Publications by authors named "Jeffrey Becker"

99 Publications

A Paradigm for Peptide Hormone-GPCR Analyses.

Molecules 2020 Sep 18;25(18). Epub 2020 Sep 18.

Department of Microbiology, University of Tennessee, 610 Ken and Blaire Mossman Building, 1311 Cumberland Avenue, Knoxville, TN 37996, USA.

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.
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http://dx.doi.org/10.3390/molecules25184272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570734PMC
September 2020

Endothelin-1-Mediated Drug Resistance in -Mutant Non-Small Cell Lung Carcinoma.

Cancer Res 2020 10 3;80(19):4224-4232. Epub 2020 Aug 3.

Department of Surgery, Division of Cardiothoracic Surgery, University of Illinois at Chicago, Chicago, Illinois.

Progression on therapy in non-small cell lung carcinoma (NSCLC) is often evaluated radiographically, however, image-based evaluation of said therapies may not distinguish disease progression due to intrinsic tumor drug resistance or inefficient tumor penetration of the drugs. Here we report that the inhibition of mutated promotes the secretion of a potent vasoconstrictor, endothelin-1 (EDN1), which continues to increase as the cells become resistant with a mesenchymal phenotype. As EDN1 and its receptor (EDNR) is linked to cancer progression, EDNR-antagonists have been evaluated in several clinical trials with disappointing results. These trials were based on a hypothesis that the EDN1-EDNR axis activates the MAPK-ERK signaling pathway that is vital to the cancer cell survival; the trials were not designed to evaluate the impact of tumor-derived EDN1 in modifying tumor microenvironment or contributing to drug resistance. Ectopic overexpression of EDN1 in cells with mutated resulted in poor drug delivery and retarded growth but not . Intratumoral injection of recombinant EDN significantly reduced blood flow and subsequent gefitinib accumulation in xenografted -mutant tumors. Furthermore, depletion of EDN1 or the use of endothelin receptor inhibitors bosentan and ambrisentan improved drug penetration into tumors and restored blood flow in tumor-associated vasculature. Correlatively, these results describe a simplistic endogenous yet previously unrealized resistance mechanism inherent to a subset of -mutant NSCLC to attenuate tyrosine kinase inhibitor delivery to the tumors by limiting drug-carrying blood flow and the drug concentration in tumors. SIGNIFICANCE: EDNR antagonists can be repurposed to improve drug delivery in VEGFA-secreting tumors, which normally respond to TKI treatment by secreting EDN1, promoting vasoconstriction, and limiting blood and drug delivery.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541638PMC
October 2020

Transforming Nursing Education Through Interprofessional Collaborative Innovation: A Project Story.

Comput Inform Nurs 2020 Apr;38(4):176-182

Author Affiliations: Department of Nursing, Otterbein University (Drs Haverkamp, Chovan, Ball, Ballard, Batross, Butz, Chavez, Garrett, Hummer, Prusinski, and Shoemaker and Mss Johnson, Justice, Smith, and Zamaripa), Westerville; Otterbein University / OhioHealth Nurse Anesthesia Program (Drs Ballard and Garrett), Columbus; and edgeThingZ (Mr Becker), Westerville, OH.

This project story is about transforming nursing education through interprofessional collaborative innovation to develop and use a complement of technology-based portable simulation devices collectively known as the Healthcare Education Simulation Station. This collection of inexpensive, simulated point-of-care instruments controlled wirelessly by an instructor or simulation operator were developed and field tested by an interdisciplinary team to enhance learning experiences in several configurations, including those using standardized patients and those using static and low-, mid-, and high-fidelity manikins. The core feature of this project story is the collaboration of students and faculty from two unrelated disciplines, nursing and engineering. The story includes a description of the development, field testing, and initial deployment of a simulated pulse oximeter, capnograph, automated sphygmomanometer, cardiac monitor, thermometer, and fetal monitor. Underpinning this project story is Rogers' Diffusion of Innovation theory and how the characteristics of the innovation, the personnel, and the environment worked together to enable this project and the innovation's subsequent diffusion into nursing education. The aspiration to improve learning experiences for students in multiple disciplines was paramount. The desire to acquire high-quality, dynamic educational tools for nursing educators, coupled with an environment that encourages collaboration, led to an innovation that can transform nursing preparation and ultimately improve patient care, while minimizing cost.
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http://dx.doi.org/10.1097/CIN.0000000000000587DOI Listing
April 2020

THE USE OF CITALOPRAM HYDROBROMIDE TO MANAGE AGGRESSION IN A MALE CHIMPANZEE ().

