Inactivation of a CRF-dependent amygdalofugal pathway reverses addiction-like behaviors in alcohol-dependent rats.

The activation of a neuronal ensemble in the central nucleus of the amygdala (CeA) during alcohol withdrawal has been hypothesized to induce high levels of alcohol drinking in dependent rats. In the present study we describe that the CeA neuronal ensemble that is activated by withdrawal from chronic alcohol exposure contains ~80% corticotropin-releasing factor (CRF) neurons and that the optogenetic inactivation of these CeA CRF+ neurons prevents recruitment of the neuronal ensemble, decreases the escalation of alcohol drinking, and decreases the intensity of somatic signs of withdrawal. Optogenetic dissection of the downstream neuronal pathways demonstrates that the reversal of addiction-like behaviors is observed after the inhibition of CeA CRF projections to the bed nucleus of the stria terminalis (BNST) and that inhibition of the CRFCeA-BNST pathway is mediated by inhibition of the CRF-CRF1 system and inhibition of BNST cell firing. These results suggest that the CRFCeA-BNST pathway could be targeted for the treatment of excessive drinking in alcohol use disorder

Examining the Health Action Process Approach for Predicting Physical Activity Behavior in Adults with Back Pain

This study investigated the appropriateness of the Health Action Process Approach (HAPA) as it relates to physical activity (PA) behavior in the back pain population. The motivational and volitional constructs of the HAPA, PA, and back pain-related disability variables were assessed in a sample of 350 men and women with back pain. HAPA model fit was satisfactory accounting for 21% of the variance in PA intentions and 28% of PA behavior. All motivational phase constructs relate to PA intention. Action/coping planning and recovery self-efficacy do not relate to PA behavior. PA intentions are the strongest predictor of PA behavior. An expanded model, including disability-specific variables, satisfactorily fit the data, accounting for 32% of PA intentions and 29% of PA participation. These data partially support assumptions of the HAPA for the back pain population. For the back pain population, interventions designed to affect PA behavior must account for disability-specific variables

A Transgenic Rat for Investigating the Anatomy and Function of Corticotrophin Releasing Factor Circuits.

Corticotrophin-releasing factor (CRF) is a 41 amino acid neuropeptide that coordinates adaptive responses to stress. CRF projections from neurons in the central nucleus of the amygdala (CeA) to the brainstem are of particular interest for their role in motivated behavior. To directly examine the anatomy and function of CRF neurons, we generated a BAC transgenic Crh-Cre rat in which bacterial Cre recombinase is expressed from the Crh promoter. Using Cre-dependent reporters, we found that Cre expressing neurons in these rats are immunoreactive for CRF and are clustered in the lateral CeA (CeL) and the oval nucleus of the BNST. We detected major projections from CeA CRF neurons to parabrachial nuclei and the locus coeruleus, dorsal and ventral BNST, and more minor projections to lateral portions of the substantia nigra, ventral tegmental area, and lateral hypothalamus. Optogenetic stimulation of CeA CRF neurons evoked GABA-ergic responses in 11% of non-CRF neurons in the medial CeA (CeM) and 44% of non-CRF neurons in the CeL. Chemogenetic stimulation of CeA CRF neurons induced Fos in a similar proportion of non-CRF CeM neurons but a smaller proportion of non-CRF CeL neurons. The CRF1 receptor antagonist R121919 reduced this Fos induction by two-thirds in these regions. These results indicate that CeL CRF neurons provide both local inhibitory GABA and excitatory CRF signals to other CeA neurons, and demonstrate the value of the Crh-Cre rat as a tool for studying circuit function and physiology of CRF neurons

Heterogeneous Chemistry Involving Methanol in Tropospheric Clouds

In this report we analyze airborne measurements to suggest that methanol in biomass burning smoke is lost heterogeneously in clouds. When a smoke plume intersected a cumulus cloud during the SAFARI 2000 field project, the observed methanol gas phase concentration rapidly declined. Current understanding of gas and aqueous phase chemistry cannot explain the loss of methanol documented by these measurements. Two plausible heterogeneous reactions are proposed to explain the observed simultaneous loss and production of methanol and formaldehyde, respectively. If the rapid heterogeneous processing of methanol, seen in a cloud impacted by smoke, occurs in more pristine clouds, it could affect the oxidizing capacity of the troposphere on a global scale

