Concurrent MEK targeted therapy prevents MAPK pathway reactivation during BRAFV600E targeted inhibition in a novel syngeneic murine glioma model.

Inhibitors of BRAFV600E kinase are currently under investigations in preclinical and clinical studies involving BRAFV600E glioma. Studies demonstrated clinical response to such individualized therapy in the majority of patients whereas in some patients tumors continue to grow despite treatment. To study resistance mechanisms, which include feedback activation of mitogen-activated protein kinase (MAPK) signaling in melanoma, we developed a luciferase-modified cell line (2341luc) from a BrafV600E mutant and Cdkn2a- deficient murine high-grade glioma, and analyzed its molecular responses to BRAFV600E- and MAPK kinase (MEK)-targeted inhibition. Immunocompetent, syngeneic FVB/N mice with intracranial grafts of 2341luc were tested for effects of BRAFV600E and MEK inhibitor treatments, with bioluminescence imaging up to 14-days after start of treatment and survival analysis as primary indicators of inhibitor activity. Intracranial injected tumor cells consistently generated high-grade glioma-like tumors in syngeneic mice. Intraperitoneal daily delivery of BRAFV600E inhibitor dabrafenib only transiently suppressed MAPK signaling, and rather increased Akt signaling and failed to extend survival for mice with intracranial 2341luc tumor. MEK inhibitor trametinib delivered by oral gavage daily suppressed MAPK pathway more effectively and had a more durable anti-growth effect than dabrafenib as well as a significant survival benefit. Compared with either agent alone, combined BRAFV600E and MEK inhibitor treatment was more effective in reducing tumor growth and extending animal subject survival, as corresponding to sustained MAPK pathway inhibition. Results derived from the 2341luc engraftment model application have clinical implications for the management of BRAFV600E glioma

Pan-cancer analysis of the extent and consequences of intratumor heterogeneity

Intratumor heterogeneity (ITH) drives neoplastic progression and therapeutic resistance. We used the bioinformatics tools ‘expanding ploidy and allele frequency on nested subpopulations’ (EXPANDS) and PyClone to detect clones that are present at a >= 10% frequency in 1,165 exome sequences from tumors in The Cancer Genome Atlas. 86% of tumors across 12 cancer types had at least two clones. ITH in the morphology of nuclei was associated with genetic ITH (Spearman’s correlation coefficient, rho = 0.24-0.41; P 2 clones coexisted in the same tumor sample (HR = 1.49, 95% CI: 1.20-1.87). In two independent data sets, copy-number alterations affecting either 75% of a tumor’s genome predicted reduced risk (HR = 0.15, 95% CI: 0.08-0.29). Mortality risk also declined when > 4 clones coexisted in the sample, suggesting a trade-off between the costs and benefits of genomic instability. ITH and genomic instability thus have the potential to be useful measures that can universally be applied to all cancers

6-thioguanine treatment in inflammatory bowel disease: A critical appraisal by a European 6-TG working party

Recently, the suggestion to use 6-thioguanine (6-TG) as an alternative thiopurine in patients with inflammatory bowel disease (IBD) has been discarded due to reports about possible (hepato) toxicity. During meetings arranged in Vienna and Prague in 2004, European experts applying 6-TG further on in IBD patients presented data on safety and efficacy of 6-TG. After thorough evaluation of its risk-benefit ratio, the group consented that 6-TG may still be considered as a rescue drug in stringently defined indications in IBD, albeit restricted to a clinical research setting. As a potential indication for administering 6-TG, we delineated the requirement for maintenance therapy as well as intolerance and/or resistance to aminosalicylates, azathioprine, 6-mercaptopurine, methotrexate and infliximab. Furthermore, indications are preferred in which surgery is thought to be inappropriate. The standard 6-TG dosage should not exceed 25 mg daily. Routine laboratory controls are mandatory in short intervals. Liver biopsies should be performed after 6-12 months, three years and then three-yearly accompanied by gastroduodenoscopy, to monitor for potential hepatotoxicity, including nodular regenerative hyperplasia (NRH) and veno-occlusive disease (VOD). Treatment with 6-TG must be discontinued in case of overt or histologically proven hepatotoxicity. Copyright (c) 2006 S. Karger AG, Basel