J Zoo Wildl Med 2020 Jan;50(4):1005-1007

10850 Wilshire Boulevard, Los Angeles, CA 90024, USA.

At times severe, and occasionally fatal, aggression plays an intrinsic role in chimpanzee behavior and social dynamics, particularly among male chimpanzees in both managed and free-ranging troops. At the Los Angeles Zoo, one adult male's natural aggressive behavior developed into unmanageable violence during a period of social and emotional instability consequent to the lack of an established alpha male in the colony. The severity and duration of resulting attacks on a subdominant member of the community, despite environmental and behavioral modification, indicated the need for psychopharmaceutical intervention. Prior treatment of this animal with haloperidol and gabapentin had produced undesirable side effects. Administration of citalopram hydrobromide, a selective serotonin reuptake inhibitor, successfully reduced both the intensity and duration of this male chimpanzee's attacks upon a conspecific animal with minimal observable side effects or adverse behavioral changes.
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http://dx.doi.org/10.1638/2018-0147DOI Listing
January 2020

CXCR7 Reactivates ERK Signaling to Promote Resistance to EGFR Kinase Inhibitors in NSCLC.

Cancer Res 2019 09 4;79(17):4439-4452. Epub 2019 Jul 4.

Department of Surgery, Division of Cardiothoracic Surgery, University of Illinois at Chicago, Chicago, Illinois.

Although EGFR mutant-selective tyrosine kinase inhibitors (TKI) are clinically effective, acquired resistance can occur by reactivating ERK. We show using models of acquired EGFR TKI resistance with a mesenchymal phenotype that CXCR7, an atypical G protein-coupled receptor, activates the MAPK-ERK pathway via β-arrestin. Depletion of CXCR7 inhibited the MAPK pathway, significantly attenuated EGFR TKI resistance, and resulted in mesenchymal-to-epithelial transition. CXCR7 overexpression was essential in reactivation of ERK1/2 for the generation of EGFR TKI-resistant persister cells. Many patients with non-small cell lung cancer (NSCLC) harboring an EGFR kinase domain mutation, who progressed on EGFR inhibitors, demonstrated increased CXCR7 expression. These data suggest that CXCR7 inhibition could considerably delay and prevent the emergence of acquired EGFR TKI resistance in EGFR-mutant NSCLC. SIGNIFICANCE: Increased expression of the chemokine receptor CXCR7 constitutes a mechanism of resistance to EGFR TKI in patients with non-small cell lung cancer through reactivation of ERK signaling.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746175PMC
September 2019

UV Laser-Induced, Time-Resolved Transcriptome Responses of .

G3 (Bethesda) 2019 08 8;9(8):2549-2560. Epub 2019 Aug 8.

Department of Microbiology, University of Tennessee, Knoxville, TN 37996 and

We determined the effect on gene transcription of laser-mediated, long-wavelength UV-irradiation of by RNAseq analysis at times T15, T30, and T60 min after recovery in growth medium. Laser-irradiated cells were viable, and the transcriptional response was transient, with over 400 genes differentially expressed at T15 or T30, returning to basal level transcription by T60. Identification of transcripts exhibiting enhanced differential expression that were unique to UV laser-irradiation were identified by imposing a stringent significance cut-off ( < 0.05, log difference >2) then filtering out genes known as environmental stress response (ESR) genes. Using these rigorous criteria, 56 genes were differentially expressed at T15; at T30 differential expression was observed for 57 genes, some of which persisted from T15. Among the highly up-regulated genes were those supporting amino acid metabolic processes sulfur amino acids, methionine, aspartate, cysteine, serine), sulfur regulation (hydrogen sulfite metabolic processes, sulfate assimilation, sulfate reduction), proteasome components, amino acid transporters, and the iron regulon. At T30, the expression profile shifted to expression of transcripts related to catabolic processes (oxidoreductase activity, peptidase activity). Transcripts common to both T15 and T30 suggested an up-regulation of catabolic events, including UV damage response genes, and protein catabolism via proteasome and peptidase activity. Specific genes encoding tRNAs were among the down-regulated genes adding to the suggestion that control of protein biosynthesis was a major response to long-wave UV laser irradiation. These transcriptional responses highlight the remarkable ability of the yeast cell to respond to a UV-induced environmental insult.
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http://dx.doi.org/10.1534/g3.119.400291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686910PMC
August 2019

a-Factor Analogues Containing Alkyne- and Azide-Functionalized Isoprenoids Are Efficiently Enzymatically Processed and Retain Wild-Type Bioactivity.

Bioconjug Chem 2018 02 20;29(2):316-323. Epub 2017 Dec 20.

Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.

