Browsing by Subject "Peptide Fragments"

Now showing 1 - 18 of 18

Open Access
A flexible statistical model for alignment of label-free proteomics data--incorporating ion mobility and product ion information.
(BMC Bioinformatics, 2013-12-16) Benjamin, Ashlee M; Thompson, J Will; Soderblom, Erik J; Geromanos, Scott J; Henao, Ricardo; Kraus, Virginia B; Moseley, M Arthur; Lucas, Joseph E
BACKGROUND: The goal of many proteomics experiments is to determine the abundance of proteins in biological samples, and the variation thereof in various physiological conditions. High-throughput quantitative proteomics, specifically label-free LC-MS/MS, allows rapid measurement of thousands of proteins, enabling large-scale studies of various biological systems. Prior to analyzing these information-rich datasets, raw data must undergo several computational processing steps. We present a method to address one of the essential steps in proteomics data processing--the matching of peptide measurements across samples. RESULTS: We describe a novel method for label-free proteomics data alignment with the ability to incorporate previously unused aspects of the data, particularly ion mobility drift times and product ion information. We compare the results of our alignment method to PEPPeR and OpenMS, and compare alignment accuracy achieved by different versions of our method utilizing various data characteristics. Our method results in increased match recall rates and similar or improved mismatch rates compared to PEPPeR and OpenMS feature-based alignment. We also show that the inclusion of drift time and product ion information results in higher recall rates and more confident matches, without increases in error rates. CONCLUSIONS: Based on the results presented here, we argue that the incorporation of ion mobility drift time and product ion information are worthy pursuits. Alignment methods should be flexible enough to utilize all available data, particularly with recent advancements in experimental separation methods.
Open Access
A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.
(Neuron, 2015-11-04) Gu, Bin; Huang, Yang Zhong; He, Xiao-Ping; Joshi, Rasesh B; Jang, Wonjo; McNamara, James O
The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.
Open Access
Characterization of the ubiquitin-modified proteome regulated by transient forebrain ischemia.
(Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2014-03) Iwabuchi, Masahiro; Sheng, Huaxin; Thompson, J Will; Wang, Liangli; Dubois, Laura G; Gooden, David; Moseley, Marthur; Paschen, Wulf; Yang, Wei
Ubiquitylation is a posttranslational protein modification that modulates various cellular processes of key significance, including protein degradation and DNA damage repair. In animals subjected to transient cerebral ischemia, ubiquitin-conjugated proteins accumulate in Triton-insoluble aggregates. Although this process is widely considered to modulate the fate of postischemic neurons, few attempts have been made to characterize the ubiquitin-modified proteome in these aggregates. We performed proteomics analyses to identify ubiquitylated proteins in postischemic aggregates. Mice were subjected to 10 minutes of forebrain ischemia and 4 hours of reperfusion. The hippocampi were dissected, aggregates were isolated, and trypsin-digested after spiking with GG-BSA as internal standard. K-ɛ-GG-containing peptides were immunoprecipitated and analyzed by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. We identified 1,664 peptides to 520 proteins containing at least one K-ɛ-GG. Sixty-six proteins were highly ubiquitylated, with 10 or more K-ɛ-GG peptides. Based on selection criteria of greater than fivefold increase and P<0.001, 763 peptides to 272 proteins were highly enriched in postischemic aggregates. These included proteins involved in important neuronal functions and signaling pathways that are impaired after ischemia. Results of this study could serve as an important platform to uncover the mechanisms linking insoluble ubiquitin aggregates to the functions of postischemic neurons.
Open Access
Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.
(Proc Natl Acad Sci U S A, 1994-12-20) Koch, WJ; Hawes, BE; Allen, LF; Lefkowitz, RJ
Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.
Open Access
Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin.
(PLoS One, 2009) Xu, J; Bae, E; Zhang, Q; Annis, DS; Erickson, HP; Mosher, DF
BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. CONCLUSIONS: These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.
Open Access
Effects of an antisense oligonucleotide inhibitor of C-reactive protein synthesis on the endotoxin challenge response in healthy human male volunteers.