The Drosophila GIPC Homologue Can Modulate Myosin Based Processes and Planar Cell Polarity but Is Not Essential for Development

Epithelia often show, in addition to the ubiquitous apico-basal (A/B) axis, a polarization within the plane of the epithelium, perpendicular to the A/B axis. Such planar cell polarity (PCP) is for example evident in the regular arrangement of the stereocilia in the cochlea of the mammalian inner ear or in (almost) all Drosophila adult external structures. GIPCs (GAIP interacting protein, C terminus) were first identified in mammals and bind to the Gαi GTPase activating protein RGS-GAIP. They have been proposed to act in a G-protein coupled complex controlling vesicular trafficking. Although GIPCs have been found to bind to numerous proteins including Frizzled receptors, which participate in PCP establishment, there is little in vivo evidence for the functional role(s) of GIPCs. We show here that overexpressed Drosophila dGIPC alters PCP generation in the wing. We were however unable to find any binding between dGIPC and the Drosophila receptors Fz1 and Fz2. The effect of overexpressed dGIPC is likely due to an effect on the actin cytoskeleton via myosins, since it is almost entirely suppressed by removing a genomic copy of the Myosin VI/jaguar gene. Surprisingly, although dGIPC can interfere with PCP generation and myosin based processes, the complete loss-of-function of dGIPC gives viable adults with no PCP or other detectable defects arguing for a non-essential role of dGIPC in viability and normal Drosophila development

CIP2A expression predicts recurrences of tamoxifen-treated breast cancer

CIP2A is emerging as an oncoprotein overexpressed commonly across many tumours and generally correlated with higher tumour grade and therapeutic resistance. CIP2A drives an oncogenic potential through inhibiting protein phosphatase 2A, stabilizing MYC, and promoting epithelial-to-mesenchymal transition, although further biological mechanisms for CIP2A are yet to be defined. CIP2A protein expression was studied by immunohistochemistry in oestrogen receptor–positive primary breast cancers (n = 250) obtained from the Leeds Tissue Bank. In total, 51 cases presented with a relapse or metastasis during adjuvant treatment with tamoxifen and were regarded as tamoxifen resistant. CIP2A expression was scored separately for cytoplasmic, nuclear, or membranous staining, and scores were tested for statistically significant relationships with clinicopathological features. Membranous CIP2A was preferentially expressed in cases who experienced a recurrence during tamoxifen treatment thus predicting a worse overall survival (log rank = 8.357, p = 0.004) and disease-free survival (log rank = 21.766, p < 0.001). Cox multivariate analysis indicates that it is an independent prognostic indicator for overall survival (hazard ratio = 4.310, p = 0.013) and disease-free survival (hazard ratio = 5.449, p = 0.002). In this study, we propose the assessment of membranous CIP2A expression as a potential novel prognostic and predictive indicator for tamoxifen resistance and recurrence within oestrogen receptor–positive breast cancer

Litterfall, litter decomposition and associated nutrient fluxes in Pinus halepensis: influence of tree removal intensity in a Mediterranean forest