Developmental differences in myocyte contractile response after cardioplegic arrest

AbstractAlthough developmental differences in left ventricular function after cardioplegic arrest and rewarming have been postulated, whether differences exist at the level of the myocyte remains unexplored. This project tested the hypothesis that there is a differential effect of hypothermic hyperkalemic cardioplegic arrest with subsequent rewarming on contractile function of immature compared with adult ventricular myocytes. Myocytes were isolated from the left ventricular free wall of five immature and five adult rabbits and incubated for 2 hours in hyperkalemic modified Ringer's solution at 4° C (cardioplegia) or for 2 hours in cell culture medium at 37° C (normothermia). Myocytes were resuspended (“rewarmed”) in 37° C cell culture medium after the incubation protocol. Normothermic baseline contractile performance was lower in immature, compared with adult, myocytes. Specifically, myocyte shortening velocity was 62 ± 4 μm/sec in immature and 112 ± 6 μm/sec in adult myocytes (p < 0.01). After cardioplegia and rewarming, immature myocyte contractile function was unchanged, whereas adult myocyte contractile function was significantly diminished. For example, myocyte shortening velocity was 65 ± 4 μm/sec in immature and 58 ± 3 μm/sec in adult myocytes (p < 0.01 versus normothermic). Myocyte surface area, which reflects myocyte volume, was increased after cardioplegia and rewarming in adults (3582 ± 55 versus 3316 ± 46 μm2, p < 0.01), but remained unchanged in immature myocytes (2212 ± 27 versus 2285 ± 28 μm2, p = not significant). These unique findings demonstrate a preservation of myocyte contractile function and volume regulation in immature myocytes after cardioplegic arrest and rewarming. Thus this study directly demonstrates that developmental differences exist in myocyte responses to hypothermic hyperkalemic cardioplegic arrest with subsequent rewarming. (J THORAC CARDIOVASC SURG 1996;111:1257-66

Investigating the relationship between mitochondrial genetic variation and cardiovascular-related traits to develop a framework for mitochondrial phenome-wide association studies

BACKGROUND: Mitochondria play a critical role in the cell and have DNA independent of the nuclear genome. There is much evidence that mitochondrial DNA (mtDNA) variation plays a role in human health and disease, however, this area of investigation has lagged behind research into the role of nuclear genetic variation on complex traits and phenotypic outcomes. Phenome-wide association studies (PheWAS) investigate the association between a wide range of traits and genetic variation. To date, this approach has not been used to investigate the relationship between mtDNA variants and phenotypic variation. Herein, we describe the development of a PheWAS framework for mtDNA variants (mt-PheWAS). Using the Metabochip custom genotyping array, nuclear and mitochondrial DNA variants were genotyped in 11,519 African Americans from the Vanderbilt University biorepository, BioVU. We employed both polygenic modeling and association testing with mitochondrial single nucleotide polymorphisms (mtSNPs) to explore the relationship between mtDNA variants and a group of eight cardiovascular-related traits obtained from de-identified electronic medical records within BioVU. RESULTS: Using polygenic modeling we found evidence for an effect of mtDNA variation on total cholesterol and type 2 diabetes (T2D). After performing comprehensive mitochondrial single SNP associations, we identified an increased number of single mtSNP associations with total cholesterol and T2D compared to the other phenotypes examined, which did not have more significantly associated SNPs than would be expected by chance. Among the mtSNPs significantly associated with T2D we identified variant mt16189, an association previously reported only in Asian and European-descent populations. CONCLUSIONS: Our replication of previous findings and identification of novel associations from this initial study suggest that our mt-PheWAS approach is robust for investigating the relationship between mitochondrial genetic variation and a range of phenotypes, providing a framework for future mt-PheWAS

Radiative effect of clouds on tropospheric chemistry in a global three‐dimensional chemical transport model

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94988/1/jgrd12465.pd

-Tetrachlorodibenzo-p-dioxin-Mediated Impairment of B Cell Differentiation Involves Dysregulation of Paired Box 5 (Pax5) Isoform, Pax5a

