Search

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphasepromoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor–induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK. [ABSTRACT FROM AUTHOR]

Research has been conducted on the receptor systems for the hemopoietic cytokines GM-CSF, IL-3, and IL-5 consisting of the ligand-specific alpha receptor subunits which activate the shared beta(c) subunit. The structure of the complete beta(c) extracellular domain has been investigated and the results are reported.

Protein kinases are present in all domains of life and play diverse roles in cellular signaling. Whereas the impact of substrate phosphorylation by protein kinases has long been appreciated, it is becoming increasingly clear that protein kinases also play other, noncatalytic, functions. Here, we review recent developments in understanding the noncatalytic functions of protein kinases. Many noncatalytic activities are best exemplified by protein kinases that are devoid of enzymatic activity altogether--known as pseudokinases. These dead proteins illustrate that, beyond conventional notions of kinase function, catalytic activity can be dispensable for biological function. Through key examples we illustrate diverse mechanisms of noncatalytic kinase activity: as allosteric modulators; protein-based switches; scaffolds for complex assembly; and as competitive inhibitors in signaling pathways. In common, these noncatalytic mechanisms exploit the nature of the protein kinase fold as a versatile protein-protein interaction module. Many examples are also intrinsically linked to the ability of the protein kinase to switch between multiple states, a function shared with catalytic protein kinases. Finally, we consider the contemporary landscape of small molecules to modulate noncatalytic functions of protein kinases, which, although challenging, has significant potential given the scope of noncatalytic protein kinase function in health and disease. [ABSTRACT FROM AUTHOR]

We generalize classical results in spectral graph theory and linear algebra more broadly, from the case where the underlying matrix is Hermitian to the case where it is non-Hermitian. New admissibility conditions are introduced to replace the Hermiticity condition. We prove new variational estimates of the Rayleigh quotient for non-Hermitian matrices. As an application, a new Delsarte-Hoffman-type bound on the size of the largest independent pair in a directed graph is developed. Our techniques consist in quantifying the impact of breaking the Hermitian symmetry of a matrix and are broadly applicable. [ABSTRACT FROM AUTHOR]

Pseudoenzymes were first described more than 50 years ago, when it was recognised that a subset of proteins that are structurally homologous to active enzymes lack amino acids necessary for catalytic activity. Recently, interest in pseudoenzymes has surged as it has become apparent that they constitute ∼10% of proteomes and perform essential metabolic and signalling functions that can be experimentally distinguished from catalytic outputs of enzymes. Here, we highlight recent structural studies of pseudoenzymes, which have revealed the molecular basis for roles as allosteric regulators of conventional enzymes, as molecular switches and integrators, as hubs for assembling protein complexes, and as competitors of substrate availability and holoenzyme assembly. As structural studies continue to illuminate pseudoenzyme molecular mechanisms, we anticipate that our knowledge of the breadth of their biological functions will expand in parallel. [ABSTRACT FROM AUTHOR]

Juvenile Chinook Salmon ( Oncorhynchus tshawytscha ) abundance in the northern Bering Sea is used to provide insight into future returns and fisheries in the Yukon River. The status of Yukon River Chinook Salmon is of concern due to recent production declines and subsequent closures of commercial, sport, and personal use fisheries, and severe restrictions on subsistence fisheries in the Yukon River. Surface trawl catch data, mixed layer depth adjustments, and genetic stock mixtures are used to estimate juvenile abundance for the Canadian-origin stock group from the Yukon River. Abundance ranged from a low of 0.62 million in 2012 to a high of 2.58 million in 2013 with an overall average of 1.5 million from 2003 to 2015. Although abundance estimates indicate that average survival is relatively low (average of 5.2%), juvenile abundance was significantly correlated ( r =0.87, p =0.005) with adult returns, indicating that much of the variability in survival occurs during early life-history stages (freshwater and initial marine). Juvenile abundance in the northern Bering Sea has increased since 2013 due to an increase in early life-history survival (average juveniles-per-spawner increased from 29 to 59). The increase in juvenile abundance is projected to produce larger runs and increased subsistence fishing opportunities for Chinook Salmon in the Yukon River as early as 2016. [ABSTRACT FROM AUTHOR]

Summary CCAAT-enhancer binding proteins (C/EBPs) are transcription factors that play a central role in the differentiation of myeloid cells and adipocytes. Tribbles pseudokinases govern levels of C/EBPs by recruiting them to the COP1 ubiquitin ligase for ubiquitination. Here, we present the first crystal structure of a Tribbles protein, which reveals a catalytically inactive TRIB1 pseudokinase domain with a unique adaptation in the αC helix. A second crystal structure and biophysical studies of TRIB1 with its C-terminal extension, which includes the COP1-binding motif, show that the C-terminal extension is sequestered at a site formed by the modified TRIB1 αC helix. In addition, we have identified and characterized the TRIB1 substrate-recognition sequence within C/EBPα, which is evolutionarily conserved in C/EBP transcription factors. Binding studies indicate that C/EBPα recruitment is weaker in the presence of the C-terminal COP1-binding motif, but the magnitude of this effect suggests that the two bind distinct rather directly overlapping binding sites. [ABSTRACT FROM AUTHOR]

The past decade has seen the emergence of the necroptosis programmed cell death pathway as an important contributor to the pathophysiology of myriad diseases. The receptor interacting protein kinase (RIPK)1 and RIPK3, and the pseudokinase executioner protein, mixed lineage kinase domain-like (MLKL), have grown to prominence as the core pathway components. Depending on cellular context, these proteins also serve as integrators of signals, such as post-translational modifications and protein or metabolite interactions, adding layers of complexity to pathway regulation. Here, we describe the emerging picture of the web of proteins that tune necroptotic signal transduction and how these events have diverged across species, presumably owing to selective pressures of pathogens upon the RIPK3–MLKL protein pair. Necroptosis is a lytic programmed cell death pathway initiated by death receptors and pathogen receptors, which is believed to have ancestrally evolved for host defense, but is frequently dysregulated in human disease. The core components of the pathway are the protein kinases, receptor interacting protein kinase (RIPK)1 and RIPK3, and the pseudokinase mixed lineage kinase domain-like (MLKL), which are responsible for membrane perturbation and cell death. The past decade has seen increasing complexity and implication of additional layers of regulation, through protein and metabolite interactions and post-translational modifications, to tune the activities and necroptotic functions of RIPK1, RIPK3, and MLKL. Many RIPK1, RIPK3, and MLKL modifications and interactions appear to be dependent on the cellular context, the necroptotic stimulus, and the species in question, which limits reductionist approaches to understanding the pathway. [ABSTRACT FROM AUTHOR]

Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broad range of hematopoietic cells and in the nervous system and appears to be important in several chronic inflammatory diseases. In this study, alanine substitutions were used to investigate the role of residues of the human β-common (hβc) receptor and the murine IL-3-specific (βIL-3) receptor in IL-3 binding. We show that the domain 1 residues, Tyr15 and Phe79, of the hβc receptor are important for high affinity IL-3 binding and receptor activation as shown previously for the related cytokines, interleukin-5 and granulocyte-macrophage colony-stimulating factor, which also signal through this receptor subunit. From the x-ray structure of hβc, it is clear that the domain 1 residues cooperate with domain 4 residues to form a novel ligand-binding interface involving the two protein chains of the intertwined homodimer receptor. We demonstrate by ultracentrifugation that the βIL-3 receptor is also a homodimer. Its high sequence homology with hβc suggests that their structures are homologous, and we identified an analogous binding interface in βIL-3 for direct IL-3 binding to the high affinity binding site in hβc. Tyr21 (A-B loop), Phe85, and Asn87 (E-F loop) of domain 1; Ile320 of the interdomain loop; and Tyr348 (B′-C′ loop) and Tyr401 (F′-G′ loop) of domain 4 were shown to have critical individual roles and Arg84 and Tyr317 major secondary roles in direct murine IL-3 binding to the β IL-3 receptor. Most surprising, none of the key residues for direct IL-3 binding were critical for high affinity binding in the presence of the murine IL-3 α receptor, indicating a fundamentally different mechanism of high affinity binding to that used by hβc. [ABSTRACT FROM AUTHOR]

Reports on the use of site-directed mutagenesis, in conjunction with ligand binding and proliferation studies, to demonstrate the critical requirement of the domain 1 residues, Tyr[sup 15](A-B loop) and Phe[sup 79](E-F loop), in high affinity complex formation and receptor activation. Novel ligand-receptor interface formed between domains 1 and 4; Class I cytokine receptor interface composed of two noncontiguous fibronectin III domains.

Homeodomain-interacting protein kinases (HIPKs) are kinases that phosphorylate transcription factors involved in cell proliferation, differentiation, and apoptosis. Their structures have been long sought because of their potential as drug targets in cancers and fibrosis. Agnew and colleagues present the first crystal structure of the HIPK2 kinase domain, complexed with the small-molecule inhibitor CX-4945, revealing important structural differences from related protein kinases of the DYRK family. This structure provides a starting point to exploit HIPK2's distinct structural features to develop selective smallmolecule inhibitors of this kinase. [ABSTRACT FROM AUTHOR]

Necroptosis is a lytic form of programmed cell death implicated in inflammatory pathologies, leading to intense interest in the underlying mechanisms and therapeutic prospects. Here, we review our current structural understanding of how the terminal executioner of the pathway, the dead kinase, mixed lineage kinase domain-like (MLKL), is converted from a dormant to killer form by the upstream regulatory kinase, RIPK3. RIPK3-mediated phosphorylation of MLKL's pseudokinase domain toggles a molecular switch that induces dissociation from a cytoplasmic platform, assembly of MLKL oligomers, and trafficking to the plasma membrane, where activated MLKL accumulates and permeabilises the lipid bilayer to induce cell death. We highlight gaps in mechanistic knowledge of MLKL's activation, how mechanisms diverge between species, and the power of modelling in advancing structural insights. [ABSTRACT FROM AUTHOR]

Life-history and life-cycle models of Pink salmon (Oncorhynchus gorbuscha) are developed to provide insight into production dynamics of northern Bering Sea Pink salmon. Arctic ecosystems, including freshwater and marine ecosystems in the northern Bering Sea, are warming at a rapid rate. Due to their short, two-year life cycle, Pink salmon are well known to respond rapidly to ecosystem change and can provide unique insight into ecosystem impacts of warming Arctic conditions. Life-cycle models suggest a lack of density-dependence for adult Pink salmon spawners in the Yukon River and potential for some density-dependence for adult Pink salmon spawners in the Norton Sound region. Life-history models identify a positive and significant relationship between the abundance index for juvenile Pink salmon and average Nome air temperature during their freshwater residency (August to June). This relationship supports the notion that warming air temperatures in this region (as a proxy for river and stream temperatures) are contributing to improved freshwater survival or increased capacity of freshwater habitats to support Pink salmon production. Life-history models also identify the number of adult Pink salmon returning to Norton Sound and the Yukon River is significantly related to the juvenile abundance in the northern Bering Sea. This result indicates that much of the variability in survival for northern Bering Sea Pink salmon occurs during early life-history stages and that juvenile abundance is an informative leading indicator of Pink salmon runs to this region. • Life-cycle and life-history models investigate cricital stages for Pink salmon survival. • Relates juvenile Pink salmon abundance to adult returns. • Models environmental parameters to understand mechanisms affecting Pink salmon survival. [ABSTRACT FROM AUTHOR]

Necroptotic cell death has been implicated in many human pathologies and is thought to have evolved as an innate immunity mechanism. The pathway relies on two key effectors: the kinase receptor-interacting protein kinase 3 (RIPK3) and the terminal effector, the pseudokinase mixed-lineage kinase-domain-like (MLKL). We identify proteins with high sequence similarity to the pseudokinase domain of MLKL in poxvirus genomes. Expression of these proteins from the BeAn 58058 and Cotia poxviruses, but not swinepox, in human and mouse cells blocks cellular MLKL activation and necroptotic cell death. We show that viral MLKL-like proteins function as dominant-negative mimics of host MLKL, which inhibit necroptosis by sequestering RIPK3 via its kinase domain to thwart MLKL engagement and phosphorylation. These data support an ancestral role for necroptosis in defense against pathogens. Furthermore, mimicry of a cellular pseudokinase by a pathogen adds to the growing repertoire of functions performed by pseudokinases in signal transduction. • Some poxviruses encode proteins with homology to mammalian MLKL pseudokinase domains • BAV and Cotia viral MLKL proteins inhibit necroptotic death of human and mouse cells • These viral MLKL proteins target RIPK3 to block activation of cellular MLKL • Viral MLKL proteins inhibit RIPK3 via species-specific mechanisms Petrie et al. identify proteins encoded by some poxviruses with homology to the pseudokinase domain of the terminal necroptosis effector, MLKL. Via species-specific mechanisms, viral MLKL proteins block necroptotic death in human and mouse cells by sequestering RIPK3 to prevent phosphorylation and activation of MLKL. [ABSTRACT FROM AUTHOR]