The online version of this article (doi:10.1007/s10342-015-0893-z) contains supplementary material, which is available to authorized users[EN] Our knowledge about the influence of silvicultural treatments on nutrient cycling processes in Mediterranean forests is still limited. Four levels of tree removal were compared in an Aleppo pine forest in eastern Spain to determine the effects on litterfall, litter decomposition and the associated nutrient fluxes after 12 years. Removal treatments included clearfelling, two shelterwood intensities (60 and 75 % of basal area removed) and untreated controls. Twelve years later, the basal area removed still explained 60 % of litterfall mass variance and 60 % of C, 52 % of N, 45 % of P, 17 % of K, 47 % of Ca and 60 % of Mg return variances. Litter decomposed somewhat more slowly in clearfellings compared to controls (p = 0.049), accumulated more Ca and released less K compared to the other three treatments. This was explained by contamination with mineral particles due to the poorly developed O horizon in clearfellings. We conclude that the management practices reduced the nutrient return via litterfall, but the nutrient release through decomposition seems poorly sensitive to canopy disturbance. In order to accurately quantify the harvesting impacts on nutrient cycling in this Mediterranean forest system, it is necessary to measure the litterfall of the understory layer.This work has been supported by a fellowship from the Generalitat Valenciana, Conselleria de Educacion, Formacion y Empleo awarded to L. Lado-Monserrat (BFPI/2008/041). Silvicultural treatments were carried out by the Mediterranean Centre for Environmental Studies (CEAM) through programme "I + D en relacion con la restauracion de la cubierta vegetal y otros aspectos de investigacion forestal". Dataloggers and probes were provided by the Generalitat Valenciana through Project "Efecto de diferentes sistemas de aclareo de masa forestal sobre la disponibilidad de agua, nutrientes y la regeneracion de la masa arborea y arbustiva en parcelas de pinar" (GV06/126). We acknowledge Joana Oliver, Ruth M. Tavera and Daniel Fortanet for their help in the laboratory and in the field. The authors wish to thank Francisco Galiana for his assistance, including help in fieldwork and providing information about the experimental design of the silvicultural treatments. Thanks also go to Rafael Herrera from the Centro de Ecologia, Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela and two anonymous reviewers for critically reviewing the manuscript.Lado Monserrat, L.; Lidón, A.; Bautista, I. (2015). Litterfall, litter decomposition and associated nutrient fluxes in Pinus halepensis: influence of tree removal intensity in a Mediterranean forest. European Journal of Forest Research. 134(5):833-844. https://doi.org/10.1007/s10342-015-0893-zS8338441345Almagro M, Martínez-Mena M (2012) Exploring short-term leaf-litter decomposition dynamics in a Mediterranean ecosystem: dependence on litter type and site conditions. Plant Soil 358:323–335Alvarez A, Gracia M, Vayreda J, Retana J (2012) Patterns of fuel types and crown fire potential in Pinus halepensis forests in the Western Mediterranean Basin. For Ecol Manage 270:282–290Austin AT, Vivanco L (2006) Plant litter decomposition in a semi-arid ecosystem controlled by photodegradation. Nature 442:555–558Bates JD, Svejcar TS, Miller RF (2007) Litter decomposition in cut and uncut western juniper woodlands. J Arid Environ 70:222–236Binkley D (2008) Three key points in the design of forest experiments. For Ecol Manage 255:2022–2023Blair JM, Crossley DA Jr (1988) Litter decomposition, nitrogen dynamics and litter microarthropods in a southern Appalachian hardwood forest 8 years following clearcutting. J Appl Ecol 25:683–698Blanco JA, Zavala MA, Imbert JB, Castillo FJ (2005) Sustainability of forest management practices: evaluation through a simulation model of nutrient cycling. For Ecol Manage 213:209–228Blanco JA, Imbert JB, Castillo FJ (2006) Influence of site characteristics and thinning intensity on litterfall production in two Pinus sylvestris L. forests in the western Pyrenees. For Ecol Manage 237:342–352Blanco JA, Imbert JB, Castillo FJ (2008) Nutrient return via litterfall in two contrasting Pinus sylvestris forests in the Pyrenees under different thinning intensities. For Ecol Manage 256:1840–1852Blanco JA, Imbert JB, Castillo FJ (2011) Thinning affects Pinus sylvestris needle decomposition rates and chemistry differently depending on site conditions. Biogeochemistry 106:397–414Caldentey J, Ibarra M, Hernández J (2001) Litter fluxes and decomposition in Nothofagus pumilio stands in the region of Magallanes, Chile. For Ecol Manage 148:145–157Christensen JH, Krishna Kumar K, et al. (2013) Climate phenomena and their relevance for future regional climate change. In: Stocker TF, Qin D, Plattner G-K et al (Eds.) Climate change 2013: the physical science basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USACortina J, Vallejo VR (1994) Effects of clearfelling on forest floor accumulation and litter decomposition in a radiata pine plantation. For Ecol Manage 70:299–310Entry JA, Rose CL, Cromack K Jr (1991) Litter decomposition and nutrient release in ectomycorrhizal mat soils of a Douglas fir ecosystem. Soil Biol Biochem 23:285–290Fabbio G, Merlo M, Tosi V (2003) Silvicultural management in maintaining biodiversity and resistance of forests in Europe—the Mediterranean region. J Environ Manage 67:67–76Galiana F, Pérez-Badía R, Camarero E, Estruch V, Currás R (2001) Estimación de la Radiación solar incidente en pinares de Pinus halepensis sometidos a tratamientos selvícolas de cortas finales. In: Junta de Andalucía. Consejería de Medio Ambiente (Ed.) Actas del III Congreso Forestal Español. Junta de Andalucía. Granada (Original in Spanish)García-Plé C, Vanrell P, Morey M (1995) Litter fall and decomposition in a Pinus halepensis forest on Mallorca. J Veg Sci 6:17–22González Utrillas N, González Pérez E, Galiana F (2005) Variación del crecimiento diametral de la masa de pinar de carrasco en cortas finales experimentales, en los montes de Tuejar y Chelva (Valencia). IV Congreso Forestal Español. Zaragoza. Soc. Esp. Cien. For. (Original in Spanish)Guo LB, Sims REH (1999) Litter decomposition and nutrient release via litter decomposition in New Zealand eucalypt short rotation forests. Agric Ecosyst Environ 75:133–140GVA (1995) Mapa de Suelos de la Comunidad Valenciana. Chelva (666). Proyecto LUCDEME (Icona), Centro de Investigaciones sobre Desertificación y Conselleria d’Agricultura i Mig Ambient. Generalitat Valenciana. Valencia, Spain. (Original in Spanish)Hennessey TC, Dougherty PM, Cregg BM, Wittwer RF (1992) Annual variation in needle fall of a loblolly pine stand in relation to climate and stand density. For Ecol Manage 51:329–338Inagaki Y, Kuramoto S, Torii A, Shinomiya Y, Fukata H (2008) Effects of thinning on leaf-fall and leaf-litter nitrogen concentration in hinoki cypress (Chamaecyparis obtusa Endlicher) plantation stands in Japan. For Ecol Manage 255:1859–1867Jonard M, Misson L, Ponette Q (2006) Long-term thinning effects on the forest floor and the foliar nutrient status of Norway spruce stands in the Belgian Ardennes. Can J For Res 36:2684–2695Kim C, Sharik TL, Jurgensen MF (1996a) Canopy cover effects on mass loss, and nitrogen and phosphorus dynamics from decomposing litter in oak and pine stands in northern Lower Michigan. For Ecol Manage 80:13–20Kim C, Sharik TL, Jurgensen MF (1996b) Litterfall, nitrogen and phosphorus inputs at various levels of canopy removal in oak and pine stands in northern lower Michigan. Am Midl Nat 135:195–204Kim C, Son Y, Lee WK, Jeong J, Noh NJ, Kim SR, Yang AR, Ju NG (2012) Influence of forest tending (Soopkakkugi) works