119 research outputs found
Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence
Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment
Development of cryogenic correlated light electron microscopy methods to study mechanisms of intracellular trafficking and their relationships to the secretory pathway
The application of cryogenic electron microscopy (cryoâEM) to the study of cellular ultrastructure provides a resolution several orders of magnitude better than light microscopy. Although this approach is increasingly applied in situ, it suffers from limitations in our ability to target imaging to specific intracellular features including the subcellular localization of specific events of interest. Cryogenic correlated light and electron microscopy (cryoâCLEM) helps to overcome this problem by spatially locating areas of interest inside cells using fluorescence from genetically tagged or stained cellular
molecules and allows for the visualization of localized fluorescentlyâtagged proteins down to the level of individual organelles. Here, we attempted to study the secretory pathway in a specialized mammalian cell line, insulinâsecreting INSâ1E cells, expressing geneticallyâencoded fluorophores as a model system to develop a cryoâCLEM methodology. We discovered that there are many bright sources of autofluorescence in frozen cells. Based on our initial observations and the current understanding in the
field, we hypothesized that autofluorescence from endogenous cellular substrates exhibits a broader spectrum of fluorescence than the fluorescence range of our expressed fluorescent proteins. To test this, we developed a quantitative approach to discriminate between autofluorescence and the fluorescent signal from geneticallyâencoded fluorophores by measuring fluorescent intensities across different bandwidths. To validate this new methodology, we visualized multiple fluorophoreâtagged
organelle markers in our experimental cell system. We found that DsRed2âcytochrome c oxidase and chromogranin AâGFP proteins were targeted in INSâ1E cells to mitochondria and secretory granules by cryoâCLEM, consistent with their respective wellâestablished intracellular localizations. Moreover, these fluorescent signals were clearly distinguishable from autofluorescence emanating from endogenous structures including insulin crystals and multilamellar bodies. Overall, our novel cryoâCLEM methods
open the door to the study of cellular phenomena and structures with a new degree of specificity
Metropolis simulations of Met-Enkephalin with solvent-accessible area parameterizations
We investigate the solvent-accessible area method by means of Metropolis
simulations of the brain peptide Met-Enkephalin at 300. For the energy
function ECEPP/2 nine atomic solvation parameter (ASP) sets are studied. The
simulations are compared with one another, with simulations with a distance
dependent electrostatic permittivity , and with vacuum
simulations (). Parallel tempering and the biased Metropolis
techniques RM are employed and their performance is evaluated. The measured
observables include energy and dihedral probability densities (pds), integrated
autocorrelation times, and acceptance rates. Two of the ASP sets turn out to be
unsuitable for these simulations. For all other systems selected configurations
are minimized in search of the global energy minima, which are found for the
vacuum and the system, but for none of the ASP models. Other
observables show a remarkable dependence on the ASPs. In particular, we find
three ASP sets for which the autocorrelations at 300K are considerably
smaller than for vacuum simulations.Comment: 10 pages and 8 figure
Multi-Overlap Simulations for Transitions between Reference Configurations
We introduce a new procedure to construct weight factors, which flatten the
probability density of the overlap with respect to some pre-defined reference
configuration. This allows one to overcome free energy barriers in the overlap
variable. Subsequently, we generalize the approach to deal with the overlaps
with respect to two reference configurations so that transitions between them
are induced. We illustrate our approach by simulations of the brainpeptide
Met-enkephalin with the ECEPP/2 energy function using the global-energy-minimum
and the second lowest-energy states as reference configurations. The free
energy is obtained as functions of the dihedral and the root-mean-square