Summary We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist -HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation. Graphical Abstract Highlights • Smchd1 does not bind endogenous RNA with detectable sequence specificity • Smchd1 depends on the Xist-HnrpnK-PRC1 pathway for recruitment to the inactive X • Smchd1 depends on histone H2A lysine 119 ubiquitination for inactive X localization • Smchd1 protein stability depends on histone H2A lysine 119 ubiquitination Jansz et al. report that the chromatin protein Smchd1 depends on polycomb repressive complex 1-mediated ubiquitylation of histone H2A for its recruitment to the inactive X chromosome and for its protein stability. These data have implications for Smchd1 targeting genome-wide. [ABSTRACT FROM AUTHOR]

The recent implication of the cell death pathway, necroptosis, in innate immunity and a range of human pathologies has led to intense interest in the underlying molecular mechanism. Unlike the better-understood apoptosis pathway, necroptosis is a caspase-independent pathway that leads to cell lysis and release of immunogens downstream of death receptor and Toll-like receptor (TLR) ligation. Here we review the role of recent structural studies of the core machinery of the pathway, the protein kinases receptor-interacting protein kinase (RIPK)1 and RIPK3, and the terminal effector, the pseudokinase mixed lineage kinase domain-like protein (MLKL), in shaping our mechanistic understanding of necroptotic signaling. Structural studies have played a key role in establishing models that describe MLKL's transition from a dormant monomer to a killer oligomer and revealing important interspecies differences. Highlights Structures of component signaling modules in all known necroptosis effectors – RIPK1, RIPK3, and MLKL – have been reported over the past 5 years. Recent NMR studies have provided insights into how RIPK1 and RIPK3 form high-molecular-weight fibrils via their RHIM motifs. Recent structural and biophysical data highlight interspecies differences in the terminal effector MLKL. MLKL assembles into higher-order complexes: mouse MLKL forms trimers while the human protein is tetrameric. Structural differences in the executioner 4HB domain and the regulatory pseudokinase domain between mouse and human MLKL may reflect mechanistic differences. Several models have been proposed to describe the mechanism by which MLKL permeabilizes the plasma membrane, leading to debate about the nature of the membrane aperture. Recent studies suggest a role for necroptotic coeffectors in regulating the susceptibility of membranes to MLKL-mediated permeabilization. [ABSTRACT FROM AUTHOR]

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation. [ABSTRACT FROM AUTHOR]

It has very recently become clear that the epigenetic modifier SMCHD1 has a role in two distinct disorders: facioscapulohumoral muscular dystrophy (FSHD) and Bosma arhinia and micropthalmia (BAMS). In the former there are heterozygous loss-of-function mutations, while both gain- and loss-of-function mutations have been proposed to underlie the latter. These findings have led to much interest in SMCHD1 and how it works at the molecular level. We summarise here current understanding of the mechanism of action of SMCHD1, its role in these diseases, and what has been learnt from study of mouse models null for Smchd1 in the decade since the discovery of SMCHD1. [ABSTRACT FROM AUTHOR]

Climate change in the Arctic has implications for influences on juvenile Chum Salmon Oncorhynchus keta early life-history patterns, such as altered timing of marine entry and/or early marine growth. Sagittal otoliths were used to estimate marine entry dates and daily growth rates of juvenile Chum Salmon collected during surface trawl surveys in summers 2007, 2012, and 2013 in the Chukchi and northern Bering seas. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to discriminate between freshwater and marine sagittal growth on the otoliths, and daily growth increments were counted to determine marine-entry dates and growth rates to make temporal and regional comparisons of juvenile Chum Salmon characteristics. Marine-entry dates ranged from mid-June to mid-July, with all region and year combinations exhibiting similar characteristics in entry timing (i.e. larger individuals at the time of capture entered the marine environment earlier in the growing season than smaller individuals in the same region/year), as well as similar mean marine-entry dates. Juvenile Chum Salmon growth rates were on average 4.9% body weight per day in both regions in summers 2007 and 2012, and significantly higher (6.8% body weight per day) in the Chukchi Sea in 2013. These results suggest that juvenile Chum Salmon in the northern Bering and Chukchi seas currently exhibit consistent marine-entry timing and early marine growth rates, despite some differences in environmental conditions between regions and among years. This study also provides a baseline of early marine life-history characteristics of Chum Salmon for comparisons with future climate change studies in these regions. [ABSTRACT FROM AUTHOR]

An abstract of the article "IL-3, IL-5, and GM-CSF Signaling: Crystal Structure of the Human Beta-Common Receptor," by James M. Murphy and Ian G. Young is presented.