ABSTRACT The persistent environmental contaminant and immunotoxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), markedly suppresses humoral immune responses. We recently reported impaired down-regulation of paired box 5 (Pax5), a repressor of B cell differentiation and concomitant suppression of the IgM response by TCDD in the murine CH12.LX B cell line. The objectives of the current study were to determine the impact of TCDD treatment on molecular outcomes characteristic of terminal B cell differentiation and to assess the role that Pax5 isoforms plays in the suppression of B cell differentiation by TCDD. In this study, we show that the highly abundant fulllength Pax5 isoform, Pax5a, and at least two additional modestly expressed Pax5 isoforms were expressed in CH12.LX and splenic B cells. In lipopolysaccharide (LPS)-activated B cells, all of the identified Pax5 isoforms were synchronously down-regulated, and in the presence of TCDD cotreatment they were abnormally and synchronously elevated, suggesting a common mechanism of regulation. Furthermore, B cell differentiation markers X-box protein-1 and major histocompatibility complex class II showed that the levels to which Pax5 was derepressed by TCDD were sufficient to impair B cell differentiation and immunoglobulin gene expression. Confirming the involvement of Pax5, ectopic expression of Pax5a in the LPS-activated CH12.LX cells closely mimicked the suppression of the IgM response by TCDD. In summary, our results demonstrate that Pax5a has a critical role in both the TCDD-mediated impairment of B cell differentiation and the suppression of the humoral immune response. Suppression of primary humoral immune responses is one of the most sensitive sequela associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous environmental contaminant. This suppression is characterized by a striking reduction in plasma cell formation and IgM secretion, and it is mediated through a direct effect by TCDD on B cells Activation by antigen or via polyclonal activators (e.g., 463 lipopolysaccharide; LPS) induces B cells to undergo cycles of intense proliferation (i.e., clonal expansion) followed by terminal differentiation into plasma cells. Terminally differentiated plasma cells specialize in secretion of antigen-specific Ig. A number of phenotypic changes distinguish terminally differentiated plasma cells from the resting B cells Materials and Methods Chemicals. TCDD, in 100% dimethyl sulfoxide (DMSO), was purchased from AccuStandard (New Heaven, CT). DMSO and LPS were purchased from Sigma-Aldrich (St. Louis, MO). Mice. Virus-free, female B6C3F1mice (six weeks old) were purchased from Charles River (Portage, MI). On arrival, mice were randomly grouped with five per plastic cage on sawdust bedding. Mice had free access to food (Purina Certified Laboratory Chow) and water at all times. The mouse holding rooms were maintained at 21 to 24°C with 40 to 60% relative humidity on a 12-h light/dark cycle. All of the experimental procedures and conditions were performed according to the guidelines of the All University Committee on Animal Use and Care at Michigan State University (East Lansing, MI). Cell Line. The CH12.LX B cell line was derived from the murine B cell lymphoma, CH12, which arose in B10.H-2aH-4bP/Wts mice (B10.A ϫ B10.129) and has been characterized previously Flow Cytometric Analysis. Cells were harvested from culture at the indicated times by centrifugation at 300g for 10 min at 4°C, washed twice in ice-cold 1ϫ Hanks&apos; balanced-salt solution, and stained using the BD Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA) according to the manufacturer&apos;s instructions. In brief, cells were incubated with 1 g/10 6 cells of purified rat anti-mouse CD16/CD32 monoclonal antibody (BD Pharmingen) for 15 min at 4°C to prevent nonspecific binding and then stained with surface marker detection antibody [anti-fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse I-A P or anti-allophycocyanin-conjugated anti-mouse CD19] or a respective isotype control (BD Pharmingen). To exclude nonviable cells, 2 l of 7-aminoactinomycin D (7-AAD) solution (Sigma-Aldrich) containing 1 mg of 7-AAD, 50 l methanol, and 950 l of Hanks&apos; balanced-salt solution were added simultaneously with detection antibodies to the cells in 50 l of staining buffer. The cells were then fixed, washed, and maintained in staining buffer containing 10 mM actinomycin D (Sigma-Aldrich) to prevent 7-AAD leakage from fixed cells. For the detection of nuclear Pax5 protein, cells were fixed and permeabilized before staining with anti-Pax5 antibody or isotype control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Fluorescence detection was performed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) on 10,000 viable cells per sample. Data analysis was performed using BD CellQuest Pro software (BD Biosciences). In Vitro Polyclonal IgM Antibody-Forming