Alcohols increase calmodulin affinity for Ca2+ and decrease target affinity for calmodulin

AbstractIt has been proposed that alcohols and anesthetics selectively inhibit proteins containing easily disrupted motifs, e.g., α-helices. In this study, the calcineurin/calmodulin/Ca2+ enzyme system was used to examine the effects of alcohols on calmodulin, a protein with a predominantly α-helical structure. Calcineurin phosphatase activity and Ca2+ binding were monitored as indicators of calmodulin function. Alcohols inhibited enzyme activity in a concentration-dependent manner, with two-, four- and five-carbon n-alcohols exhibiting similar leftward shifts in the inhibition curves for calmodulin-dependent and -independent activities; the former was slightly more sensitive than the latter. Ca2+ binding was measured by flow dialysis as a direct measure of calmodulin function, whereas, with the addition of a binding domain peptide, measured calmodulin–target interactions. Ethanol increased the affinity of calmodulin for Ca2+ in the presence and absence of the peptide, indicating that ethanol stabilizes the Ca2+ bound form of calmodulin. An increase in Ca2+ affinity was detected in a calmodulin binding assay, but the affinity of calmodulin for calcineurin decreased at saturating Ca2+. These data demonstrate that although specific regions within proteins may be more sensitive to alcohols and anesthetics, the presence of α-helices is unlikely to be a reliable indicator of alcohol or anesthetic potency

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111127/1/pmic8033.pd

Effects of Impurity Content on the Sintering Characteristics of Plasma-Sprayed Zirconia

Yttria-stabilized zirconia powders, containing different levels of SiO2 and Al2O3, have been plasma sprayed onto metallic substrates. The coatings were detached from their substrates and a dilatometer was used to monitor the dimensional changes they exhibited during prolonged heat treatments. It was found that specimens containing higher levels of silica and alumina exhibited higher rates of linear contraction, in both in-plane and through-thickness directions. The in-plane stiffness and the through-thickness thermal conductivity were also measured after different heat treatments and these were found to increase at a greater rate for specimens with higher impurity (silica and alumina) levels. Changes in the pore architecture during heat treatments were studied using Mercury Intrusion Porosimetry (MIP). Fine scale porosity (<_50 nm) was found to be sharply reduced even by relatively short heat treatments. This is correlated with improvements in inter-splat bonding and partial healing of intra-splat microcracks, which are responsible for the observed changes in stiffness and conductivity, as well as the dimensional changes

Genomic sequencing of meningiomas identifies oncogenic SMO and AKT1 mutations

Meningiomas are the most common primary nervous system tumor. The tumor suppressor NF2 is disrupted in approximately half of meningiomas1 but the complete spectrum of genetic changes remains undefined. We performed whole-genome or whole-exome sequencing on 17 meningiomas and focused sequencing on an additional 48 tumors to identify and validate somatic genetic alterations. Most meningiomas exhibited simple genomes, with fewer mutations, rearrangements, and copy-number alterations than reported in other adult tumors. However, several meningiomas harbored more complex patterns of copy-number changes and rearrangements including one tumor with chromothripsis. We confirmed focal NF2 inactivation in 43% of tumors and found alterations in epigenetic modifiers among an additional 8% of tumors. A subset of meningiomas lacking NF2 alterations harbored recurrent oncogenic mutations in AKT1 (E17K) and SMO (W535L) and exhibited immunohistochemical evidence of activation of their pathways. These mutations were present in therapeutically challenging tumors of the skull base and higher grade. These results begin to define the spectrum of genetic alterations in meningiomas and identify potential therapeutic targets

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic β-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic β-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand

Sporadic hemangioblastomas are characterized by cryptic VHL inactivation

Abstract Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas.http://deepblue.lib.umich.edu/bitstream/2027.42/110224/1/40478_2014_Article_167.pd

Identification of Shell Colour Pigments in Marine Snails Clanculus pharaonius and C. margaritarius (Trochoidea; Gastropoda)

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. https://creativecommons.org/publicdomain/zero/1.0/ The attached file is the published version of the article

A Directed Molecular Evolution Approach to Improved Immunogenicity of the HIV-1 Envelope Glycoprotein

A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences

Sporadic hemangioblastomas are characterized by cryptic VHL inactivation

Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas

Filamin A phosphorylation by Akt promotes cell migration in response to arsenic

We had previously reported that trivalent arsenic (As3+), a well-known environmental carcinogen, induces phosphorylation of several putative Akt substrates. In the present report, we characterized one of these substrates by immunoprecipitation and proteomics analysis. The results indicate that a cytoskeleton remodeling protein, filamin A, with a molecular weight around 280 kDa, is phosphorylated by Akt in HEK-293 cells treated with As3+, which was also confirmed in human bronchial epithelial cell line, BEAS-2B cells. Additional biochemical and biological studies revealed that serine 2152 (S2152) of filamin A is phosphorylated by activated Akt in the cells treated with As3+. To further confirm the importance of Akt-dependent filamin A S2152 phosphorylation in As3+-induced cell migration, we over-expressed either wild type filamin A or the mutated filamin A in which the S2152 was substituted with alanine (S2152A). The capability of cell migration was reduced significantly in the cells expressing the mutated filamin A (S2152A). Clinically, we found that increased expression of filamin A predicts poorer overall survival of the lung cancer patients with adenocarcinoma. Thus, these data suggest that Akt dependent filamin A phosphorylation is one of the key events in mediating As3+-induced carcinogenesis. Antagonizing Akt signaling can ameliorate As3+-induced filamin A phosphorylation and cell migration, which may serve as a molecular targeting strategy for malignancies associated with environmental As3+ exposure