Highlights: [•] RIP3 kinase activation causes programmed necrosis, referred to as necroptosis. [•] The RIP3 kinase substrate MLKL is essential for necroptotic cell death. [•] RIP3 signaling has been implicated in common inflammatory diseases. [•] Caspase-8 is a critical negative regulator of necroptosis. [•] RIP3 can also activate the NLRP3 inflammasome and regulate caspase-8 activity. [Copyright &y& Elsevier]

Patients who recover from nosocomial pneumonia oftentimes exhibit long-lasting cognitive impairment comparable with what is observed in Alzheimer's disease patients. We previously hypothesized that the lung endothelium contributes to infection-related neurocognitive dysfunction, because bacteria-exposed endothelial cells release a form(s) of cytotoxic tau that is sufficient to impair long-term potentiation in the hippocampus. However, the full-length lung and endothelial tau isoform(s) have yet to be resolved and it remains unclear whether the infection-induced endothelial cytotoxic tau triggers neuronal tau aggregation. Here, we demonstrate that lung endothelial cells express a big tau isoform and three additional tau isoforms that are similar to neuronal tau, each containing four microtubule-binding repeat domains, and that tau is expressed in lung capillaries in vivo. To test whether infection elicits endothelial tau capable of causing transmissible tau aggregation, the cells were infected with Pseudomonas aeruginosa. The infection-induced tau released from endothelium into the medium-induced neuronal tau aggregation in reporter cells, including reporter cells that express either the four microtubule-binding repeat domains or the full-length tau. Infection-induced release of pathological tau variant(s) from endothelium, and the ability of the endothelial-derived tau to cause neuronal tau aggregation, was abolished in tau knockout cells. After bacterial lung infection, brain homogenates from WT mice, but not from tau knockout mice, initiated tau aggregation. Thus, we conclude that bacterial pneumonia initiates the release of lung endothelial-derived cytotoxic tau, which is capable of propagating a neuronal tauopathy. [ABSTRACT FROM AUTHOR]

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the "SP2" isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, βIL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, βIL-3, not only precluded direct binding of mouse 1L-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and β βIL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling. [ABSTRACT FROM AUTHOR]

Abstract: Past research has indicated that chronic ethanol exposure enhances dopamine (DA) neurotransmission in several brain regions. The present study examined the effects of chronic ethanol drinking on dopamine transporter (DAT) function in the nucleus accumbens (Acb) of High-Alcohol-Drinking replicate line 1 (HAD-1) rats. HAD rats were given concurrent 24-h access to 15% ethanol and water or water alone for 8 weeks. Subsequently, DA uptake and the V max of the DAT were compared between the two groups using homogenates of the nucleus accumbens. DA uptake was measured following a 2min incubation at 37°C in the presence of 8nM [3H]DA. For kinetic analyses, DA uptake was assessed in the presence of 5 concentrations of [3H]DA ranging from 8nM to 500nM. Analyses of the data revealed a significant increase in DA uptake in the ethanol group compared to water controls. Kinetic analyses revealed the change in DA uptake to be a consequence of an increase in the V max of transport. These findings demonstrate that chronic free-choice oral ethanol consumption in HAD-1 female rats increases DA uptake in the Acb by increasing the V max of the transporter. However, it is not known whether the ethanol-induced change in V max is caused by differences in the actual number of available transporter sites or from a difference in the velocity of operation of a similar number of transporters. Overall, the data indicate that chronic ethanol consumption by HAD-1 rats produces prolonged neuroadaptations within the mesolimbic DA system, which may be important for the understanding of the neurobiological basis of alcoholism. [Copyright &y& Elsevier]

Abstract: Neuropeptide Y (NPY) suppresses ethanol drinking in alcohol-preferring (P) rats, an effect which is augmented following a single ethanol abstinence period. The present experiment tests both ethanol drinking and feeding in P rats following multiple abstinence periods. Female P rats had continuous access to 15% (v/v) ethanol and water for 6 weeks followed by 3 ethanol access cycles of 2 weeks with no ethanol and 2 weeks with ethanol. Following intracerebroventricular cannula implantation during the third period of ethanol abstinence, groups (n =12–13/dose) were infused with NPY (2.5, 5.0, 10.0 μg) or aCSF prior to ethanol reinstatement. Two additional groups (n =11–12/dose) were treated similarly except that ethanol access was uninterrupted, and they were infused with a single NPY dose (10.0 μg) or aCSF. NPY increased food intake in all groups, and this effect was greater following ethanol abstinence. NPY suppressed ethanol intake, and this suppression lasted longer (24 h post-infusion) in rats with a history of ethanol abstinence periods than rats with a history of continuous ethanol access (4 h post-infusion). These results confirm past findings and indicate that global dysregulation of brain NPY systems during ethanol abstinence may render P rats more sensitive to the behavioral effects of NPY. [Copyright &y& Elsevier]

The multifunctional glycoprotein fibronectin influences several crucial cellular processes and contributes to multiple pathologies. While a link exists between fibronectin-associated pathologies and the receptor tyrosine kinase EphA2, the mechanism by which EphA2 promotes fibronectin matrix remodeling remains unknown. We previously demonstrated that EphA2 deletion reduces smooth muscle fibronectin deposition and blunts fibronectin deposition in atherosclerosis without influencing fibronectin expression. We now show that EphA2 expression is required for contractility-dependent elongation of tensin- and α5β1 integrin-rich fibrillar adhesions that drive fibronectin fibrillogenesis. Mechanistically, EphA2 localizes to integrin adhesions where focal adhesion kinase mediates ligand-independent Y772 phosphorylation, and mutation of this site significantly blunts fibrillar adhesion length. EphA2 deficiency decreases smooth muscle cell contractility by enhancing p190RhoGAP activation and reducing RhoA activity, whereas stimulating RhoA signaling in EphA2 deficient cells rescues fibrillar adhesion elongation. Together, these data identify EphA2 as a novel regulator of fibrillar adhesion elongation and provide the first data identifying a role for EphA2 signaling in integrin adhesions. [Display omitted] • EphA2 expression is required for efficient elongation of tensin- and α5β1-rich fibrillar adhesions in vascular smooth muscle cells. • EphA2 undergoes ligand-independent Y772 phosphorylation within integrin adhesions through focal adhesion kinase-dependent transphosphorylation. • EphA2 Y772 phosphorylation, but not EphA2 ligand-binding, promotes fibrillar adhesion elongation and fibronectin deposition by enhancing RhoA-mediated contractility. • EphA2 expression limits p190RhoGAP activation to promote contractility-dependent fibrillar adhesion formation and fibronectin deposition. [ABSTRACT FROM AUTHOR]

Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1) plays important roles in epigenetic silencing and normal mammalian development. Recently, heterozygous mutations in SMCHD1 have been reported in two disparate disorders: facioscapulohumeral muscular dystrophy type 2 (FSHD2) and Bosma arhinia microphthalmia syndrome (BAMS). FSHD2-associated mutations lead to loss of function; however, whether BAMS is associated with loss- or gain-of-function mutations in SMCHD1 is unclear. Here, we have assessed the effect of SMCHD1 missense mutations from FSHD2 and BAMS patients on ATP hydrolysis activity and protein conformation and the effect of BAMS mutations on craniofacial development in a Xenopus model. These data demonstrated that FSHD2 mutations only result in decreased ATP hydrolysis, whereas many BAMS mutations can result in elevated ATPase activity and decreased eye size in Xenopus. Interestingly, a mutation reported in both an FSHD2 patient and a BAMS patient results in increased ATPase activity and a smaller Xenopus eye size. Mutations in the extended ATPase domain increased catalytic activity, suggesting critical regulatory intramolecular interactions and the possibility of targeting this region therapeutically to boost SMCHD1's activity to counter FSHD. [ABSTRACT FROM AUTHOR]

Climate warming has impacted the southern extent of sea ice in the eastern Bering Sea (EBS) ecosystem, leading to many changes in ocean conditions and food webs there. We explore how these changes have affected two key forage fish species, capelin ( Mallotus villosus ) and Pacific herring ( Clupea pallasii ), examining the effects of climate change on this commercially important ecosystem in the EBS. Catch per unit effort (CPUE) data from surface trawls, size, and diet of capelin and Pacific herring were collected during a series of warm and cold years by fisheries oceanographic surveys conducted from mid-August to early October 2003 through 2011. Overall, mean CPUE for both species was higher in the northeastern Bering Sea [NEBS; capelin=1.2 kg/km 2 (warm) and 40.0 kg/km 2 (cold); herring=141.1 kg/km 2 (warm) and 132.4 kg/km 2 (cold)] relative to the southeastern Bering Sea [SEBS; capelin=0.2 kg/km 2 (warm) and 5.8 kg/km 2 (cold); herring=15.8 kg/km 2 (warm) and 24.5 kg/km 2 (cold)], irrespective of temperature conditions. Capelin mean CPUE was significantly lower during warm years than during cold years [ p <0.001; 0.6 kg/km 2 (warm), 19.0 kg/km 2 (cold)]. Pacific herring mean CPUE was less variable between warm and cold years [ p <0.001; 63.8 kg/km 2 (warm), 66.2 kg/km 2 (cold)], but was still significantly less during warm years than cold. Capelin and herring lengths remained relatively constant between climate periods. Capelin lengths were similar among oceanographic domains [104 mm (South Inner domain), 112 mm (South Middle domain), 107 mm (North Inner domain), and 104 mm (North Middle domain)], while herring were larger in domains further offshore [123 mm (South Inner domain), 232 mm (South Middle domain), 260 mm (South Outer domain), 129 mm (North Inner domain), and 198 mm (North Middle domain)]. Diets for both species were significantly different between climate periods. Large crustacean prey comprised a higher proportion of the diets in most regions during cold years. Age-0 walleye pollock ( Gadus chalcogrammus ) contributed >60% to the diets of Pacific herring in southern Middle Domain and >30% in the northern Middle domain during warm years. A switch to less energetic prey for these forage fishes during warm years may have implications for fitness and future recruitment. The shifts in the distribution and lower biomass of capelin in the EBS during warm years could lead to disruptions in energy pathways in this complex marine ecosystem. [ABSTRACT FROM AUTHOR]

Tribbles proteins (TRIB1–3) are pseudokinases that recruit substrates to the COP1 ubiquitin ligase. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukemia oncogene, because it recruits the C/EBPα transcription factor for ubiquitination by COP1. Here we report identification of nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of the TRIB2-Nb4.103 complex identified the nanobody to bind the N-terminal lobe of TRIB2, enabling specific recognition of TRIB2 in an activated conformation that is similar to the C/EBPα-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilize a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors and nanobody tools for future investigation of TRIB2 function. [Display omitted] • Nanobodies identified from synthetic library that bind TRIB2 with nanomolar affinity • Structure of Nb4.103-bound TRIB2 reveals an activated conformation • Nb4.103 binding mode to N-terminal lobe underlies Tribbles specificity • Nanobodies have potential to stabilize TRIB2 dimer, or be used as functional tools Tribbles pseudokinases (TRIB1–3) control transcription factor levels and differentiation. Jamieson et al. report the selection and characterization of nanobodies that specifically recognize TRIB2, and the structure of one of these nanobodies in complex with TRIB2. Nanobodies will enable future work analyzing and inhibiting Tribbles function. [ABSTRACT FROM AUTHOR]

Alcohol drinking during adolescence is associated in adulthood with heavier alcohol drinking and an increased rate of alcohol dependence. Past research in our laboratory has indicated that peri-adolescent ethanol consumption can enhance the acquisition and reduce the rate of extinction of ethanol selfadministration in adulthood. Caveats of the past research include reinforcer specificity, increased oral consumption during peri-adolescence, and a lack of quantitative assessment of the reinforcing properties of ethanol. The current experiments were designed to determine the effects of peri-adolescent ethanol or saccharin drinking on acquisition and extinction of oral ethanol self-administration and ethanol seeking, and to quantitatively assess the reinforcing properties of ethanol (progressive ratio). Ethanol or saccharin access by alcohol-preferring (P) rats occurred during postnatal day (PND) 30-60. Animals began operant self-administration of ethanol or saccharin after PND 85. After 10 weeks of daily operant selfadministration, rats were tested in a progressive ratio paradigm. Two weeks later, self-administration was extinguished in all rats. Peri-adolescent ethanol consumption specifically enhanced the acquisition of ethanol self-administration, reduced the rate of extinction for ethanol self-administration, and quantitatively increased the reinforcing properties of ethanol during adulthood. Peri-adolescent saccharin consumption was without effect. The data indicate that ethanol consumption during periadolescence results in neuroadaptations that may specifically enhance the reinforcing properties of ethanol during adulthood. This increase in the reinforcing properties of ethanol could be a part of biological sequelae that are the basis for the effects of adolescent alcohol consumption on the increase in the rate of alcoholism during adulthood. [ABSTRACT FROM AUTHOR]