Protein prenylation is a post-translational modification that involves the addition of one or two isoprenoid groups to the C-terminus of selected proteins using either farnesyl diphosphate or geranylgeranyl diphosphate. Three crucial enzymatic steps are involved in the processing of prenylated proteins to yield the final mature product. The farnesylated dodecapeptide, a-factor, is particularly useful for studies of protein prenylation because it requires the identical three-step process to generate the same C-terminal farnesylated cysteine methyl ester substructure present in larger farnesylated proteins. Recently, several groups have developed isoprenoid analogs bearing azide and alkyne groups that can be used in metabolic labeling experiments. Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. Herein, we describe the preparation and use of prenylated a-factor analogs, and precursor peptides, to investigate two key questions. First, a-factor analogues containing modified isoprenoids were prepared to evaluate whether the non-natural lipid group interferes with the biological activity of the a-factor. Second, a-factor-derived precursor peptides were synthesized to evaluate whether they can be efficiently processed by the yeast proteases Rce1 and Ste24 as well as the yeast methyltransferase Ste14 to yield mature a-factor analogues. Taken together, the results reported here indicate that metabolic labeling experiments with azide- and alkyne-functionalized isoprenoids can yield prenylated products that are fully processed and biologically functional. Overall, these observations suggest that the isoprenoids studied here that incorporate bio-orthogonal functionality can be used in metabolic labeling experiments without concern that they will induce undesired physiological changes that may complicate data interpretation.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824361PMC
February 2018

Uptake Assay for Radiolabeled Peptides in Yeast.

Bio Protoc 2016 Nov;6(22)

Department of Microbiology, University of Tennessee, Knoxville, USA.

We describe an assay for measuring the uptake of radioactive peptides into the yeast . The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.
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http://dx.doi.org/10.21769/BioProtoc.2026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669370PMC
November 2016

Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry.

J Mol Recognit 2018 02 10;31(2). Epub 2017 Oct 10.

Department of Microbiology, University of Tennessee, Knoxville, TN, USA.

We are developing a rapid, time-resolved method using laser-activated cross-linking to capture protein-peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding-yeast mating pheromone (α-factor) and the decapeptide human gonadotropin-releasing hormone (GnRH). Cross-linking of α-factor, using a biotinylated, photoactivatable p-benzoyl-L-phenylalanine (Bpa)-modified analog, was energy-dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA-peptide complex. The cross-linked complex was trypsinized and then interrogated with nano-LC-MS/MS to identify the peptide cross-links. Cross-linking was greatly facilitated by Bpa in the peptide, but some cross-linking occurred at higher laser powers and high concentrations of a non-Bpa-modified α-factor. This was supported by experiments using GnRH, a peptide with sequence homology to α-factor, which was likewise found to be cross-linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α-factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser-activation to facilitate cross-linking of Bpa-containing molecules to proteins. The rapid cross-linking procedure and high performance of MS/MS to identify cross-links provides a method to interrogate protein-peptide interactions in a living cell in a time-resolved manner.
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http://dx.doi.org/10.1002/jmr.2680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766399PMC
February 2018

GPCR-Gα protein precoupling: Interaction between Ste2p, a yeast GPCR, and Gpa1p, its Gα protein, is formed before ligand binding via the Ste2p C-terminal domain and the Gpa1p N-terminal domain.

Biochim Biophys Acta Biomembr 2017 Dec 27;1859(12):2435-2446. Epub 2017 Sep 27.

Department of Biological Sciences, Middle East Technical University, Universiteler Mah. Dumlupinar Blv. No: 1, Çankaya, Ankara, 06800, Turkey. Electronic address:

G protein coupled receptors bind ligands that initiate intracellular signaling cascades via heterotrimeric G proteins. In this study, involvement of the N-terminal residues of yeast G-alpha (Gpa1p) with the C-terminal residues of a full-length or C-terminally truncated Ste2p were investigated using bioluminescence resonance energy transfer (BRET), a non-radiative energy transfer phenomenon where protein-protein interactions can be quantified between a donor bioluminescent molecule and a suitable acceptor fluorophore. Constitutive and position-dependent BRET signal was observed in the absence of agonist (α-factor). Upon the activation of the receptors with α-factor, no significant change in BRET signal was observed. The location of Ste2p-Gpa1p heterodimer was investigated using confocal fluorescence microscopy and bimolecular fluorescence complementation (BiFC) assay, a technique where two non-fluorescent fragments of a fluorescent protein reassemble in vivo to restore fluorescence property thereby directly reporting a protein-protein interaction. BiFC experiments resulted in a dimerization signal intracellularly during biosynthesis on the endoplasmic reticulum (ER) and on the plasma membrane (PM). The constitutive BRET and BiFC signals observed on ER between Ste2p and Gpa1p in their quiescent and activated states are indicative of pre-coupling between these two proteins. This study is the first to show that the extreme N-terminus of yeast G protein alpha subunit is in close proximity to its receptor. The data suggests a pre-coupled heterodimer prior to receptor activation. The images presented in this study are the first direct in vivo evidence showing the localization of receptor - G protein heterodimers during biosynthesis and before reaching the plasma membrane.
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http://dx.doi.org/10.1016/j.bbamem.2017.09.022DOI Listing
December 2017

Dynamic roles for the N-terminus of the yeast G protein-coupled receptor Ste2p.