Cell Response. Single-cell suspensions of splenocytes from naive mice were adjusted to 1 ϫ 10 6 cells/ml in RPMI 1640 medium (Invitrogen) supplemented with 10% bovine calf serum (HyClone Laboratories), 100 units/ml penicillin, 100 g/ml streptomycin, and 50 M 2-mercaptoethanol. Spleen cells were transferred to 48-well culture plates in 500-l aliquots with four wells per treatment group. TCDD (3, 10, or 30 nM) and/or vehicle (0.01% DMSO) were added directly to each well in 5-l aliquots. The splenocytes were sensitized with 10 g/ml LPS and cultured for 3 days in a pressurized chamber at 5.0 psi containing 10% O 2 , 7% CO 2 , and 83% N 2 gas mixture with continuous rocking at 37°C. Enumeration of the IgM antibody-forming cell (AFC) response was performed using trinitrophenol-haptenated sheep red blood cells as described previously the cell mixture was poured onto a 100 ϫ 15-mm Petri dish and immediately covered with a 45 ϫ 50-mm glass coverslip. Upon solidification of the agar, the Petri dishes were placed in a humidified 37°C incubator overnight to allow for plaque formation. The number of splenocytes from each spleen was determined using a Coulter Counter (Beckman Coulter, Fullerton, CA). Results are expressed as AFC/10 6 viable splenocytes Ϯ S.E. Splenocyte viability was measured using pronase (EMD Biosciences, San Diego, CA) as described previously Purification of Splenic B Cells. Spleens were removed aseptically and made into a single-cell suspension. B cells were then isolated using the B Cell Isolation Kit (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer&apos;s protocol. In brief, 40 l of MACS buffer (phosphate-buffered saline solution, pH 7.2, supplemented with 0.5% bovine serum albumin and 2 mM EDTA, 4°C) per 10 7 cells was used to suspend the splenocytes, and 10 l of Biotin-Antibody Cocktail (Miltenyi Biotec Inc.) per 10 7 cells was added to label non-B cells. After incubation at 4 to 8°C for 10 min, 30 l of buffer and 20 l of Anti-Biotin Microbeads (Miltenyi Biotec Inc.) per 10 7 cells were added. The cell suspension was incubated for another 15 min at 4 to 8°C, washed with 10 to 20 times the labeling volume, and then centrifuged at 300g for 5 min. The cell pellet was finally resuspended in 500 l of buffer per 1 ϫ 10 8 cells, and passed through the prerinsed LS column (Miltenyi Biotec Inc.), followed by four washes of the column with 3 ml of buffer. The entire effluent containing the purified B cell fraction was collected. The purity of the isolated B cells was evaluated using flow cytometry to enumerate the percentage of CD19 ϩ cells through directly labeling with FITC-conjugated anti-CD19 antibody (BD Biosciences). Real-Time Polymerase Chain Reaction. Total RNA was isolated from naive or LPS-activated cells using a SV Total RNA Isolation kit (Promega, Madison, WI). To synthesize cDNA, 1000 ng/ sample of total RNA was incubated with 600 ng of random primer (Invitrogen) in 10 l of endonuclease-free water at 70°C for 10 min, cooled on ice for 10 min, and reverse transcribed in 20 l of 1ϫ First-Strand Synthesis buffer (Invitrogen), containing 0.2 mM deoxynucleotide triphosphates, 10 mM dithiothreitol, and 200 units of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42°C for 60 min, and the reaction was stopped by incubation at 75°C for 15 min. Real-time polymerase chain reaction (PCR) detection was performed using TaqMan primers and probes Isolation of Pax5a cDNA. cDNA was obtained by PCR amplification of total RNA isolated from CH12.LX cells. Total RNA was extracted with the SV Total RNA Isolation kit (Promega), and firststrand cDNA was synthesized by reverse transcription (RT) using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). PCR was carried out as follows: one initial 2-min denaturing step at 94°C followed by denaturation for 30 s at 94°C, annealing for 30 s at 58°C, and extension for 90 s at 72°C during 30 cycles in the presence of Pfu polymerase (Stratagene, La Jolla, CA) under the conditions suggested by the supplier (at a final primer concentration of 0.5 M). Sequences of the primers designed for this PCR reaction were as follows: 5Ј-CTGTCCATTTCATCAAGTCCTGA-3Ј and 5Ј-ACC-GTCACTACCCTCAGAG-3Ј. The resulting PCR product of 1.2 kilobase was separated in 1% agarose gel electrophoresis in 1ϫ TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3) and stained with ethidium bromide at 1 g/ml. The PCR fragment was eluted from agarose using the QIAquick gel extraction kit (QIAGEN, Valencia, CA). Cloning and Ectopic Expression for Pax5a cDNA. Pax5a cDNA, generated as described above, was inserted into the pcDNA 3.1 V5-His-TOPO vector (Invitrogen) according to the manufacturer&apos;s protocol. The new Pax5a-V5-His tag vector sequence was confirmed by sequencing. Additional PCR reactions, in which Pax5a-V5-His vector was used as template, were performed to generate a new Pax5a expression plasmid, Pax5a-V5-His-GFP. The primer used for 5Ј-end cloning