The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific α-subunit and common β-subunit (βc; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a β-subunit that specifically binds IL-3 (βIL-3; present in mice but not humans). We recently identified two splice variants of the α-subunit of the IL -3 receptor (IL-3Rα) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length ("SP1" isoform) and a novel isoform (denoted "SP2") lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) Rα isoform can direct mIL-3 binding to two distinct sites on the βIL -3 subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of βIL-3 recognition and whether the human IL-3Rα SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human βc subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu23, in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the βIL-3 and mIL-3Rα SP2 subunits, whereas an overlapping cluster was required for binding and activation of βIL-3 in the presence of mIL-3Rα SP1. Similarly, our studies of human IL-3 indicate that two different modes of βc binding are utilized in the presence of the hIL-3Rα SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes. [ABSTRACT FROM AUTHOR]

Abstract: Several studies indicated the involvement of serotonin-3 ([5-hydroxy tryptamine] 5-HT3) receptors in regulating alcohol-drinking behavior. The objective of this study was to determine the involvement of 5-HT3 receptors within the ventral tegmental area (VTA) in regulating ethanol self-administration by alcohol-preferring (P) rats. Standard two-lever operant chambers (Coulbourn Instruments, Allentown, PA) were used to examine the effects of seven consecutive bilateral microinfusions of ICS 205-930 (ICS), a 5-HT3 receptor antagonist, directly into the posterior VTA on the acquisition and maintenance of 15% (vol/vol) ethanol self-administration. P rats readily acquired ethanol self-administration by the fourth session. The three highest doses (0.125, 0.25, and 1.25μg) of ICS prevented acquisition of ethanol self-administration. During the acquisition postinjection period, all rats treated with ICS demonstrated higher responding on the ethanol lever, with the highest dose producing the greatest effect. In contrast, during the maintenance phase, the three highest doses (0.75, 1.0, and 1.25μg) of ICS significantly increased responding on the ethanol lever; after the 7-day dosing regimen, responding on the ethanol lever returned to control levels. Microinfusion of ICS into the posterior VTA did not alter the low responding on the water lever and did not alter saccharin (0.0125% wt/v) self-administration. Microinfusion of ICS into the anterior VTA did not alter ethanol self-administration. Overall, the results of this study suggest that 5-HT3 receptors in the posterior VTA of the P rat may be involved in regulating ethanol self-administration. In addition, chronic operant ethanol self-administration and/or repeated treatments with a 5-HT3 receptor antagonist may alter neuronal circuitry within the posterior VTA. [Copyright &y& Elsevier]

Abstract: A bioenergetic model of juvenile pink salmon (Oncorhynchus gorbuscha) was used to estimate daily prey consumption and growth potential of four ocean habitats in the Gulf of Alaska during 2001 and 2002. Growth potential was not significantly higher in 2002 than in 2001 at an alpha level of 0.05 (P=0.073). Average differences in growth potential across habitats were minimal (slope habitat=0.844gd−1, shelf habitat=0.806gd−1, offshore habitat=0.820gd−1, and nearshore habitat=0.703gd−1) and not significantly different (P=0.630). Consumption demand differed significantly between hatchery and wild stocks (P=0.035) when examined within year due to the interaction between hatchery verses wild origin and year. However, the overall effect of origin across years was not significant (P=0.705) due to similar total amounts of prey consumed by all juvenile pink salmon in both study years. We anticipated that years in which ocean survival was high would have had high growth potential, but this relationship did not prove to be true. Therefore, modeled growth potential may not be useful as a tool for forecasting survival of Prince William Sound hatchery pink salmon stocks. Significant differences in consumption demand and a two-fold difference in nearshore abundance during 2001 of hatchery and wild pink salmon confirmed the existence of strong and variable interannual competition and the importance of the nearshore region as being a potential competitive bottleneck. [Copyright &y& Elsevier]

lnterleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor α (LL-3Rα), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with βIL-3 mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBPα and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that lL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in Ml cells, although the activation was more prolonged for the SP2 isoform. [ABSTRACT FROM AUTHOR]

Abstract: The present study examined changes in heart rate (HR) prior to and during limited access ethanol drinking in adult female P rats. P rats were implanted with radio-telemetric transmitters to measure HR. Daily testing involved a 90-min pre-test period (water only available) and a subsequent 90-min test period [either water (W) or ethanol available]. After a week of habituation, one ethanol group had access to ethanol for 7 weeks (CE), and another ethanol group had access for 4 weeks, was deprived for 2 weeks and then had access for a final week (DEP). Analyses of HR revealed that CE and DEP rats had significantly higher HR than W rats during test periods that ethanol was present and that DEP rats displayed higher HR during the early test period of the ethanol deprivation interval, as well. These data indicate that ethanol drinking induces HR activation in adult female P rats, and that this activation can be conditioned to the test cage environment, paralleling reports on contextual conditioning and cue-reactivity in alcoholics exposed to alcohol-associated stimuli. Therefore, this behavioral test may prove advantageous in screening pharmacotherapies for reducing craving and relapse, which are associated with cue-reactivity in abstinent alcoholics. [Copyright &y& Elsevier]

Abstract: Alcoholics generally display cycles of excessive ethanol intake, abstinence and relapse behavior. Using an animal model of relapse-like drinking, the alcohol deprivation effect (ADE), our laboratory has shown that repeated 2-week cycles of ethanol deprivation and re-exposure, following an initial 6-week access period, result in a robust ADE by alcohol-preferring (P) and high alcohol-drinking (HAD-1 and HAD-2) rats. These rat lines have been selectively bred to prefer a 10% ethanol solution over water. The present study examined whether P and HAD rats would display an ADE using much shorter ethanol deprivation and re-exposure intervals. Rats were given either continuous or periodic concurrent access to multiple concentrations (10%, 20%, and 30% [vol/vol]) of ethanol. The periodic protocol involved access to ethanol for 12 days followed by four cycles of 4 days of deprivation and 4 days of re-exposure to ethanol access. High–alcohol-drinking rats displayed a robust 24-h ADE upon first re-exposure (HAD-1: ∼5 vs. 8g/kg/day; HAD-2: ∼6 vs. 9g/kg/day, baseline vs. re-exposure), whereas P rats (∼7 vs. 8g/kg/day) displayed a modest, nonsignificant, increase in 24-h intake. In a separate group of rats, ethanol intake and blood alcohol concentrations after the first hour of the fourth re-exposure cycle were HAD-1: 2.0g/kg and 97 mg%, HAD-2: 2.3g/kg and 73 mg%, and P: 1.2g/kg and 71 mg%; with all three lines displaying a robust first hour ADE. These findings suggest that (a) an ADE may be observed with short ethanol deprivation and re-exposure intervals in HAD rats, and (b) the genetic make-up of the P and HAD rats influences the expression of this ADE. [Copyright &y& Elsevier]

