The N-Terminus of Apolipoprotein A-V Adopts a Helix-Bundle Molecular Architecture

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic R-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein

A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans

Small molecule metabolites play important roles in Caenorhabditis elegans biology, but effective approaches for identifying their chemical structures are lacking. Recent studies revealed that a family of glycosides, the ascarosides, differentially regulate C. elegans development and behavior. Low concentrations of ascarosides attract males and thus appear to be part of the C. elegans sex pheromone, whereas higher concentrations induce developmental arrest at the dauer stage, an alternative, nonaging larval stage. The ascarosides act synergistically, which presented challenges for their identification via traditional activity-guided fractionation. As a result the chemical characterization of the dauer and male attracting pheromones remained incomplete. Here, we describe the identification of several additional pheromone components by using a recently developed NMR-spectroscopic approach, differential analysis by 2D NMR spectroscopy (DANS), which simplifies linking small molecule metabolites with their biological function. DANS-based comparison of wild-type C. elegans and a signaling-deficient mutant, daf-22, enabled identification of 3 known and 4 previously undescribed ascarosides, including a compound that features a p-aminobenzoic acid subunit. Biological testing of synthetic samples of these compounds revealed additional evidence for synergy and provided insights into structure–activity relationships. Using a combination of the three most active ascarosides allowed full reconstitution of the male-attracting activity of wild-type pheromone extract. Our results highlight the efficacy of DANS as a method for identifying small-molecule metabolites and placing them within a specific genetic context. This study further supports the hypothesis that ascarosides represent a structurally diverse set of nematode signaling molecules regulating major life history traits

Exploring the Population Dynamics of Wintering Bald Eagles Through Long-Term Data

THE ECOLOGICAL QUESTION How does a bald eagle population change over time at a winter migratory stopover and which factors influence its abundance? ECOLOGICAL CONTENT Bald eagle biology, conservation biology, endangered species, population ecology, and migration ecology (stopover) WHAT STUDENTS DO Guided Approach: Students will generate questions about bald eagle numbers influenced by weather and food availability. Students will then use graphing software (JMP or Excel) to compile the data in a graphical form to answer their questions. Open-ended Approach: Students will generate their own hypotheses of interest from the larger bald eagle data set. This approach is encouraged for upper division ecology students in conservation biology, wildlife management, or population ecology classes. SKILLS Generation of a hypothesis, critical thinking, experimental design, data management using a spreadsheet, graph preparation, data analysis and interpretation, and/or written or oral presentation. STUDENT-ACTIVE APPROACHES Brainstorming, critical thinking, concept mapping, cooperative learning, guided inquiry, and/or open-ended inquiry ASSESSABLE OUTCOMES Proposal of research, figures from spreadsheet data, written interpretation of data, short or full research reports, and/or oral reports SOURCE U.S. Department of the Interior, Bureau of Land Management, Coeur d\u27Alene Field Office, archived data

Construction of Species-Specific PCR Primers for Detection of Coccidia Parasites in Captive-Reared Northern Bobwhites

Captive rearing and subsequent release of game birds, including northern bobwhites (Colinus virginianus), has become common in certain areas. In this practice, bobwhites are often raised in confinement to ‘flight ready’ and subsequently released for hunting. It is estimated that 30–40 million bobwhites are raised in captivity annually and some farms in the USA produce upwards of 1 million birds annually for this market. Raising game birds in these densities greatly facilitates the transmission of pathogenic organisms. Coccidiosis has been previously identified as an important disease in captive bobwhites and infection can lead to weight loss, diarrhea, poor feather growth, dehydration and, in severe cases, death. Eimeria lettyae, E. colini, and E. dispersa are the three described coccidia species from bobwhites. We investigated the prevalence and distribution of species of coccidia in captive bobwhite facilities throughout the United States. We collected litter or intestinal samples from 31captive bobwhite facilities originating from 13 states. Species-specific PCR primers were constructed against the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene of the various Eimeria spp. to aid in parasite detection and distinction. Primers were used to detect the specific Eimeria spp. in the collected samples. All 31 samples were positive for coccidia. Results of the primer survey disclosed E. lettyae, E. dispersa, and an unidentified Eimeria sp. in 20 (64.5%), 22 (72%), and 29 (93.5%) of the samples, respectively. Thirteen (41.9%) samples had 3 Eimeria spp. detected, 14 (45.2%) samples had 2 spp. detected, and 4 (12.9%) samples had 1 sp. detected. Flock age or geographical location was not associated with the presence of any particular Eimeria spp. To our knowledge, this is the first study of coccidia in captive bobwhites. Previous studies of Eimeria spp. in wild northern bobwhite are rare and disclosed variable prevalence rates ranging from 0 to 36%; no efforts were made to distinguish the coccidia species in these studies It would be helpful to use the species-specific primers constructed in this study to examine the prevalence and distribution of the Eimeria spp. in wild bobwhites from throughout their range to investigate the potential for captive-raised bobwhites to be a source of coccidiosis for wild bobwhites