Biochim Biophys Acta Biomembr 2017 Oct 25;1859(10):2058-2067. Epub 2017 Jul 25.

Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996, United States. Electronic address:

The Saccharomyces cerevisiae α-factor receptor Ste2p has been used extensively as a model to understand the molecular mechanism of signal transduction by G protein-coupled receptors (GPCRs). Single and double cysteine mutants of Ste2p were created and served as surrogates to detect intramolecular interactions and dimerization of Ste2p using disulfide cross-linking methodology. When a mutation was introduced into the phylogenetically conserved tyrosine residue at position 26 (Y26C) in the N-terminus of Ste2p, dimerization was increased greatly. The amount of dimer formed by this Y26C mutant was greatly reduced by ligand binding even though the ligand binding site is far removed from the N-terminus; the lowering of the dimer formation was consistent with a conformational change in the N-terminus of the receptor upon activation. Dimerization was decreased by double mutations Y26C/V109C or Y26C/T114C indicating that Y26 is in close proximity to V109 and T114 of extracellular loop 1 in native Ste2p. Combined with earlier studies, these results indicate previously unrecognized roles for the N-terminus of Ste2p, and perhaps of GPCRs in general, and reveal a specific N-terminus residue or region, that is involved in GPCR signaling, intrareceptor interactions, and receptor dimerization.
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http://dx.doi.org/10.1016/j.bbamem.2017.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582982PMC
October 2017

Three-dimensional spatiotemporal tracking of fluorine-18 radiolabeled yeast cells via positron emission particle tracking.

PLoS One 2017 6;12(7):e0180503. Epub 2017 Jul 6.

Department of Nuclear Engineering, University of Tennessee-Knoxville, Knoxville, Tennessee, United States of America.

A method for Positron Emission Particle Tracking (PEPT) based on optical feature point identification techniques is demonstrated for use in low activity tracking experiments. A population of yeast cells of approximately 125,000 members is activated to roughly 55 Bq/cell by 18F uptake. An in vitro particle tracking experiment is performed with nearly 20 of these cells after decay to 32 Bq/cell. These cells are successfully identified and tracked simultaneously in this experiment. This work extends the applicability of PEPT as a cell tracking method by allowing a number of cells to be tracked together, and demonstrating tracking for very low activity tracers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180503PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500330PMC
October 2017

Halo Assay for Toxic Peptides and Other Compounds in Microorganisms.

Bio Protoc 2016 Nov;6(22)

Department of Microbiology, University of Tennessee, Knoxville, USA.

We describe an assay for determination of toxicity in involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of any small compound toward another microorganism by replacing with the target microbe and modifying growth conditions accordingly.

Background: Di-/tripeptides are one of the major sources of nitrogen, carbon, and amino acids for all organisms. Synthetic peptides containing a toxic amino acid residue provide an experimental approach to measure peptide transport and/or utilization in . Hydrolysis of internalized peptides by intracellular peptidases or proteases releases the toxic residue leading to an easily detectable zone (halo) of growth arrest on a lawn of cells plated in a Petri plate. For example, upon intracellular hydrolysis the toxic peptide Ala-Eth releases ethionine (Eth), a methionine antagonist which interferes with the incorporation of amino acids into proteins and with the normal methylation of DNA and other methylation pathways, thereby leading to cell death. When spotted onto a lawn of yeast cells, the transported dipeptide Ala-Eth will inhibit growth, and a clear 'halo' will form in the lawn of cells around the region where the Eth-containing toxic peptide is spotted (Figure 1A). The assay described here for determination of peptide toxicity in may be generalized as follows: (1) it may be modified to determine toxicity of any substrate by simply using a putative toxic compound in place of a peptide containing a toxic amino acid, or (2) it may be modified to determine toxicity of a substrate toward any microorganism by replacing in the assay with the target organism. It is a simple, inexpensive and relatively rapid method for determining substrate toxicities as modified for the specific organism and toxic moiety assayed.
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http://dx.doi.org/10.21769/BioProtoc.2025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448412PMC
November 2016

The yeast Ste2p G protein-coupled receptor dimerizes on the cell plasma membrane.