Abstract: Consumption of sweet solutions has been associated with a reduction in withdrawal symptoms and alcohol craving in humans. The objective of the present study was to determine the effects of ethanol and saccharin (SACC) deprivations on operant oral self-administration. Alcohol-preferring (P) rats were allowed to lever press concurrently self-administer ethanol (15% vol/vol) and SACC (0.0125% g/vol) for 8 weeks. Rats were then maintained on daily operant access (nondeprived), deprived of both fluids (2 weeks), deprived of SACC and given 2ml of ethanol daily, or deprived of ethanol and given 2ml of SACC daily. All groups were then given 2 weeks of daily operant access to ethanol and SACC, followed by an identical second deprivation period. P rats responded more for ethanol than SACC. All deprived groups increased responding on the ethanol lever, but not on the SACC lever. Daily consumption of 2ml ethanol decreased the duration of the alcohol deprivation effect (ADE). Home cage access to 2ml of SACC also decreased the ADE but to a lesser extent than access to ethanol. A second deprivation period further increased and prolonged the expression of an ADE. These results show ethanol is a more salient reinforcer than SACC. With concurrent access to ethanol and SACC, P rats do not display a saccharin deprivation effect. Depriving P rats of both ethanol and SACC had the most pronounced effect on the magnitude and duration of the ADE, suggesting that there may be some interactions between ethanol and SACC in their CNS reinforcing effects. [Copyright &y& Elsevier]

Abstract: Metabotropic glutamate (mGlu) receptors have been shown to mediate a number of behaviors including emotionality and responsivity to stress as demonstrated by efficacy in preclinical and clinical studies. The objective of this study was to assess the effects of the mGlu2/3 receptor agonist LY404039 (LY) on operant ethanol (EtOH) self-administration during alcohol seeking (pavlovian spontaneous recovery, PSR), alcohol relapse (alcohol deprivation effect, ADE), and maintenance responding for alcohol. Adult alcohol-preferring (P) rats were trained in 2-lever operant chambers to self-administer 15% EtOH (v/v) and water on a concurrent fixed-ratio 5–fixed-ratio 1 (FR5–FR1) schedule of reinforcement in daily 1h sessions. After at least 10 weeks of daily 1h sessions, rats underwent seven extinction sessions, followed by 2 weeks of no manipulation, and then rats were tested for the expression of an EtOH PSR for four sessions. Rats were then given a week in their home cage before being returned to the operant chambers with access to EtOH and water (alcohol relapse). Finally, the effects of LY upon maintenance EtOH and water responding were assessed once stable responding was reestablished. The mGlu2/3 receptor agonist LY404039 reduced responding on the EtOH in the PSR test. LY also reduced the expression of an alcohol deprivation effect (ADE) during relapse, but did not reduce EtOH responding under maintenance conditions. The results of this study demonstrate that activating mGlu2/3 receptors inhibits the expression of alcohol seeking and relapse behavior without altering alcohol self-administration behavior. [Copyright &y& Elsevier]

Abstract: Previously, we reported that the expression of an alcohol deprivation effect (ADE) under 24-h free-choice alcohol-drinking access in high-alcohol-drinking (HAD) replicate lines of rats is dependent upon repeated cycles of alcohol access and forced abstinence. In the present study, operant techniques (including progressive ratio measures) were used to examine the effects of initial deprivation length and number of deprivation cycles on the magnitude and duration of the ADE in HAD rats to test the hypothesis that repeated deprivations increase the reinforcing effects of ethanol. Adult male HAD-1 and HAD-2 rats were trained in two-lever operant chambers to concurrently self-administer 15% ethanol (v/v) on a fixed-ratio (FR)-5 schedule and water on an FR-1 schedule of reinforcement in daily 1-h sessions. Following 10 weeks of daily 1-h sessions, the HAD-1 and HAD-2 rats were randomly assigned to one of four groups (n =6–8/group/line): nondeprived, or deprived of alcohol for 2, 5, or 8 weeks. Following this initial period, the deprived groups were given 15% ethanol again in the operant chambers for a 2-week period, following which they were deprived again for 2 weeks (all three deprived groups). Following the fifth deprivation, the rats underwent a progressive ratio test to determine the breakpoints for the nondeprived and deprived groups. The expression of an ADE under operant conditions in HAD rats was dependent upon exposure to repeated cycles of ethanol access and abstinence. Additionally, repeated deprivations increased both the magnitude and the duration of the ADE as indicated by increased responding on the ethanol lever for more sessions. Breakpoint values for the deprived groups were 1.5-fold and twofold higher than the value for the nondeprived group for the HAD-1 and HAD-2 rats, respectively. The results suggest that repeated alcohol deprivations increased the expression of an ADE and the reinforcing effects of ethanol in both HAD replicate lines of rats, and these effects were more pronounced in the HAD-2 line than the HAD-1 line. [Copyright &y& Elsevier]

Abstract: Alcohol abuse among adolescents continues to be a major health problem for our society. Our laboratory has used the peri-adolescent alcohol-preferring, P, rat as an animal model of adolescent alcohol abuse. Even though peri-adolescent P rats consume more alcohol (g/kg/day) than their adult counterparts, it is uncertain whether their drinking is sufficiently aggregated to result in measurable blood ethanol concentrations (BECs). The objectives of this study were to examine daily alcohol drinking patterns of adolescent and adult, male and female P rats, and to determine whether alcohol drinking episodes were sufficiently aggregated to result in meaningful BECs. Male and female P rats were given 30 days of 24 h free-choice access to alcohol (15%, v / v) and water, with ad lib access to food, starting at the beginning of adolescence (PND 30) or adulthood (PND 90). Water and alcohol drinking patterns were monitored 22 h/day with a “lickometer” set-up. The results indicated that (a) peri-adolescent P rats consumed more water and total fluids than adult P rats, (b) female P rats consumed more water and total fluids than male P rats, (c) there were differences in alcohol, and water, licking patterns between peri-adolescent and adult and female and male P rats, (d) individual licking patterns revealed that alcohol was consumed in bouts often exceeding the amount required to self-administer 1 g/kg of alcohol, and (e) BECs at the end of the dark cycle, on the 30th day of alcohol access, averaged 50 mg%, with alcohol intakes during the last 1 to 2 h averaging 1.2 g/kg. Overall, these findings indicate that alcohol drinking patterns differ across the age and sex of P rats. This suggests that the effectiveness of treatments for reducing excessive alcohol intake may vary depending upon the age and/or sex of the subjects being tested. [Copyright &y& Elsevier]