on litterfall and nutrient inputs in a Pinus densiflora stand. For Sci Technol 8:83–88Kimmins JP (2004) Forest ecology, a foundation for sustainable management and environmental ethics in forestry. Prentice-Hall, New JerseyKimmins JP, Mailly D, Seely B (1999) Modelling forest ecosystem net primary production: the hybrid simulation approach used in FORECAST. Ecol Modell 122:195–224Klemmedson JO, Meier CE, Campbell RE (1990) Litter fall transfers of dry matter and nutrients in ponderosa pine stands. Can J For Res 20:1105–1115Kunhamu TK, Kumar BM, Viswanath S (2009) Does thinning affect litterfall, litter decomposition, and associated nutrient release in Acacia mangium stands of Kerala in peninsular India? Can J For Res 39:792–801Lytle DE, Cronan CS (1998) Comparative soil CO2 evolution, litter decay, and root dynamics in clearcut and uncut spruce–fir forest. For Ecol Manage 103:121–128Molina AJ, Del Campo AD (2012) The effects of experimental thinning on throughfall and stemflow: a contribution towards hydrology-oriented silviculture in Aleppo pine plantations. For Ecol Manage 269:206–213Navarro FB, Romero-Freire A, Del Castillo T, Foronda A, Jiménez MN, Ripoll MA, Sánchez-Miranda A, Hutsinger L, Fernández-Ondoño E (2013) Effects of thinning on litterfall were found after years in a Pinus halepensis afforestation area at tree and stand levels. For Ecol Manage 289:354–362Olson JS (1963) Energy storage and the balance of producers and decomposers in ecological systems. Ecology 44:322–331Pérez Cueva AJ (1994) Atlas Climático de la Comunidad Valenciana. Colección Territori nº 4. Generalitat Valenciana. Conselleria d’Obres Publiques, Urbanisme i Transport, ValenciaPetritsch R, Hasenauer H, Pietsch SA (2007) Incorporating forest growth response to thinning within biome-BGC. For Ecol Manage 242:324–336Prescott CE (1997) Effects of clearcutting and alternative silvicultural systems on rates of decomposition and nitrogen mineralization in a coastal montane coniferous forest. For Ecol Manage 95:253–260Prescott CE (2002) The influence of the forest canopy on nutrient cycling. Tree Physiol 22:1193–1200Prescott CE, Blevins LL, Staley CL (2000) Effects of clear-cutting on decomposition rates of litter and forest floor in forests of British Columbia. Can J For Res 30:1751–1757Roig S, Del Río M, Cañellas I, Montero G (2005) Litter fall in Mediterranean Pinus pinaster Ait. stands under different thinning regimes. For Ecol Manage 206:179–190Sardans J, Peñuelas J, Rodà F (2005) Changes in nutrient use efficiency, status and retranslocation in young post-fire regeneration Pinus halepensis in response to sudden N and P input, irrigation and removal of competing vegetation. Trees 19:233–250Scarascia-Mugnozza G, Oswald H, Piussi P, Radoglou K (2000) Forests of the Mediterranean region: gaps in knowledge and research needs. For Ecol Manage 132:97–109Slovik S (1997) Tree physiology. In: Hüttl RF, Schaaf W (eds) Magnesium deficiency in forest ecosystems. Kluwer Academic Publishers, London, pp 101–214Taylor BR, Parkinson D (1988) Does repeated freezing and thawing accelerate decay of leaf litter? Soil Biol Biochem 20:657–665Torras O, Saura S (2008) Effects of silvicultural treatments on forest biodiversity indicators in the Mediterranean. For Ecol Manage 255:3322–3330Trofymow JA, Barclay HJ, McCullough KM (1991) Annual rates and elemental concentrations of litter fall in thinned and fertilized Douglas-fir. Can J For Res 21:1601–1615Wallace ES, Freedman B (1986) Forest floor dynamics in a chronosequence of hardwood stands in central Nova Scotia. Can J For Res 16:293–302Whitford WG, Meentemeyer V, Seastedt TR, Cromack Jr K, Crossley Jr DA, Santos P, Todd RL, Waide JB (1981) Exceptions to the AET model: deserts and clear-cut forest. Ecology 62:275–277Yin X, Perry JA, Dixon RK (1989) Influence of canopy removal on oak forest floor decomposition. Can J For Res 19:204–21