(J Am Heart Assoc, 2014-07-10) Noveck, Robert; Stroes, Erik SG; Flaim, JoAnn D; Baker, Brenda F; Hughes, Steve; Graham, Mark J; Crooke, Rosanne M; Ridker, Paul M
BACKGROUND: C-reactive protein (CRP) binds to damaged cells, activates the classical complement pathway, is elevated in multiple inflammatory conditions, and provides prognostic information on risk of future atherosclerotic events. It is controversial, however, as to whether inhibiting CRP synthesis would have any direct anti-inflammatory effects in humans. METHODS AND RESULTS: A placebo-controlled study was used to evaluate the effects of ISIS 329993 (ISIS-CRPR x) on the acute-phase response after endotoxin challenge in 30 evaluable subjects. Healthy adult males were randomly allocated to receive 6 injections over a 22-day period of placebo or active therapy with ISIS 329993 at 400- or 600-mg doses. Eligible subjects were subsequently challenged with a bolus of endotoxin (2 ng/kg). Inflammatory and hematological biomarkers were measured before and serially after the challenge. ISIS-CRPR x was well tolerated with no serious adverse events. Median CRP levels increased more than 50-fold from baseline 24 hours after endotoxin challenge in the placebo group. In contrast, the median increase in CRP levels was attenuated by 37% (400 mg) and 69% (600 mg) in subjects pretreated with ISIS-CRPR x (P<0.05 vs. placebo). All other aspects of the acute inflammatory response were similar between treatment groups. CONCLUSION: Pretreatment of subjects with ISIS-CRPR x selectively reduced the endotoxin-induced increase in CRP levels in a dose-dependent manner, without affecting other components of the acute-phase response. These data demonstrate the specificity of antisense oligonucleotides and provide an investigative tool to further define the role of CRP in human pathological conditions.
Open Access
Effects of nociceptin (13-17) in pain modulation at supraspinal level in mice.
(Neurosci Lett, 2002-10-11) Chen, Li-Xiang; Wang, Zhuan-Zi; Wu, Hua; Fang, Quan; Chen, Yong; Wang, Rui
This work was designed to observe the effects of nociceptin(13-17), one of the main metabolites of nociceptin (also termed orphanin FQ), in pain modulation at supraspinal level in mice. Intracerebroventricular (i.c.v.) administration of nociceptin/orphanin FQ(13-17) (N/OFQ(13-17)) (5, 0.5, 0.05, 0.005 nmol/mouse) dose-dependently induced potent hyperalgesic effects in the 48 degrees C warm-water tail-flick test in mice. I.c.v. pretreatment with N/OFQ(13-17) (5, 0.5, 0.05 nmol/mouse) potentiated the analgesic effects induced by morphine (i.p., 2 mg/kg) and reversed the hyperalgesic effects induced by N/OFQ (i.c.v., 5 nmol/mouse). The hyperalgesic effects induced by N/OFQ(13-17) could not be antagonized by [Nphe((1))]N/OFQ(1-13)NH((2)) or naloxone. These findings suggest that N/OFQ(13-17) may play important roles in pain modulation at supraspinal level in mice and elicits these effects through a novel mechanism independent of the N/OFQ receptor and the mu, delta and kappa opioid receptors.
Open Access
G protein signaling and vein graft intimal hyperplasia: reduction of intimal hyperplasia in vein grafts by a Gbetagamma inhibitor suggests a major role of G protein signaling in lesion development.
(Arterioscler Thromb Vasc Biol, 1998-08) Davies, MG; Fulton, Gregory J; Hagen, Per-Otto Frode; Huynh, Tam; Koch, Walter J; Lefkowitz, Robert J; Svendsen, E
Vein grafting results in the development of intimal hyperplasia with accompanying changes in guanine nucleotide-binding (G) protein expression and function. Several serum mitogens that act through G protein-coupled receptors, such as lysophosphatidic acid, stimulate proliferative pathways that are dependent on the G protein betagamma subunit (Gbetagamma)-mediated activation of p21ras. This study examines the role of Gbetagamma signaling in intimal hyperplasia by targeting a gene encoding a specific Gbetagamma inhibitor in an experimental rabbit vein graft model. This inhibitor, the carboxyl terminus of the beta-adrenergic receptor kinase (betaARK(CT)), contains a Gbetagamma-binding domain. Vein graft intimal hyperplasia was significantly reduced by 37% (P<0.01), and physiological studies demonstrated that the normal alterations in G protein coupling phenotypically seen in this model were blocked by betaARK(CT) treatment. Thus, it appears that Gbetagamma-mediated pathways play a major role in intimal hyperplasia and that targeting inhibitors of Gbetagamma signaling offers novel intraoperative therapeutic modalities to inhibit the development of vein graft intimal hyperplasia and subsequent vein graft failure.