Abstract: Previous studies have identified deficiencies in the mesocorticolimbic dopamine (DA) systems of alcohol-preferring (P) rats. This study uses quantitative microdialysis to compare the extracellular levels of DA in the medial prefrontal cortex (MPF) of P rats and outbred Wistar rats and also compares the effects of systemic ethanol administration on DA levels in the MPF using traditional microdialysis. In experiment 1, male Wistar and P rats were implanted with loop-style microdialysis probes and later perfused at 0.5μl/min with artificial cerebrospinal fluid for 120min prior to five baseline (20-min) sample collections. Three concentrations (5, 10, and 20nM) of DA were then perfused in random order for 100min each. Samples (20-min) were collected and stored at −70°C until assayed using high performance liquid chromatography/electrochemical detection (HPLC/EC), and the data were analyzed using the quantitative no-net-flux (NNF) method. In experiment 2, male Wistar and P rats were implanted with dialysis probes aimed at the MPF. After collecting four baseline samples, all rats were injected (i.p.) with one dose of either 0.9% saline or 2.0g/kg ethanol. Microdialysis samples were collected at 20-min intervals and stored at −70°C until analyzed by HPLC/EC. NNF microdialysis yielded significantly (P <.05) lower extracellular DA concentrations in the MPF of P rats compared to Wistar rats (2.0±0.4 vs. 4.8±0.4nM, respectively). The extraction fractions were not different between the P and Wistar groups (69±3 vs. 65±3%, respectively). No significant change in extracellular DA levels was observed in P rats or Wistar rats after either saline or 2g/kg ethanol. The lower extracellular concentrations of DA in the MPF of P rats compared to Wistar rats, without a difference in the extraction fraction, suggest that DA neurotransmission is lower in the MPF of the P rat. This lower DA neurotransmission could be a result of reduced activity of the DA neurons projecting to the MPF, reduced excitatory or increased inhibitory tone occurring locally within the MPF, and/or reduced DA innervation to the MPF. The lack of effect of systemic EtOH administration on extracellular DA levels in the MPF suggests that unlike the mesolimbic DA system, the mesocortical DA system is not responsive to acute EtOH administration. [Copyright &y& Elsevier]

Abstract: Sufficient evidence exists for the inclusion of the ventral pallidum (VP) into the category of a dopaminoceptive brain region. The effects of inhibiting dopamine D1- or D2-like receptors in the VP on (a) ethanol intake and (b) extracellular levels of dopamine, were investigated in the alcohol-preferring (P) rat. The D1-like antagonist, SCH-23390, and the D2-like antagonist, sulpiride (0.25–2μg/0.5μl) were bilaterally injected into the VP and ethanol (15%, v/v) intake was assessed in a 1h limited access paradigm. The results indicate that microinjections of sulpiride significantly increased ethanol consumption (65% increase at the 2.0μg dose). Whereas the D1 antagonists SCH-23390 tended to decrease ethanol intake, the effect was not statistically significant. In a separate group of rats, reverse microdialysis of sulpiride and SCH-23390 (10–200μM) were conducted in the VP of P rats. Local perfusion of only the 200μM sulpiride dose significantly increased the extracellular levels of dopamine (maximal increase: 250% of baseline). On the other hand, local perfusion of SCH-23390 (10–200μM) dose dependently increased the extracellular levels of dopamine 180–640% of baseline. Overall, the results of this study suggest that (a) tonic activation of D2 postsynaptic receptors in VP imposes a limit on ethanol intake in the P rat; (b) there are few D2 autoreceptors functioning in the VP; (c) there is tonic D1-like receptor mediated inhibitory feedback regulation of VP-dopamine release. [Copyright &y& Elsevier]

Abstract: Animal models designed to examine different facets of alcohol-related behaviors have been developed to study genetic and neurobiological factors underlying alcoholism and alcohol abuse. One goal has been to develop valid, congruent, complementary animal models of alcohol craving and relapse, with the ultimate objective of assessing the effectiveness of pharmacological agents with these models. Animal models of alcohol craving include drug-induced responding (drug reinstatement), cue-induced responding, Pavlovian Spontaneous Recovery (PSR), and appetitive/consummatory responding. A primary experimental approach to study alcohol relapse has been through expression of the Alcohol Deprivation Effect (ADE) following a single deprivation or multiple deprivations. To date, five selectively bred lines of rats have been developed to study alcohol-drinking behavior. These are the ALKO/Alcohol (AA), alcohol-preferring (P), high alcohol-drinking (HAD-1 and HAD-2 replicates), and the Sardinian alcohol-preferring (sP) lines of rats. Findings thus far indicate that only the P line of rats meets all the criteria established for a valid animal model of alcoholism, with progress having been made in characterizing the AA, HAD and sP lines of rats. The focus of the current review will be to analyze the various models of alcohol craving, emphasizing the use of the Indiana University selected rat lines (P and HADs). Overall, the findings indicate substantial progress has been made in developing animal models of alcohol abuse, relapse and craving using these selectively bred rat lines, as well as outbred rats. [Copyright &y& Elsevier]

The effect of housing conditions on sulpiride-induced increases in extracellular dopamine (DA) levels was investigated in the nucleus accumbens (NAC) of P rats. Rats were double-housed (DH) in plastic tubs, or single-housed (SH) in hanging wire cages for 12 weeks. Microdialysis in the NAC showed greater sulpiride-induced DA increases in the NAC of SH vs. DH rats, with no difference in basal levels. The data indicate that housing conditions can alter DA D2 receptor function in the NAC. [Copyright &y& Elsevier]