene polymorphisms with prostate cancer risk in Japanese men who are considered to be much less influenced by environmental risk factors for prostate cancer. We studied 222 prostate cancer patients, 209 benign prostatic hyperplasia (BPH) patients, 128 male controls who were over 60 years old and without any evidence of prostate cancer or BPH, and 198 female controls. A PCR-RFLP method was used to determine three VDR gene polymorphisms in the 3'UTR characterized by restriction enzymes BsmI, ApaI and TaqI. In the BsmI polymorphism, heterozygosity or homozygosity for the absence of the BsmI restriction site was associated with one-third the risk of prostate cancer (P0.0001; odds ratio, 3.31; 95% confidence interval, 2.05-5.32) and with one-half the risk of BPH (P0.005; odds ratio, 2.07; 95% confidence interval, 1.33-3.22) compared with the male controls. The TaqI and ApaI polymorphisms did not show any significant association with either prostate cancer or BPH. The results indicate that the BsmI polymorphism in the VDR gene plays a significant role in protection against prostate cancer and BPH. Because of the racial difference in the strength of the linkage disequilibrium between the three polymorphisms, additional studies are required to apply the present results to other racial-ethnic groups.

Interferon alpha-2b therapy for Chronic Hepatitis C patients has been unsatisfactory. Recombinant Granulocyte Macrophage Colony-Stimulating Factor has been shown to have anti-viral effects in vivo and in vitro via cytokines release. Recently its effects on chronic hepatitis B and possibly chronic hepatitis C were reported. We, decided to conduct a pilot study to evaluate the anti-viral effects of recombinant human GM-CSF mono-therapy in patients with chronic hepatitis C and to assess its side effects.A total of 10 patients (male/female: 5/5) (age: 34-60, mean: 45) seen in our center between 2/95 to 2/96 were randomly selected to receive recombinant human Granulocyte Macrophage Colony-Stimulating-Factor at 125 ug/m2 subcutaneously daily for two weeks followed by three times weekly for another 8 weeks. Biochemical (ALT) and viral (HCV-RNA) responses were measured prior to treatment and at weeks four and eight. Side effects were recorded.Six out of the ten patients treated had significant viral reduction but none became negative. Eight out the ten patients treated showed biochemical improvement and three out of the eight had normalized liver enzymes. Age, sex, stage of the disease did not influence the response but there seems to be a tendency for patients with higher pre-treatment viral level to respond virally. Side effects are minimal and well-tolerated.Recombinant human Granulocyte Macrophage Colony-Stimulating-Factor in the dose used has anti-viral effects in the majority of the chronic hepatitis C patients studied. Side effects are minimal and well tolerated. Further study with higher doses and longer duration is needed to prove its clinical efficacy in treating patients with chronic hepatitis C.

Recombinant human erythropoietin (Epo) is used frequently through intravenous (i.v.) and subcutaneous (s.c.) administration for the clinical treatment of the last stage of renal anemia. We encapsulated Epo in liposomes to develop an alternative administration route. The purpose of our study was to evaluate the pharmacokinetics and the pharmacological effects of liposomal Epo in comparison with the Epo after i.v. and s.c. administration to rats.Epo was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterol mixture (SS) prepared by the reversed-phase evaporation vesicle method. After filtration through a 0.1 micron polycarbonate membrane, liposomes were gel filtered (Epo/liposomes).Epo/liposomes showed higher pharmacological activity than Epo/liposomes before gel filtration after i.v. administration to rats. Non-encapsulated Epo lost its activity, whereas encapsulated Epo in liposomes retained it. The pharmacological effects of Epo/liposomes were greater than those of Epo after i.v. administration. Epo/liposomes afforded 3-9 times higher AUC, lower clearance and lower steady-state volume of distribution than Epo after both i.v. and s.c. administrations. Epo/liposomes had an improved pharmacokinetics profile compared with Epo. S.c. administration of Epo/liposomes at 7 h may penetrate primarily (40% of dose) through the blood as a liposome and partly (7% of dose) in lymph. CONCLUSIONS. Epo/liposomes may reduce the frequency of injections required for a certain reticulocyte effect in comparison to Epo. The lower clearance of Epo/liposomes may increase the plasma concentrations of Epo, which increases the efficacy.