Biochim Biophys Acta Biomembr 2017 May 8;1859(5):698-711. Epub 2017 Jan 8.

Department of Biological Sciences, Middle East Technical University, Universiteler Mah. Dumlupinar Blv. No: 1, 06800 Cankaya, Ankara, Turkey. Electronic address:

Dimerization of G protein-coupled receptors (GPCR) may play an important role in maturation, internalization, signaling and/or pharmacology of these receptors. However, the location where dimerization occurs is still under debate. In our study, variants of Ste2p, a yeast mating pheromone GPCR, were tagged with split EGFP (enhanced green fluorescent protein) fragments inserted between transmembrane domain seven and the C-terminus or appended to the C-terminus. Bimolecular Fluorescence Complementation (BiFC) assay was used to determine where receptor dimerization occurred during protein trafficking by monitoring generation of EGFP fluorescence, which occurred upon GPCR dimerization. Our results suggest that these tagged receptors traffic to the membrane as monomers, undergo dimerization or higher ordered oligomerization predominantly on the plasma membrane, and are internalized as dimers/oligomers. This study is the first to provide direct in vivo visualization of GPCR dimerization/oligomerization, during trafficking to and from the plasma membrane.
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http://dx.doi.org/10.1016/j.bbamem.2017.01.008DOI Listing
May 2017

The N-terminus of the yeast G protein-coupled receptor Ste2p plays critical roles in surface expression, signaling, and negative regulation.

Biochim Biophys Acta 2016 Apr 17;1858(4):715-24. Epub 2015 Dec 17.

Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States. Electronic address:

G protein-coupled receptors (GPCRs) are found in all eukaryotic cells examined to date where they function as membrane-bound proteins that bind a multitude of extracellular ligands to initiate intracellular signal transduction systems controlling cellular physiology. GPCRs have seven heptahelical membrane spanning domains connected by extracellular and intracellular loops with an extracellular N-terminus and an intracellular C-terminus. The N-terminus has been the least studied domain of most GPCRs. The yeast Ste2p protein, the receptor for the thirteen amino acid peptide pheromone α-factor, has been used extensively as a model to study GPCR structure and function. In this study we constructed a number of deletions of the Ste2p N-terminus and uncovered an unexpected function as a negative regulatory domain. We examined the role of the N-terminus in expression, signaling function and ligand-binding properties and found that the residues 11-30 play a critical role in receptor expression on the cell surface. The studies also indicated that residues 2-10 of the N-terminus are involved in negative regulation of signaling as shown by the observation that deletion of these residues enhanced mating and gene induction. Furthermore, our results indicated that the residues 21-30 are essential for optimal signaling. Overall, we propose that the N-terminus of Ste2p plays multiple regulatory roles in controlling receptor function.
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http://dx.doi.org/10.1016/j.bbamem.2015.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779653PMC
April 2016

Intratumoral Heterogeneity in EGFR-Mutant NSCLC Results in Divergent Resistance Mechanisms in Response to EGFR Tyrosine Kinase Inhibition.

Cancer Res 2015 Oct 17;75(20):4372-83. Epub 2015 Aug 17.

Department of Molecular Pharmacology and Therapeutics, Oncology Research Institute, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois.

Non-small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548796PMC
October 2015

Synthesis of Peptides Containing C-Terminal Esters Using Trityl Side-Chain Anchoring: Applications to the Synthesis of C-Terminal Ester Analogs of the Saccharomyces cerevisiae Mating Pheromone a-Factor.

J Org Chem 2015 Nov 24;80(22):11266-74. Epub 2015 Aug 24.

Department of Microbiology, University of Tennessee , Knoxville, Tennessee 37996, United States.

Peptides containing C-terminal esters are an important class of bioactive molecules that includes a-factor, a farnesylated dodecapeptide, involved in the mating of Saccharomyces cerevisiae. Here, results that expand the scope of solid-phase peptide synthetic methodology that uses trityl side-chain anchoring for the preparation of peptides with C-terminal cysteine alkyl esters are described. In this method, Fmoc-protected C-terminal cysteine esters are anchored to trityl chloride resin and extended by standard solid-phase procedures followed by acidolytic cleavage and HPLC purification. Analysis using a Gly-Phe-Cys-OMe model tripeptide revealed minimal epimerization of the C-terminal cysteine residue under basic conditions used for Fmoc deprotection. (1)H NMR analysis of the unfarnesylated a-factor precursor peptide confirmed the absence of epimerization. The side-chain anchoring method was used to produce wild-type a-factor that contains a C-terminal methyl ester along with ethyl-, isopropyl-, and benzyl-ester analogs in good yield. Activity assays using a yeast-mating assay demonstrate that while the ethyl and isopropyl esters manifest near-wild-type activity, the benzyl ester-containing analog is ca. 100-fold less active. This simple method opens the door to the synthesis of a variety of C-terminal ester-modified peptides that should be useful in studies of protein prenylation and other structurally related biological processes.
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http://dx.doi.org/10.1021/acs.joc.5b01376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035043PMC
November 2015

Cross-linking strategies to study peptide ligand-receptor interactions.