Open Access
GRK2 blockade with βARKct is essential for cardiac β2-adrenergic receptor signaling towards increased contractility.
(Cell communication and signaling : CCS, 2013-08) Salazar, Norma C; Vallejos, Ximena; Siryk, Ashley; Rengo, Giuseppe; Cannavo, Alessandro; Liccardo, Daniela; De Lucia, Claudio; Gao, Erhe; Leosco, Dario; Koch, Walter J; Lymperopoulos, Anastasios
Background
β1- and β2-adrenergic receptors (ARs) play distinct roles in the heart, e.g. β1AR is pro-contractile and pro-apoptotic but β2AR anti-apoptotic and only weakly pro-contractile. G protein coupled receptor kinase (GRK)-2 desensitizes and opposes βAR pro-contractile signaling by phosphorylating the receptor and inducing beta-arrestin (βarr) binding. We posited herein that GRK2 blockade might enhance the pro-contractile signaling of the β2AR subtype in the heart. We tested the effects of cardiac-targeted GRK2 inhibition in vivo exclusively on β2AR signaling under normal conditions and in heart failure (HF).
Results
We crossed β1AR knockout (B1KO) mice with cardiac-specific transgenic mice expressing the βARKct, a known GRK2 inhibitor, and studied the offspring under normal conditions and in post-myocardial infarction (MI). βARKct expression in vivo proved essential for β2AR-dependent contractile function, as β2AR stimulation with isoproterenol fails to increase contractility in either healthy or post-MI B1KO mice and it only does so in the presence of βARKct. The main underlying mechanism for this is blockade of the interaction of phosphodiesterase (PDE) type 4D with the cardiac β2AR, which is normally mediated by the actions of GRK2 and βarrs on the receptor. The molecular "brake" that PDE4D poses on β2AR signaling to contractility stimulation is thus "released". Regarding the other beneficial functions of cardiac β2AR, βARKct increased overall survival of the post-MI B1KO mice progressing to HF, via a decrease in cardiac apoptosis and an increase in wound healing-associated inflammation early (at 24 hrs) post-MI. However, these effects disappear by 4 weeks post-MI, and, in their place, upregulation of the other major GRK in the heart, GRK5, is observed.
Conclusions
GRK2 inhibition in vivo with βARKct is absolutely essential for cardiac β2AR pro-contractile signaling and function. In addition, β2AR anti-apoptotic signaling in post-MI HF is augmented by βARKct, although this effect is short-lived.
Open Access
In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.
(Circulation, 1999-08-10) Akhter, SA; Eckhart, AD; Rockman, HA; Shotwell, K; Lefkowitz, RJ; Koch, WJ
BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.
Unknown
Influence of HLA-C expression level on HIV control.
(Science, 2013-04-05) Apps, Richard; Qi, Ying; Carlson, Jonathan M; Chen, Haoyan; Gao, Xiaojiang; Thomas, Rasmi; Yuki, Yuko; Del Prete, Greg Q; Goulder, Philip; Brumme, Zabrina L; Brumme, Chanson J; John, Mina; Mallal, Simon; Nelson, George; Bosch, Ronald; Heckerman, David; Stein, Judy L; Soderberg, Kelly A; Moody, M Anthony; Denny, Thomas N; Zeng, Xue; Fang, Jingyuan; Moffett, Ashley; Lifson, Jeffrey D; Goedert, James J; Buchbinder, Susan; Kirk, Gregory D; Fellay, Jacques; McLaren, Paul; Deeks, Steven G; Pereyra, Florencia; Walker, Bruce; Michael, Nelson L; Weintrob, Amy; Wolinsky, Steven; Liao, Wilson; Carrington, Mary
A variant upstream of human leukocyte antigen C (HLA-C) shows the most significant genome-wide effect on HIV control in European Americans and is also associated with the level of HLA-C expression. We characterized the differential cell surface expression levels of all common HLA-C allotypes and tested directly for effects of HLA-C expression on outcomes of HIV infection in 5243 individuals. Increasing HLA-C expression was associated with protection against multiple outcomes independently of individual HLA allelic effects in both African and European Americans, regardless of their distinct HLA-C frequencies and linkage relationships with HLA-B and HLA-A. Higher HLA-C expression was correlated with increased likelihood of cytotoxic T lymphocyte responses and frequency of viral escape mutation. In contrast, high HLA-C expression had a deleterious effect in Crohn's disease, suggesting a broader influence of HLA expression levels in human disease.