The effects of recombinant human growth hormone (GH) and insulin-like growth factor-1 (IGF-1) were investigated in burned rats. Sprague-Dawley rats were fed exclusively by total parenteral nutrition and were subjected to 20% third-degree scald burns. The rats were then divided into the following three groups: (1) the GH group received GH at a dose of 1 IU.kg-1.d-1 for 2d (n = 10); (2) the IGF group received IGF-1 at a dose of 4 mg.kg-1.d-1 for 2d (n = 19); and (3) the control group received saline (n = 17). Cumulative nitrogen balance increased significantly in the GH (P0.01) and IGF (P0.01) groups as compared with the control group. There were no differences in nitrogen balance between the GH and IGF groups. Blood glucose was decreased in the IGF group (P0.01) and increased in the GH group (P0.05) as compared with the control group. The intestinal villus height and wall thickness of the GH and IGF groups were significantly greater than those of the control group. Delayed-type hypersensitivity was enhanced in both the GH and the IGF groups as compared with the control group (both P0.01). Furthermore, the increase in the IGF group was significantly greater than that in the GH group (P0.05). It was concluded that both GH and IGF-1 improve protein metabolism and immune responsiveness, as well as promote proliferation of the intestinal mucosa.

We have measured the thermal conductivity of a Ni20Cr / 8% yttria-partially-stabilized-zirconia functionally graded 1.1 mm thick coating on a substrate of 403 stainless steel. We measured thermal conductivity of the as-received coated specimen, then thermally shocked the specimen and measured thermal conductivity again. The measurements were done using an absolute, steady-state technique over a temperature range from 400 K to 1200 K. The specimen was thermally shocked by heating in a furnace to 475 K, then quenching in water at 295 K. We discuss the effect of moderate thermal shock on the thermal conductivity of the coating.

Over the past two decades there has been an increase in the incidence of obesity and metabolic diseases due to sedentary lifestyles, high caloric intake, genetics and environmental factors. Studies have shown that obesity is linked with multiple co-morbid pathologies, including increases in the incidence of some cancers. We have studied the effect of a nutrient-dense environment upon the mast cell, a pro-inflammatory immunocyte. Our recently published work shows that these immunocytes are sites for ectopic lipid deposition (steatosis) under conditions that mimic high nutrient availability and hyperinsulineamia. Moreover, the steatotic phenotype is associated with phenotypic change in the mast cells, with relative over-production of bioactive eicosanoids and other pro-inflammatory lipid mediators, but suppression of some aspects of the acute Type I hypersensitivity pathway. In the current study we sought to further explore the impact of nutrient abundance upon the cell biology of the mast cell. We observe the induction of ER stress pathways, which is associated with the re-programming of the ER towards lipid synthesis and deposition in lipid bodies. Moreover, we observe the induction of autophagy pathways, which likely reflect cellular attempts to access the dysregulated ER and lipid bodies in an effort to return to homeostasis. These studies deepen our understanding of the degree to which altered nutrient environments can, in turn, cause immunological dysregulation. Of particular interest for future studies will be the links between mast cell dysregulation and the systemic and local inflammatory pathology that is associated with metabolic syndrome. Citation Format: Januaria M. Balajadia, Lori Shimoda, William Greineisen, Helen Turner. ER stress and autophagy induced in pro-inflammatory mast cells exposed to stimuli associated with a positive energy balance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2884. doi:10.1158/1538-7445.AM2013-2884

To further investigate the role of calbindin D28k in Alzheimer's disease (AD); hippocampus, superior temporal gyrus and cerebellum from control and AD cases were examined by quantitative in situ hybridization. We report here a decrease in CaBD28k mRNA in the CA2 region of AD hippocampus compared to control subjects. There were no significant differences between AD and control subjects in the other regions studied.