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

HIF- and Non-HIF-Regulated Hypoxic Responses Require the Estrogen-Related Receptor in Drosophila melanogaster

Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. Here, we investigate how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs

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

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

Soil Moisture and Fungi Affect Seed Survival in California Grassland Annual Plants

Survival of seeds in the seed bank is important for the population dynamics of many plant species, yet the environmental factors that control seed survival at a landscape level remain poorly understood. These factors may include soil moisture, vegetation cover, soil type, and soil pathogens. Because many soil fungi respond to moisture and host species, fungi may mediate environmental drivers of seed survival. Here, I measure patterns of seed survival in California annual grassland plants across 15 species in three experiments. First, I surveyed seed survival for eight species at 18 grasslands and coastal sage scrub sites ranging across coastal and inland Santa Barbara County, California. Species differed in seed survival, and soil moisture and geographic location had the strongest influence on survival. Grasslands had higher survival than coastal sage scrub sites for some species. Second, I used a fungicide addition and exotic grass thatch removal experiment in the field to tease apart the relative impact of fungi, thatch, and their interaction in an invaded grassland. Seed survival was lower in the winter (wet season) than in the summer (dry season), but fungicide improved winter survival. Seed survival varied between species but did not depend on thatch. Third, I manipulated water and fungicide in the laboratory to directly examine the relationship between water, fungi, and survival. Seed survival declined from dry to single watered to continuously watered treatments. Fungicide slightly improved seed survival when seeds were watered once but not continually. Together, these experiments demonstrate an important role of soil moisture, potentially mediated by fungal pathogens, in driving seed survival

Genome-wide regulation of innate immunity by juvenile hormone and 20-hydroxyecdysone in the Bombyx fat body

<p>Abstract</p> <p>Background</p> <p>Insect innate immunity can be affected by juvenile hormone (JH) and 20-hydroxyecdysone (20E), but how innate immunity is developmentally regulated by these two hormones in insects has not yet been elucidated. In the silkworm, <it>Bombyx mori</it>, JH and 20E levels are high during the final larval molt (4 M) but absent during the feeding stage of 5^thinstar (5 F), while JH level is low and 20E level is high during the prepupal stage (PP). Fat body produces humoral response molecules and hence is considered as the major organ involved in innate immunity.</p> <p>Results</p> <p>A genome-wide microarray analysis of <it>Bombyx </it>fat body isolated from 4 M, 5 F and PP uncovered a large number of differentially-expressed genes. Most notably, 6 antimicrobial peptide (AMP) genes were up-regulated at 4 M versus PP suggesting that <it>Bombyx </it>innate immunity is developmentally regulated by the two hormones. First, JH treatment dramatically increased AMP mRNA levels and activities. Furthermore, 20E treatment exhibited inhibitory effects on AMP mRNA levels and activities, and RNA interference of the 20E receptor <it>EcR</it>-<it>USP </it>had the opposite effects to 20E treatment.</p> <p>Conclusion</p> <p>Taken together, we demonstrate that JH acts as an immune-activator while 20E inhibits innate immunity in the fat body during <it>Bombyx </it>postembryonic development.</p

A Rapid and Highly Sensitive Method of Non Radioactive Colorimetric In Situ Hybridization for the Detection of mRNA on Tissue Sections

Background: Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels. Methodology/Principal Findings: A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50–60 % shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFAfixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method. Conclusions/Significance: Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highl

Comprehensive in vivo Mapping of the Human Basal Ganglia and Thalamic Connectome in Individuals Using 7T MRI

Basal ganglia circuits are affected in neurological disorders such as Parkinson's disease (PD), essential tremor, dystonia and Tourette syndrome. Understanding the structural and functional connectivity of these circuits is critical for elucidating the mechanisms of the movement and neuropsychiatric disorders, and is vital for developing new therapeutic strategies such as deep brain stimulation (DBS). Knowledge about the connectivity of the human basal ganglia and thalamus has rapidly evolved over recent years through non-invasive imaging techniques, but has remained incomplete because of insufficient resolution and sensitivity of these techniques. Here, we present an imaging and computational protocol designed to generate a comprehensive in vivo and subject-specific, three-dimensional model of the structure and connections of the human basal ganglia. High-resolution structural and functional magnetic resonance images were acquired with a 7-Tesla magnet. Capitalizing on the enhanced signal-to-noise ratio (SNR) and enriched contrast obtained at high-field MRI, detailed structural and connectivity representations of the human basal ganglia and thalamus were achieved. This unique combination of multiple imaging modalities enabled the in-vivo visualization of the individual human basal ganglia and thalamic nuclei, the reconstruction of seven white-matter pathways and their connectivity probability that, to date, have only been reported in animal studies, histologically, or group-averaged MRI population studies. Also described are subject-specific parcellations of the basal ganglia and thalamus into sub-territories based on their distinct connectivity patterns. These anatomical connectivity findings are supported by functional connectivity data derived from resting-state functional MRI (R-fMRI). This work demonstrates new capabilities for studying basal ganglia circuitry, and opens new avenues of investigation into the movement and neuropsychiatric disorders, in individual human subjects