was as follows: 5Ј-CTC ACT ATA GGG AGA TCT AAG CTG GCT AGT-3Ј (BglII restriction site is underlined); and the primer used for the 3Јend cloning was as follows: 5Ј-TGA TCA GCG GGT TTA AAA GCT TTG GGA TGG TG-3Ј (HindIII restriction site is underlined). PCR products of 1.38 kilobase were obtained and isolated as described above. The Pax-5a-V5-His PCR fragment was then cloned into the vector phCMV-C-GFP (Genlantis, San Diego, CA) by a standard ligation reaction using T4-DNA ligase (New England Biolabs, Ipswich, MA). The phCMV vector from Genlantis contains an optimized cytomegalovirus promoter and intron sequences and a kanamycin/neomycin resistance gene for generation of stable cell lines. Transfection of Pax5a-V5-His-GFP. CH12.LX cells (2.5 ϫ 10 6 ) were transfected with 5 g of each plasmid using Amaxa Nucleofector (Amaxa Inc., Gaithersburg, MD). Cells, DNA, and 100 l of 90:20 (solution V supplement) were mixed and electroporated using program A-20 following the manufacturer&apos;s recommendations. A total of TCDD Impairment of B Cell Differentiation by Altered Pax5 465 2.5 ϫ 10 7 cells was used in each experiment. The backbone plasmid, phCMV-C-GFP, was used as a control in all of the transfection experiments. After electroporation, cells were incubated at 37°C in an atmosphere of 5% CO 2 for 8 h. Eight hours after transfection, cells were collected and sorted by flow cytometry for green fluorescent protein (GFP)-expressing cells. Cells that showed fluorescence above the level of fluorescence of naive CH12.LX cells were isolated using a BD FACSCalibur. The isolated cells were counted and LPS-activated for assessments of IgM secretion as determined by enzymelinked immunosorbent assay (ELISA). An aliquot of the sorted cells was also used to prepare protein extracts to determine endogenous/ ectopic Pax5a levels by Western blotting and by flow cytometry. IgM ELISA. IgM ELISA was performed as described previously Western Blotting. Proteins were extracted from transfected cells before and after sorting by flow cytometry. Protein extracts were resolved on 4 to 12% Nu-Page gradient gel (Invitrogen) and then transferred to a nitrocellulose membrane (GE Healthcare, Piscataway, NJ). Immunoblotting was performed using anti-␤-actin and anti-Pax5 antibodies (Santa Cruz Biotechnology, Inc.). SuperSignal West Pico reagent (Pierce, Rockford, IL) was used for protein detection. Statistical Analysis of Data. Mean Ϯ S.E. was determined for each treatment group of a given PCR or ELISA experiment. Statistical differences between groups in each experiment were determined by a two-way analysis of variance (ANOVA) followed by Bonferroni&apos;s post hoc test for PCR experiments and by a one-way ANOVA followed by Bonferroni&apos;s post hoc test for ELISA experiments. Results TCDD Decreases the Levels of XBP-1 in LPS-Activated B Cells. Previous studies in LPS-activated CH12.LX cells demonstrated robust suppression of the humoral IgM response by TCDD, which correlated with a marked reduction in XBP-1 protein levels Additional studies were performed to determine whether LPS and/or TCDD treatment affects the post-transcriptional modification of XBP-1. Transcriptional activity of XBP-1 is known to be enhanced by a splicing event, which removes a 26-nucleotide-long fragment from XBP-1 mRNA, resulting in an open reading frame shift and yielding a larger XBP-1 466 Schneider et al. protein that is more potent as a transcription factor TCDD Attenuates Cell Surface MHC Class II DownRegulation in LPS-Activated CH12.LX Cells. An additional event closely associated with terminal differentiation of B cells is the down-regulation of MHC class II on the cell membrane. Levels of surface MHC class II in LPS-activated CH12.LX cells and in splenic B cells (i.e., CD19 ϩ ), in the presence and absence of TCDD, were monitored by flow cytometry. LPS activation induced a down-regulation of MHC class II on the CH12.LX cell surface between 24 and 72 h of culture compared with the time-matched naive control TCDD Attenuates the LPS-Induced Down-Regulation of Pax5 Protein in LPS-Activated CH12.LX Cells. We also examined the impact of TCDD on Pax5, a repressor of B cell differentiation. Expression of Pax5 protein was characterized in splenic B cells and CH12.LX cells by flow cytometry facilitating the evaluation of individual cells in contrast to prior Western blot studies that we performed exclusively in CH12.LX cells Characterization of Pax5 Isoforms in CH12.LX Cells. In light of the altered Pax5 expression observed by flow cytometry and by real-time PCR, studies were undertaken to further characterize the effect of TCDD on Pax5 regulation. In particular, we examined the role of alternative splicing in the deregulation of Pax5 by TCDD Additional studies performed in CH12.LX cells showed that the levels of amplicons I, II, and III were down-regulated by LPS between 24 and 72 h, as determined by real-time quantitative PC