Unknown
Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor.
(Nature, 2010-11) Sheard, Laura B; Tan, Xu; Mao, Haibin; Withers, John; Ben-Nissan, Gili; Hinds, Thomas R; Kobayashi, Yuichi; Hsu, Fong-Fu; Sharon, Michal; Browse, John; He, Sheng Yang; Rizo, Josep; Howe, Gregg A; Zheng, Ning
Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.
Unknown
Left ventricular dysfunction screening in hypertensive patients with N-terminal pro-B-type natriuretic peptide and electrocardiogram.
(Am J Emerg Med, 2012-01) Limkakeng, Alexander T; Drake, Weiying; Mani, Giselle; Freeman, Debbie; Best, Randall; Newby, L Kristin; Chandra, Abhinav
OBJECTIVE: Early recognition of left ventricular hypertrophy is important because antihypertensive treatment decreases morbidity and mortality. The ideal screening method for left ventricular hypertrophy in hypertensive emergency department (ED) patients has not been identified. Our objective was to determine the diagnostic accuracies of electrocardiogram (ECG) and N-terminal Pro-B-type natriuretic peptide (pro-BNP) for left ventricular hypertrophy individually and in combination in hypertensive ED patients. METHODS: Prospective diagnostic study in an academic urban tertiary care hospital ED with annual census of 65,000 visits. Inclusion criteria are as follows: adult ED patients with systolic blood pressure greater than or equal to 160 mm Hg or diastolic blood pressure greater than or equal to 100 mm Hg on 2 or more measurements taken 60 minutes apart. Exclusion criteria are as follows: patients with heart failure, renal insufficiency/failure, acute myocardial infarction, or without recent or scheduled echocardiograms. All patients received echocardiograms and had pro-BNP levels measured using a RAMP point-of-care device (Response Biomedical, Vancouver, BC, Canada). We calculated diagnostic test characteristics with 95% confidence intervals (CIs). RESULTS: A total of 49 patients were enrolled. The average age was 57.9 years, 26.5% were male, and 63.3% were African American. Thirty-two patients (65%) had left ventricular hypertrophy by echocardiogram. Twenty-one (43%) had ECG evidence of left ventricular hypertrophy. Median pro-BNP level was 268 pg/mL. The combination of the 2 tests provided the greatest specificity (94%; 95% CI, 69%-99.7%) and positive predictive value (94%; 95% CI, (68%-99.7%). CONCLUSIONS: The combination of ECG and pro-BNP is a promising screening algorithm for identification of hypertensive ED patients with left ventricular hypertrophy.
Open Access
Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission.
(J Clin Invest, 2015-07-01) Permar, Sallie R; Fong, Youyi; Vandergrift, Nathan; Fouda, Genevieve G; Gilbert, Peter; Parks, Robert; Jaeger, Frederick H; Pollara, Justin; Martelli, Amanda; Liebl, Brooke E; Lloyd, Krissey; Yates, Nicole L; Overman, R Glenn; Shen, Xiaoying; Whitaker, Kaylan; Chen, Haiyan; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Marshall, Dawn J; Whitesides, John F; Gurley, Thaddeus C; Von Holle, Tarra; Martinez, David R; Cai, Fangping; Kumar, Amit; Xia, Shi-Mao; Lu, Xiaozhi; Louzao, Raul; Wilkes, Samantha; Datta, Saheli; Sarzotti-Kelsoe, Marcella; Liao, Hua-Xin; Ferrari, Guido; Alam, S Munir; Montefiori, David C; Denny, Thomas N; Moody, M Anthony; Tomaras, Georgia D; Gao, Feng; Haynes, Barton F
Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1-transmitting mothers and 165 propensity score-matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1-infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3-specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.
Open Access
Potent and broad neutralizing activity of a single chain antibody fragment against cell-free and cell-associated HIV-1.
(MAbs, 2010-05) Zhang, MY; Borges, AR; Ptak, RG; Wang, Y; Dimitrov, AS; Alam, SM; Wieczorek, L; Bouma, P; Fouts, T; Jiang, S; Polonis, VR; Haynes, BF; Quinnan, GV; Montefiori, DC; Dimitrov, DS
Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A-G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG(1) b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (T-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (PS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. These results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.