Methods Enzymol 2015 20;556:527-47. Epub 2015 Mar 20.

Chemistry Department, College of Staten Island, City University of New York.

Experiments are described that allowed cross-linking of analogs of a 13-amino acid peptide into the binding site of a model G protein-coupled receptor. Syntheses of peptide analogs that were used for photochemical or chemical cross-linking were carried out using solid-phase peptide synthesis. Chemical cross-linking utilized 3,4-dihydroxy-l-phenylalanine-incorporated peptides and subsequent periodate-mediated activation, whereas photochemical cross-linking was mediated by p-benzoyl-l-phenylalanine (Bpa)-labeled peptides and UV-initiated activation. Mass spectrometry was employed to locate the site(s) in the receptor that formed the cross-links to the ligand. We also describe a method called unnatural amino acid replacement that allowed capture of a peptide ligand into the receptor. In this method, the receptor was genetically modified by replacement of a natural amino acid with Bpa. The modified receptor was UV-irradiated to capture the ligand. The approaches described are applicable to other peptide-binding proteins and can reveal the ligand-binding site in atomic detail.
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http://dx.doi.org/10.1016/bs.mie.2014.12.001DOI Listing
January 2016

Structural characterization of triple transmembrane domain containing fragments of a yeast G protein-coupled receptor in an organic : aqueous environment by solution-state NMR spectroscopy.

J Pept Sci 2015 Mar 2;21(3):212-22. Epub 2015 Feb 2.

Department of Chemistry, The College of Staten Island, City University of New York, Staten Island, NY, 10314, USA; Department of Biochemistry, The Graduate Center, City University of New York, New York, NY, 10016, USA.

This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second, and third transmembrane (TM) domains and the contiguous loops of the α-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3, was investigated by solution NMR in trifluoroethanol/water. TM1-TM3 contains helical TM domains at the predicted locations, supported by continuous sets of medium-range NOEs. In addition, a short helix N-terminal to TM1 was detected, as well as a short helical stretch in the first extracellular loop. Two 161-residue polypeptides, [Ste2p(M1-R161), NT-TM1-TM3], that contain the entire N-terminal sequence, one with a single mutation, were directly expressed and isolated from Escherichia coli in yields as high as 30 mg/L. Based on its increased stability, the L11P mutant will be used in future experiments to determine long-range interactions. The study demonstrated that 3-TM domains of a yeast G protein-coupled receptor can be produced in isotopically labeled form suitable for solution NMR studies. The quality of spectra is superior to data recorded in micelles and allows more rapid data analysis. No tertiary contacts have been determined, and if present, they are likely transient. This observation supports earlier studies by us that secondary structure was retained in smaller fragments, both in organic solvents and in detergent micelles, but that stable tertiary contacts may only be present when the protein is imbedded in lipids.
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http://dx.doi.org/10.1002/psc.2750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501492PMC
March 2015

Novobiocin and peptide analogs of α-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p.

Biochim Biophys Acta 2015 Apr 7;1848(4):916-24. Epub 2015 Jan 7.

Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States. Electronic address:

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p-[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.
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http://dx.doi.org/10.1016/j.bbamem.2014.12.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331264PMC
April 2015

Novobiocin and peptide analogs of α-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p.

Biochim Biophys Acta 2015 Apr 7;1848(4):916-24. Epub 2015 Jan 7.

Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States. Electronic address:

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p-[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.
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http://dx.doi.org/10.1016/j.bbamem.2014.12.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331264PMC
April 2015

A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

PLoS Pathog 2014 Oct 2;10(10):e1004407. Epub 2014 Oct 2.

B. Rappaport Faculty of Medicine, Technion - I.I.T. and the Rappaport Institute for Research in the Medical Sciences, Haifa, Israel.

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.
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http://dx.doi.org/10.1371/journal.ppat.1004407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183699PMC
October 2014

Gender abuse, depressive symptoms, and substance use among transgender women: a 3-year prospective study.

Am J Public Health 2014 Nov 11;104(11):2199-206. Epub 2014 Sep 11.