Neurobiological Mechanisms That Contribute to Stress-related Cocaine Use

The ability of stressful life events to trigger drug use is particularly problematic for the management of cocaine addiction due to the unpredictable and often uncontrollable nature of stress. For this reason, understanding the neurobiological processes that contribute to stress-related drug use is important for the development of new and more effective treatment strategies aimed at minimizing the role of stress in the addiction cycle. In this review we discuss the neurocircuitry that has been implicated in stress-induced drug use with an emphasis on corticotropin releasing factor actions in the ventral tegmental area (VTA) and an important pathway from the bed nucleus of the stria terminalis to the VTA that is regulated by norepinephrine via actions at beta adrenergic receptors. In addition to the neurobiological mechanisms that underlie stress-induced cocaine seeking, we review findings suggesting that the ability of stressful stimuli to trigger cocaine use emerges and intensifies in an intake-dependent manner with repeated cocaine self-administration. Further, we discuss evidence that the drug-induced neuroadaptations that are necessary for heightened susceptibility to stress-induced drug use are reliant on elevated levels of glucocorticoid hormones at the time of cocaine use. Finally, the potential ability of stress to function as a “stage setter” for drug use – increasing sensitivity to cocaine and drug-associated cues – under conditions where it does not directly trigger cocaine seeking is discussed. As our understanding of the mechanisms through which stress promotes drug use advances, the hope is that so too will the available tools for effectively managing addiction, particularly in cocaine addicts whose drug use is stress-driven

EphA2-receptor deficiency exacerbates myocardial infarction and reduces survival in hyperglycemic mice

Background We have previously shown that EphrinA1/EphA expression profile changes in response to myocardial infarction (MI), exogenous EphrinA1-Fc administration following MI positively influences wound healing, and that deletion of the EphA2 Receptor (EphA2-R) exacerbates injury and remodeling. To determine whether or not ephrinA1-Fc would be of therapeutic value in the hyperglycemic infarcted heart, it is critical to evaluate how ephrinA1/EphA signaling changes in the hyperglycemic myocardium in response to MI. Methods Streptozotocin (STZ)-induced hyperglycemia in wild type (WT) and EphA2-receptor mutant (EphA2-R-M) mice was initiated by an intraperitoneal injection of STZ (150 mg/kg) 10 days before surgery. MI was induced by permanent ligation of the left anterior descending coronary artery and analyses were performed at 4 days post-MI. ANOVAs with Student-Newman Keuls multiple comparison post-hoc analysis illustrated which groups were significantly different, with significance of at least p < 0.05. Results Both WT and EphA2-R-M mice responded adversely to STZ, but only hyperglycemic EphA2-R-M mice had lower ejection fraction (EF) and fractional shortening (FS). At 4 days post-MI, we observed greater post-MI mortality in EphA2-R-M mice compared with WT and this was greater still in the EphA2-R-M hyperglycemic mice. Although infarct size was greater in hyperglycemic WT mice vs normoglycemic mice, there was no difference between hyperglycemic EphA2-R-M mice and normoglycemic EphA2-R-M mice. The hypertrophic response that normally occurs in viable myocardium remote to the infarct was noticeably absent in epicardial cardiomyocytes and cardiac dysfunction worsened in hyperglycemic EphA2-R-M hearts post-MI. The characteristic interstitial fibrotic response in the compensating myocardium remote to the infarct also did not occur in hyperglycemic EphA2-R-M mouse hearts to the same extent as that observed in the hyperglycemic WT mouse hearts. Differences in neutrophil and pan-leukocyte infiltration and serum cytokines implicate EphA2-R in modulation of injury and the differences in ephrinA1 and EphA6-R expression in governing this are discussed. Conclusions We conclude that EphA2-mutant mice are more prone to hyperglycemia-induced increased injury, decreased survival, and worsened LV remodeling due to impaired wound healing

A Forward Genetic Screen in Mice Identifies Mutants with Abnormal Cortical Patterning