distances from these two configurations. The latter allows one to identify the
transition state and to estimate its associated free energy barrier.Comment: 12 pages, (RevTeX), 14 figures, Phys. Rev. E, submitte
Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease
In the amyloidogenic pathway associated with Alzheimer disease (AD), the
amyloid precursor protein (APP) is cleaved by beta-secretase to generate
a 99-aa C-terminal fragment (C99) that is then cleaved by c-secretase to
generate the beta-amyloid (Ab) found in senile plaques. In previous
reports, we and others have shown that c-secretase activity is enriched
in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM)
and that ER-mitochondrial connectivity and MAM function are upregulated
in AD. We now show that C99, in addition to its localization in
endosomes, can also be found in MAM, where it is normally processed
rapidly by c-secretase. In cell models of AD, however, the concentration
of unprocessed C99 increases in MAM regions, resulting in elevated
sphingolipid turnover and an altered lipid composition of both MAM and
mitochondrial membranes. In turn, this change in mitochondrial membrane
composition interferes with the proper assembly and activity of
mitochondrial respiratory supercomplexes, thereby likely contributing to
the bioenergetic defects characteristic of AD.We thank Drs. Orian Shirihai and Marc Liesa (UCLA) for assistance with
the Seahorse measurements, Dr. Huaxi Xu (Sanford Burnham Institute) for
the APP-DKO MEFs and Dr. Mark Mattson (NIH) for the PS1 knock-in mice,
Drs. Arancio and Teich for the APP-KO mice tissues used in these
studies, Dr. Hua Yang (Columbia University) for mouse husbandry, and
Drs. Marc Tambini, Ira Tabas, and Serge Przedborski for helpful
comments. This work was supported by the Fundacion Alfonso Martin
Escudero (to M.P.); the Alzheimer's Drug Discovery Foundation, the
Ellison Medical Foundation, the Muscular Dystrophy Association, the U.S.
Department of Defense W911NF-12-1-9159 and W911F-15-1-0169), and the J.
Willard and Alice S. Marriott Foundation (to E.A.S.); the U.S. National
Institutes of Health (P01-HD080642 and P01-HD032062 to E.A.S.; NS071571
and HD071593 to M.F.M.; R01-NS056049 and P50-AG008702 to G.D.P.;
1S10OD016214-01A1 to G.S.P. and F.P.M, and K01-AG045335 to E.A.-G.), the
Lucien Cote Early Investigator Award in Clinical Genetics from the
Parkinson's Disease Foundation (PDF-CEI-1364 and PDF-CEI-1240) to
C.G.-L., and National Defense Science and Engineering Graduate
Fellowship (FA9550-11-C-0028) to R.R.A.S
Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy
Single-particle analysis by electron microscopy is a well established technique
for analyzing the three-dimensional structures of biological macromolecules.
Besides its ability to produce high-resolution structures, it also provides insights
into the dynamic behavior of the structures by elucidating their conformational
variability. Here, the different image-processing methods currently available to
study continuous conformational changes are reviewedThe authors would like to acknowledge support from the
Spanish Ministry of Economy and Competitiveness through
grants BIO2013-44647-R and BIO2016-76400-R (AEI/
FEDER, UE), Comunidad Autonoma de Madrid through
grant S2017/BMD-3817, Instituto de Salud Carlos III through
grants PT13 /0001/0009 and PT17/0009/0010,the European
Union (EU) and Horizon 2020 through West-Life (EINFRA-
2015-1, Proposal 675858), CORBEL (INFRADEV-1-2014-1,
Proposal 654248), ELIXIRâEXCELERATE (INFRADEV-3-
2015, Proposal 676559), iNEXT (INFRAIA-1-2014-2015,
Proposal 653706), EOSCpilot (INFRADEV-04-2016,
Proposal 739563) and the National Institutes of Health (P41
GM 103712) (IB
β-1,3-Glucan-Induced Host Phospholipase D Activation Is Involved in Aspergillus fumigatus Internalization into Type II Human Pneumocyte A549 Cells
