Inhibition of Cholinergic Signaling Causes Apoptosis in Human Bronchioalveolar Carcinoma

Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II–induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy

An analysis of RSQE forecasts: 1971–1992

The purpose of this paper is to evaluate the accuracy of ex ante econometric model forecasts of four key macroeconomic variables: real GNP growth, the rate of price inflation measured by the GNP deflator, the civilian unemployment rate, and the Treasury Bill rate. Annual forecasts produced by the Research Seminar in Quantitative Economics (RSQE) based on the Michigan Quarterly Econometric Model of the U.S. Economy are compared with quasi ex ante forecasts from a four-variable vector autoregressive (VAR) model. Statistical tests of the equality of forecast error variances as well as univariate and multivariate forecast encompassing-type tests are conducted. The forecast error variance comparisons indicate that for three of the four variables the RSQE forecasts are more accurate than the VAR forecasts and for one of the variables (real GNP growth) only slightly less accurate. The forecast encompassing-type tests indicate that the RSQE forecasts contain information not contained in the VAR forecasts and, conversely, that VAR forecasts contain information not included in the RSQE forecasts. The scope for improving RSQE forecasts by combining them with VAR forecasts is rather limited, however.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43925/1/11293_2006_Article_BF02299030.pd

Has Macro Progressed?

There have been a number of recent papers arguing that there has been considerable convergence in macro research and to the good. This paper considers the question whether what has been converged to is good. Has progress been made in understanding how the macro economy works?

<i>Candida albicans FRE8</i> encodes a member of the NADPH oxidase family that produces a burst of ROS during fungal morphogenesis

<div><p>Until recently, NADPH oxidase (NOX) enzymes were thought to be a property of multicellularity, where the reactive oxygen species (ROS) produced by NOX acts in signaling processes or in attacking invading microbes through oxidative damage. We demonstrate here that the unicellular yeast and opportunistic fungal pathogen <i>Candida albicans</i> is capable of a ROS burst using a member of the NOX enzyme family, which we identify as Fre8. <i>C</i>. <i>albicans</i> can exist in either a unicellular yeast-like budding form or as filamentous multicellular hyphae or pseudohyphae, and the ROS burst of Fre8 begins as cells transition to the hyphal state. Fre8 is induced during hyphal morphogenesis and specifically produces ROS at the growing tip of the polarized cell. The superoxide dismutase Sod5 is co-induced with Fre8 and our findings are consistent with a model in which extracellular Sod5 acts as partner for Fre8, converting Fre8-derived superoxide to the diffusible H₂O₂ molecule. Mutants of <i>fre8</i>Δ/Δ exhibit a morphogenesis defect <i>in vitro</i> and are specifically impaired in development or maintenance of elongated hyphae, a defect that is rescued by exogenous sources of H₂O₂. A <i>fre8</i>Δ/Δ deficiency in hyphal development was similarly observed <i>in vivo</i> during <i>C</i>. <i>albicans</i> invasion of the kidney in a mouse model for disseminated candidiasis. Moreover <i>C</i>. <i>albicans fre8</i>Δ/Δ mutants showed defects in a rat catheter model for biofilms. Together these studies demonstrate that like multicellular organisms, <i>C</i>. <i>albicans</i> expresses NOX to produce ROS and this ROS helps drive fungal morphogenesis in the animal host.</p></div

Susceptibility of <i>fre8</i>Δ/Δ cells to killing by neutrophils and macrophages.

<p>(A) SC5314 WT or the isogenic <i>fre8</i>Δ/Δ mutant biofilms were grown for 24 hours in 96 well plates and the relative fungal burden was estimated using the XTT assay as described in <i>Materials and Methods</i>. Results were normalized to SC5314 biofilm allowing for averaging of 3 independent experiments. (B) 24 hour SC5314 or <i>fre8</i>Δ/Δ biofilms were incubated with or without human neutrophils (E:T ratio 1:2) for 4 hours and an XTT metabolic assay was used to estimate fungal burden. No biofilm controls were included to estimate neutrophil contribution to XTT assays. Neutrophil contributions were subtracted from biofilm XTT assays to calculate fungal inhibition. Results represent the averages of 3 independent experimental trials. The increase in inhibition of <i>fre8</i>Δ/Δ cells by neutrophils was statistically significant (P ≤ 0.04 by T-test). (C) WT SC5314 or the isogenic <i>fre8</i>Δ/Δ, <i>sod5</i>Δ/Δ or the <i>FRE8</i> complemented <i>fre8</i>Δ/Δ (<i>fre8</i>Δ/Δ Res) were tested for killing by BMDM as described in <i>Materials and Methods</i>. Results represent the averages of 9 samples over two experimental trials. The increased killing of <i>sod5</i>Δ/Δ and <i>fre8</i>Δ/Δ cells compared to WT or to the <i>fre8</i>Δ/Δ Res was statistically significant ****p<0.0001 by one-way ANOVA with Tukey post-test. There was no statistically significant difference between WT and <i>fre8</i>Δ/Δ Res, while the increased killing in <i>sod5</i>Δ/Δ compared to <i>fre8</i>Δ/Δ was significant (p = 0.004) by one-way ANOVA with Tukey post-test.</p

Effect of <i>fre8</i>Δ/Δ mutations on fungal invasion of the kidney and virulence in a mouse model of disseminated candidiasis.

<p>Mice were infected with either WT SC5314 or the isogenic <i>fre8</i>Δ/Δ mutant by the lateral tail vein injection as described in <i>Materials and Methods</i>. (A) Following 7 days of infection, kidneys were harvested from surviving mice and analyzed for fungal morphology by PAS staining as in <i>Materials and Methods</i>. Shown are the individual kidney sections from 4 independent mice for each group. (B) Quantification of the hyphal length from WT versus <i>fre8</i>Δ/Δ fungi invading the kidney. Results represent 375 and 503 fungal cells from 6 WT-infected and 4 <i>fre8</i>Δ/Δ-infected mice, respectively. The short oblong morphological forms that were prevalent with <i>fre8</i>Δ/Δ cells were included in this analysis, but not the occasional rounded yeast-forms. The difference in hyphal length between WT and <i>fre8</i>Δ/Δ cells is statistically significant as determined by t-test, ***p<0.0001. (C) Survival curves of infected mice including 10 mice from each group. There was no statistical difference between mice infected with WT <i>C</i>. <i>albicans</i> versus the <i>fre8</i>Δ/Δ mutant as determined by the log-rank (Mantel-Cox) test.</p

<i>C</i>. <i>albicans</i> ROS production as a function of <i>FRE8</i> expression.

<p>(A,B) The indicated strains were induced to form hyphae with 10% FBS for 1 hour and were tested for (A) <i>FRE8</i> and <i>SOD5</i> mRNA by qRT-PCR or (B) ROS formation by luminol chemiluminescence as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006763#ppat.1006763.g001" target="_blank">Fig 1</a>. (A) mRNA levels were compared to that of yeast-form cells prior to serum addition. Results represent the averages of 4 samples over two independent trials, error bars equal standard error. (C-E) The indicated strains engineered to express <i>FRE8</i> from the <i>MET3</i> promoter were grown to mid log phase in SC medium either containing methionine (+MET) to repress <i>FRE8</i>, or lacking methionine (-MET) to de-repress <i>FRE8</i> expression for either 1 hour (Top) or the indicated time points (Bottom). Cells were examined for (Top) ROS production by luminol chemiluminescence and (Bottom) dark field microscopy for cell morphology at 40X magnification. Images were taken of cells following the indicated times of de-repressing <i>FRE8</i> expression. All strains are in the background of SC5314.</p