Open Access
Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.
(Proc Natl Acad Sci U S A, 1997-03-18) Daaka, Y; Pitcher, JA; Richardson, M; Stoffel, RH; Robishaw, JD; Lefkowitz, RJ
The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.
Open Access
Reliable genotypic tropism tests for the major HIV-1 subtypes.
(Scientific reports, 2015-02-25) Cashin, Kieran; Gray, Lachlan R; Harvey, Katherine L; Perez-Bercoff, Danielle; Lee, Guinevere Q; Sterjovski, Jasminka; Roche, Michael; Demarest, James F; Drummond, Fraser; Harrigan, P Richard; Churchill, Melissa J; Gorry, Paul R
Over the past decade antiretroviral drugs have dramatically improved the prognosis for HIV-1 infected individuals, yet achieving better access to vulnerable populations remains a challenge. The principal obstacle to the CCR5-antagonist, maraviroc, from being more widely used in anti-HIV-1 therapy regimens is that the pre-treatment genotypic "tropism tests" to determine virus susceptibility to maraviroc have been developed primarily for HIV-1 subtype B strains, which account for only 10% of infections worldwide. We therefore developed PhenoSeq, a suite of HIV-1 genotypic tropism assays that are highly sensitive and specific for establishing the tropism of HIV-1 subtypes A, B, C, D and circulating recombinant forms of subtypes AE and AG, which together account for 95% of HIV-1 infections worldwide. The PhenoSeq platform will inform the appropriate use of maraviroc and future CCR5 blocking drugs in regions of the world where non-B HIV-1 predominates, which are burdened the most by the HIV-1 pandemic.
Open Access
Screening of Multiple Biomarkers Associated With Ischemic Stroke in Atrial Fibrillation.
(Journal of the American Heart Association, 2020-12-09) Hijazi, Ziad; Wallentin, Lars; Lindbäck, Johan; Alexander, John H; Connolly, Stuart J; Eikelboom, John W; Ezekowitz, Michael D; Granger, Christopher B; Lopes, Renato D; Pol, Tymon; Yusuf, Salim; Oldgren, Jonas; Siegbahn, Agneta
Background To explore the pathophysiological features of ischemic stroke in patients with atrial fibrillation (AF), we evaluated the association between 268 plasma proteins and subsequent ischemic stroke in 2 large AF cohorts receiving oral anticoagulation. Methods and Results A case-cohort sample of patients with AF from the ARISTOTLE (Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation) trial, including 282 cases with ischemic stroke or systemic embolism and a random sample of 4124 without these events, during 1.9 years of follow-up was used for identification. Validation was provided by a similar case-cohort sample of patients with AF from the RE-LY (Randomized Evaluation of Long-Term Anticoagulation Therapy) trial, including 149 cases with ischemic stroke/systemic embolism and a random sample of 1062 without these events. In plasma obtained before randomization, 268 unique biomarkers were measured with OLINK proximity extension assay panels (CVD II, CVD III, and Inflammation) and conventional immunoassays. The association between biomarkers and outcomes was evaluated by random survival forest and adjusted Cox regression. According to random survival forest or Cox regression analyses, the biomarkers most strongly and consistently associated with ischemic stroke/systemic embolism were matrix metalloproteinase-9, NT-proBNP (N-terminal pro-B-type natriuretic peptide), osteopontin, sortilin, soluble suppression of tumorigenesis 2, and trefoil factor-3. The corresponding hazard ratios (95% CIs) for an interquartile difference were as follows: 1.18 (1.00-1.38), 1.55 (1.28-1.88), 1.28 (1.07-1.53), 1.19 (1.02-1.39), 1.23 (1.05-1.45), and 1.19 (0.97-1.45), respectively. Conclusions In patients with AF, of 268 unique biomarkers, the 6 biomarkers most strongly associated with subsequent ischemic stroke/systemic embolism represent fibrosis/remodeling (matrix metalloproteinase-9 and soluble suppression of tumorigenesis 2), cardiac dysfunction (NT-proBNP), vascular calcification (osteopontin), metabolism (sortilin), and mucosal integrity/ischemia (trefoil factor-3). Registration URL: https://www.clinicaltrials.gov. Unique Identifiers: NCT00412984 and NCT00262600.

Browsing by Subject "Peptide Fragments"

Background

Results

Conclusions