The internalization of Aspergillus fumigatus into lung epithelial cells is a process that depends on host cell actin dynamics. The host membrane phosphatidylcholine cleavage driven by phospholipase D (PLD) is closely related to cellular actin dynamics. However, little is known about the impact of PLD on A. fumigatus internalization into lung epithelial cells. Here, we report that once germinated, A. fumigatus conidia were able to stimulate host PLD activity and internalize more efficiently in A549 cells without altering PLD expression. The internalization of A. fumigatus in A549 cells was suppressed by the downregulation of host cell PLD using chemical inhibitors or siRNA interference. The heat-killed swollen conidia, but not the resting conidia, were able to activate host PLD. Further, β-1,3-glucan, the core component of the conidial cell wall, stimulated host PLD activity. This PLD activation and conidia internalization were inhibited by anti-dectin-1 antibody. Indeed, dectin-1, a β-1,3-glucan receptor, was expressed in A549 cells, and its expression profile was not altered by conidial stimulation. Finally, host cell PLD1 and PLD2 accompanied A. fumigatus conidia during internalization. Our data indicate that host cell PLD activity induced by β-1,3-glucan on the surface of germinated conidia is important for the efficient internalization of A. fumigatus into A549 lung epithelial cells
Association of the Type 2 Diabetes Mellitus Susceptibility Gene, TCF7L2, with Schizophrenia in an Arab-Israeli Family Sample
Many reports in different populations have demonstrated linkage of the 10q24âq26 region to schizophrenia, thus encouraging further analysis of this locus for detection of specific schizophrenia genes. Our group previously reported linkage of the 10q24âq26 region to schizophrenia in a unique, homogeneous sample of Arab-Israeli families with multiple schizophrenia-affected individuals, under a dominant model of inheritance. To further explore this candidate region and identify specific susceptibility variants within it, we performed re-analysis of the 10q24-26 genotype data, taken from our previous genome-wide association study (GWAS) (Alkelai et al, 2011). We analyzed 2089 SNPs in an extended sample of 57 Arab Israeli families (189 genotyped individuals), under the dominant model of inheritance, which best fits this locus according to previously performed MOD score analysis. We found significant association with schizophrenia of the TCF7L2 gene intronic SNP, rs12573128, (pâ=â7.01Ă10â6) and of the nearby intergenic SNP, rs1033772, (pâ=â6.59Ă10â6) which is positioned between TCF7L2 and HABP2. TCF7L2 is one of the best confirmed susceptibility genes for type 2 diabetes (T2D) among different ethnic groups, has a role in pancreatic beta cell function and may contribute to the comorbidity of schizophrenia and T2D. These preliminary results independently support previous findings regarding a possible role of TCF7L2 in susceptibility to schizophrenia, and strengthen the importance of integrating linkage analysis models of inheritance while performing association analyses in regions of interest. Further validation studies in additional populations are required
Phospholipase D signaling: orchestration by PIP2 and small GTPases
Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions
Genetic regulation of Nrnx1 expression: an integrative cross-species analysis of schizophrenia candidate genes
Neurexin 1 (NRXN1) is a large presynaptic transmembrane protein that has complex and variable patterns of expression in the brain. Sequence variants in NRXN1 are associated with differences in cognition, and with schizophrenia and autism. The murine Nrxn1 gene is also highly polymorphic and is associated with significant variation in expression that is under strong genetic control. Here, we use co-expression analysis, high coverage genomic sequence, and expression quantitative trait locus (eQTL) mapping to study the regulation of this gene in the brain. We profiled a family of 72 isogenic progeny strains of a cross between C57BL/6J and DBA/2J (the BXD family) using exon arrays and massively parallel RNA sequencing. Expression of most Nrxn1 exons have high genetic correlation (r>0.6) because of the segregation of a common trans eQTL on chromosome (Chr) 8 and a common cis eQTL on Chr 17. These two loci are also linked to murine phenotypes relevant to schizophrenia and to a novel human schizophrenia candidate gene with high neuronal expression (Pleckstrin and Sec7 domain containing 3). In both human and mice, NRXN1 is co-expressed with numerous synaptic and cell signaling genes, and known schizophrenia candidates. Cross-species co-expression and protein interaction network analyses identified glycogen synthase kinase 3 beta (GSK3B) as one of the most consistent and conserved covariates of NRXN1. By using the Molecular Genetics of Schizophrenia data set, we were able to test and confirm that markers in NRXN1 and GSK3B have epistatic interactions in human populations that can jointly modulate risk of schizophrenia
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