At the time of this research, Walter Bockting was with the Division of Gender, Sexuality and Health, New York State Psychiatric Institute/Columbia University, New York, NY. Larry Nuttbrock, Andrew Rosenblum, Mona Mason, Monica Macri, and Jeffrey Becker were with the National Development and Research Institutes, New York, NY. Sel Hwahng is with Columbia University.

Objectives: We examined the effects of gender abuse (enacted stigma), depressive symptoms, and demographic, economic, and lifestyle factors on substance use among transgender women.

Methods: We conducted a 3-year prospective study (December 2004 to September 2007) of 230 transgender women aged 19 to 59 years from the New York Metropolitan Area. Statistical techniques included generalized estimating equations with logistic and linear regression links.

Results: Six-month prevalence of any substance use at baseline was 76.2%. Across assessment points, gender abuse was associated with alcohol, cannabis, cocaine, or any substance use during the previous 6 months, the number of days these substances were used during the previous month, and the number of substances used. Additional modeling associated changes in gender abuse with changes in substance use across time. Associations of gender abuse and substance use were mediated 55% by depressive symptoms. Positive associations of employment income, sex work, transgender identity, and hormone therapy with substance use were mediated 19% to 42% by gender abuse.

Conclusions: Gender abuse, in conjunction with depressive symptoms, is a pervasive and moderately strong risk factor for substance use among transgender women. Improved substance abuse treatment is sorely needed for this population.
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http://dx.doi.org/10.2105/AJPH.2014.302106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202966PMC
November 2014

Gender abuse and major depression among transgender women: a prospective study of vulnerability and resilience.

Am J Public Health 2014 Nov 12;104(11):2191-8. Epub 2013 Dec 12.

At the time of this research, Larry Nuttbrock, Andrew Rosenblum, Sel Hwahng, Mona Mason, Monica Macri, and Jeffrey Becker were with the National Development and Research Institutes, New York, NY. Walter Bockting was with the Division of Gender, Sexuality and Health, New York State Psychiatric Institute, Columbia University, New York, NY.

Objectives: We examined the social and interpersonal context of gender abuse and its effects on Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition major depression among transgender women.

Methods: We conducted a 3-year prospective study (2004-2007) among 230 transgender women aged 19 to 59 years from the New York City Metropolitan Area. Statistical techniques included generalized estimating equations (logistic regression).

Results: We observed significant associations of psychological and physical gender abuse with major depression during follow-up. New or persistent experiences of both types of abuse were associated with 4- to 7-fold increases in the likelihood of incident major depression. Employment, transgender presentation, sex work, and hormone therapy correlated across time with psychological abuse; the latter 2 variables correlated with physical abuse. The association of psychological abuse with depression was stronger among younger than among older transgender women.

Conclusions: Psychological and physical gender abuse is endemic in this population and may result from occupational success and attempts to affirm gender identity. Both types of abuse have serious mental health consequences in the form of major depression. Older transgender women have apparently developed some degree of resilience to psychological gender abuse.
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http://dx.doi.org/10.2105/AJPH.2013.301545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202964PMC
November 2014

Guided reconstitution of membrane protein fragments.

Biopolymers 2014 Jan;102(1):16-29

Department of Chemistry, The College of Staten Island, City University of New York (CUNY), Staten Island, NY, 10314.

Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of ∼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination.
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http://dx.doi.org/10.1002/bip.22349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246424PMC
January 2014

Large multiple transmembrane domain fragments of a G protein-coupled receptor: biosynthesis, purification, and biophysical studies.

Biopolymers 2012 ;98(5):485-500

Department of Chemistry, College of Staten Island, The City University of New York, Staten Island, NY 10314, USA.

To conduct biophysical analyses on large domains of GPCRs, multimilligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of four transmembrane and five transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G(31) -A(198) ] (4TMN), four C-terminal Ste2p[T(155) -L(340) ] (4TMC), or five C-terminal Ste2p[I(120) -L(340) ] (5TMC) transmembrane segments of Ste2p. 4TMN was expressed as a fusion protein using a modified pMMHa vector in L-arabinose-induced Escherichia coli BL21-AI, and cleaved with cyanogen bromide. 4TMC and 5TMC were obtained by direct expression using a pET21a vector in IPTG-induced E. coli BL21(DE3) cells. 4TMC and 5TMC were biosynthesized on a preparative scale, isolated in multimilligram amounts, characterized by MS and investigated by biophysical methods. CD spectroscopy indicated the expected highly α-helical content for 4TMC and 5TMC in membrane mimetic environments. Tryptophan fluorescence showed that 5TMC integrated into the nonpolar region of 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) micelles. HSQC-TROSY investigations revealed that [(15) N]-labeled 5TMC in 50% trifluoroethanol-d(2) /H(2) O/0.05%-trifluoroacetic acid was stable enough to conduct long multidimensional NMR measurements. The entire Ste2p GPCR was not readily reconstituted from the first two and last five or first three and last four transmembrane domains.
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http://dx.doi.org/10.1002/bip.22122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542537PMC
April 2014

Synthesis of peptides containing C-terminal methyl esters using trityl side-chain anchoring: application to the synthesis of a-factor and a-factor analogs.