Formation of a 6-layered cortical plate and axon tract patterning are key features of cerebral cortex development. Abnormalities of these processes may be the underlying cause for a range of functional disabilities seen in human neurodevelopmental disorders. To identify mouse mutants with defects in cortical lamination or corticofugal axon guidance, N-ethyl-N-nitrosourea (ENU) mutagenesis was performed using mice expressing LacZ reporter genes in layers II/III and V of the cortex (Rgs4-lacZ) or in corticofugal axons (TAG1-tau-lacZ). Four lines with abnormal cortical lamination have been identified. One of these was a splice site mutation in reelin (Reln) that results in a premature stop codon and the truncation of the C-terminal region (CTR) domain of reelin. Interestingly, this novel allele of Reln did not display cerebellar malformation or ataxia, and this is the first report of a Reln mutant without a cerebellar defect. Four lines with abnormal cortical axon development were also identified, one of which was found by whole-genome resequencing to carry a mutation in Lrp2. These findings demonstrated that the application of ENU mutagenesis to mice carrying transgenic reporters marking cortical anatomy is a sensitive and specific method to identify mutations that disrupt patterning of the developing brain

Coexpression of vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and calretinin in rat entorhinal cortex

We studied the distribution and coexpression of vesicular glutamate transporters (VGluT1, VGluT2), glutamic acid decarboxylase (GAD) and calretinin (CR, calcium-binding protein) in rat entorhinal cortex, using immunofluorescence staining and multichannel confocal laser scanning microscopy. Images were computer processed and subjected to automated 3D object recognition, colocalization analysis and 3D reconstruction. Since the VGluTs (in contrast to CR and GAD) occurred in fibers and axon terminals only, we focused our attention on these neuronal processes. An intense, punctate VGluT1-staining occurred everywhere in the entorhinal cortex. Our computer program resolved these punctae as small 3D objects. Also VGluT2 showed a punctate immunostaining pattern, yet with half the number of 3D objects per tissue volume compared with VGluT1, and with statistically significantly larger 3D objects. Both VGluTs were distributed homogeneously across cortical layers, with in MEA VGluT1 slightly more densely distributed than in LEA. The distribution pattern and the size distribution of GAD 3D objects resembled that of VGluT2. CR-immunopositive fibers were abundant in all cortical layers. In double-stained sections we noted ample colocalization of CR and VGluT2, whereas coexpression of CR and VGluT1 was nearly absent. Also in triple-staining experiments (VGluT2, GAD and CR combined) we noted coexpression of VGluT2 and CR and, in addition, frequent coexpression of GAD and CR. Modest colocalization occurred of VGluT2 and GAD, and incidental colocalization of all three markers. We conclude that the CR-containing axon terminals in the entorhinal cortex belong to at least two subpopulations of CR-neurons: a glutamatergic excitatory and a GABAergic inhibitory

Non-human immunodeficiency virus-related Kaposi’s sarcoma of the oropharynx: a case report and review of the literature

INTRODUCTION: Kaposi’s sarcoma is a malignant, slowly progressing, mesenchymal neoplasm characterized by a proliferation of connective tissue and capillaries. Clinical presentation is usually as nodules and red-purple plaques. This case report not only represents an uncommon presentation of Kaposi’s sarcoma in a non-immunocompromised patient, but also supports the role of viral infection in the pathogenesis of this disease. It provides some interesting information about this rare disease, particularly in patients who are human immunodeficiency virus negative. CASE PRESENTATION: A 48-year-old Caucasian man presented with a sensation of a foreign body in his throat, accompanied by stomatolalia. Maxillofacial and neck magnetic resonance imaging confirmed the presence of a voluminous solid mass at the base of his tongue with oropharyngeal space reduction. Histological analysis indicated that the lesion was compatible with ulcerated Kaposi’s sarcoma of the oropharynx. Results of serological tests for human immunodeficiency virus infection were negative as was the result of the human herpesvirus-8 test, but the cytomegalovirus test result was positive. CONCLUSIONS: This case is unusual because the patient had only oropharyngeal localization of disease, without evidence of immunosuppression or the typical background or risk factors suggesting the classic or endemic form of Kaposi’s sarcoma. Isolated cases of Kaposi’s sarcoma with oropharyngeal manifestations not associated with human immunodeficiency virus infection are rare, and only 15 cases have been reported to date. At present, its localization, microscopic and histological characteristics, and patterns of progression are the main tools used for differential diagnosis of Kaposi’s sarcoma from other vascular neoplasms