PED/PEA-15 Controls Fibroblast Motility and Wound Closure by ERK1/2-Dependent Mechanisms

Cell migration is dependent on the control of signaling events that play significant roles in creating contractile force and in contributing to wound closure. We evaluated wound closure in fibroblasts from mice overexpressing (TgPED) or lacking ped/pea-15 (KO), a gene overexpressed in patients with type 2 diabetes. Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts. This difference was observed both in the absence and in the presence of mytomicin C, an inhibitor of mitosis. In time-lapse experiments, TgPED fibroblasts displayed about twofold lower velocity and diffusion coefficient, as compared to controls. These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques. At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). Inhibition of ERK1/2 activity by PD98059 restored RhoA activation, cytoskeleton organization and cell motility, and almost completely rescued wound closure of TgPED fibroblasts. Interestingly, skin fibroblasts isolated from KO mice displayed an increased wound closure ability. In vivo, healing of dorsal wounds was delayed in TgPED and accelerated in KO mice. Thus, PED/PEA-15 may affect fibroblast motility by a mechanism, at least in part, mediated by ERK1/2. J. Cell. Physiol. 227: 2106–2116, 2012. © 2011 Wiley Periodicals, Inc

Effect of oral lactulose on clinical and immunohistochemical parameters in patients with inflammatory bowel disease: a pilot study

<p>Abstract</p> <p>Background</p> <p>The prebiotic potential of lactulose is well established and preclinical studies demonstrated a protective effect of lactulose in murine models of colitis. The aim of the present study was to investigate the clinical and histological efficacy of lactulose in patients with inflammatory bowel disease (IBD), for which probiotic therapy yielded promising results.</p> <p>Methods</p> <p>Patients were treated with standard medication alone or combined with 10 g lactulose daily as adjuvant therapy for 4 months. Clinical efficacy of treatment was assessed using clinical activity indices, a quality of life index (IBDQ), endoscopic scores, defecation frequency and monitoring corticosteroid medication. Orsomucoid, alpha1-antitrypsin and other laboratory parameters were determined. In addition, in some participants colonic biopsies were analyzed with haematoxylin-eosin staining or with antibodies against HLA-DR, CD68, IgA and CD3, and evaluated systematically. All measurements were performed both at enrolment and at the end of the trial.</p> <p>Results</p> <p>14 patients presenting ulcerative colitis (UC) and 17 patients presenting Crohn's disease (CD), most of them in a clinically active state, were enrolled in this pilot study. After 4 month no significant improvement of clinical activity index, endoscopic score or immunohistochemical parameters was observed in CD or UC patients receiving lactulose in comparison to the control group. However, significant improvement of quality of life was observed in UC patients receiving lactulose compared to the control group (p = 0.04).</p> <p>Conclusion</p> <p>The findings of the present pilot study indicate that oral lactulose has no beneficial effects in IBD patients in particular with regard to clinical activity, endoscopic score or immunohistochemical parameters. The importance of the beneficial effect of lactulose in UC patients regarding the quality of life needs further evaluation in larger controlled clinical trials.</p> <p>Trial registration</p> <p>Current Controlled Trials ISRCTN92101486</p

Pervasive Growth Reduction in Norway Spruce Forests following Wind Disturbance

Background: In recent decades the frequency and severity of natural disturbances by e.g., strong winds and insect outbreaks has increased considerably in many forest ecosystems around the world. Future climate change is expected to further intensify disturbance regimes, which makes addressing disturbances in ecosystem management a top priority. As a prerequisite a broader understanding of disturbance impacts and ecosystem responses is needed. With regard to the effects of strong winds – the most detrimental disturbance agent in Europe – monitoring and management has focused on structural damage, i.e., tree mortality from uprooting and stem breakage. Effects on the functioning of trees surviving the storm (e.g., their productivity and allocation) have been rarely accounted for to date. Methodology/Principal Findings: Here we show that growth reduction was significant and pervasive in a 6.79?million hectare forest landscape in southern Sweden following the storm Gudrun (January 2005). Wind-related growth reduction in Norway spruce (Picea abies (L.) Karst.) forests surviving the storm exceeded 10 % in the worst hit regions, and was closely related to maximum gust wind speed (R 2 = 0.849) and structural wind damage (R 2 = 0.782). At the landscape scale, windrelated growth reduction amounted to 3.0 million m 3 in the three years following Gudrun. It thus exceeds secondary damage from bark beetles after Gudrun as well as the long-term average storm damage from uprooting and stem breakage in Sweden

Myosin VI in PC12 cells plays important roles in cell migration and proliferation but not in catecholamine secretion

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release