ROSAT observations of X-ray emission from planetary nebulae

We have searched the entire ROSAT archive for useful observations to study X-ray emission from Galactic planetary nebulae (PNs). The search yields a sample of 63 PNs, which we call the ROSAT PN sample. About 20-25% of this sample show X-ray emission; these include 13 definite detections and three possible detections (at a 2-sigma level). All X-ray sources in these PNs are concentrated near the central stars. Only A 30, BD+30 3639, and NGC 6543 are marginally resolved by the ROSAT instruments. Three types of X-ray spectra are seen in PNs. Type 1 consists of only soft X-ray emission (<0.5 keV), peaks at 0.1-0.2 keV, and can be fitted by blackbody models at temperatures 1-2 10^5 K. Type 2 consists of harder X-ray emission, peaks at >0.5 keV, and can be fitted by thin plasma emission models at temperatures of a few 10^6 K. Type 3 is a composite of a bright Type 1 component and a fainter Type 2 component. Unresolved soft sources with Type 1 spectra or the soft component of Type 3 spectra are most likely photospheric emission from the hot central stars. Absorption cross sections are large for these soft-energy photons; therefore, only large, tenuous, evolved PNs with hot central stars and small absorption column densities have been detected. The origin of hard X-ray emission from PNs is uncertain. PNs with Type 2 spectra are small, dense, young nebulae with relatively cool (<<10^5 K) central stars, while PNs with Type 3 X-ray spectra are large, tenuous, evolved nebulae with hot central stars. The hard X-ray luminosities are also different between these two types of PNs, indicating perhaps different origins of their hard X-ray emission. Future Chandra and XMM observations with high spatial and spectral resolution will help to understand the origin of hard X-ray emission from PNs.Comment: To be published in The Astrophysical Journal Supplement Series. 21 pages, 7 figures, 5 table