Org Lett 2012 Nov 2;14(22):5648-51. Epub 2012 Nov 2.

Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.

A new cysteine anchoring method was developed for the synthesis of peptides containing C-terminal cysteine methyl esters. This method consists of attachment of Fmoc-Cys-OCH(3) to either 2-ClTrt-Cl or Trt-Cl resins (via the side-chain thiol) followed by preparation of the desired peptide using Fmoc-based SPPS. We applied this method to the synthesis of the mating pheromone a-factor and a 5-FAM labeled a-factor analog. The peptides were obtained with high yield and purity and were shown to be bioactive in a growth arrest assay.
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http://dx.doi.org/10.1021/ol302592vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622458PMC
November 2012

Gender abuse, depressive symptoms, and HIV and other sexually transmitted infections among male-to-female transgender persons: a three-year prospective study.

Am J Public Health 2013 Feb 14;103(2):300-7. Epub 2012 Jun 14.

National Development and Research Institutes, New York, NY, USA.

Objectives: We examined gender abuse and depressive symptoms as risk factors for HIV and other sexually transmitted infections (HIV/STI) among male-to-female transgender persons (MTFs).

Methods: We conducted a 3-year prospective study of factors associated with incident HIV, syphilis, hepatitis B, chlamydia, and gonorrhea among 230 MTFs from the New York Metropolitan Area. Statistical techniques included Cox proportional hazards analysis with time varying covariates.

Results: Among younger MTFs (aged 19-30 years), gender abuse predicted depressive symptoms (Center for Epidemiologic Studies Depression score ≥ 20), and gender abuse combined with depressive symptoms predicted both high-risk sexual behavior (unprotected receptive anal intercourse) and incident HIV/STI. These associations were independent of socioeconomic status, ethnicity, sexual orientation, hormone therapy, and sexual reassignment surgery.

Conclusions: Gender abuse is a fundamental distal risk factor for HIV/STI among younger MTFs. Interventions for younger MTFs are needed to reduce the psychological impact of gender abuse and limit the effects of this abuse on high-risk sexual behavior. Age differences in the impact of gender abuse on HIV/STI suggest the efficacy of peer-based interventions in which older MTFs teach their younger counterparts how to cope with this abuse.
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http://dx.doi.org/10.2105/AJPH.2011.300568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558792PMC
February 2013

Neuroprotection supports signal processing in the hippocampus of Syrian hamsters, a facultative hibernator.

Neurosci Lett 2012 Jun 9;520(1):20-5. Epub 2012 May 9.

Dept. of Neurobiology, Physiology & Behavior, University of California, Davis, One Shields Ave, Davis, CA 95616, United States.

Studies on several species of mammalian seasonal hibernators (those hibernating only in winter) show that their neurons are more tolerant to hypoxia than those in non-hibernating species. Such tolerance has not been studied in facultative hibernators [e.g., Syrian hamsters (Mesocricetus auratus)], which can hibernate at any time of year. We tested the hypotheses that, when exposed to hypoxia, hamster hippocampal pyramidal cells more effectively support signal processing than do rat hippocampal neurons and this protection is enhanced in slices from hibernating versus non-hibernating hamsters and as temperature decreases. Population spike amplitudes (PSAs) were recorded from CA1 pyramidal cells. Slices were perfused in oxygenated artificial cerebral spinal fluid (O(2)ACSF) to establish a baseline. Oxygen was then replaced by nitrogen (N(2)ACSF) for 15 min, followed by a 30-min recovery period in O(2)ACSF. Three minutes after slices were returned to O(2)ACSF, PSAs recovered to 62.4 ± 6.8% of baseline in 15 slices from 8 non-hibernating hamsters but only to 22.7 ± 5.6% in 17 slices from 5 rats. Additionally, PSA recovery was greater in slices from hibernating than non-hibernating hamsters and recovery increased as temperature decreased. These significant differences (P ≤ 0.05) suggest Syrian hamsters are a useful model for studying naturally occurring neuroprotective mechanisms.
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http://dx.doi.org/10.1016/j.neulet.2012.05.010DOI Listing
June 2012