43 results on '"Snyder JT"'
Search Results
2. Levels of purine nucleoside phosphorylase (PNP) as a viability marker of nonparenchymal cells in cold preserved livers.
- Author
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Mischinger, HJ, Rao, PN, Todo, S, Snyder, JT, Quehenberger, F, Murase, N, Starzl, TE, Mischinger, HJ, Rao, PN, Todo, S, Snyder, JT, Quehenberger, F, Murase, N, and Starzl, TE
- Published
- 1991
3. Purine nucleoside phosphorylase: A new marker for free oxygen radical injury to the endothelial cell
- Author
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Rao, PN, Walsh, TR, Makowka, L, Rubin, RS, Weber, T, Snyder, JT, Starzl, TE, Rao, PN, Walsh, TR, Makowka, L, Rubin, RS, Weber, T, Snyder, JT, and Starzl, TE
- Abstract
The effect of ischemia and reperfusion on purine nucleoside phosphorylase was studied in an isolated perfused rat liver model. This enzyme is localized primarily in the cytoplasm of the endothelial and Kupffer cells; some activity is associated with the parenchymal cells. Levels of this enzyme accurately predicted the extent of ischemia and reperfusion damage to the microvascular endothelial cell of the liver. Livers from Lewis rats were subjected to 30, 45 and 60 min of warm (37° C) no flow ischemia that was followed by a standard reperfusion period lasting 45 min. Purine nucleoside phosphorylase was measured at the end of the no flow ischemia and reperfusion periods as was superoxide generation (O2‐). Bile production was monitored throughout the no flow ischemia and reperfusion periods. Control perfusions were carried out for 120 min. A significant rise in purine nucleoside phosphorylase levels as compared with controls was observed at the end of ischemia in all the three groups. The highest level, 203.5 ± 29.2 mU/ml, was observed after 60 min of ischemia. After the reperfusion period, levels of purine nucleoside phosphorylase decreased in the 30‐ and 45‐min groups 58.17 ± 9.66 mU/ml and 67.5 ± 17.1 mU/ml, respectively. These levels were equal to control perfusions. In contrast, after 60 min of ischemia, levels of purine nucleoside phosphorylase decreased early in the reperfusion period and then rose to 127.8 ± 14.8 mU/ml by the end of reperfusion (p < 0.0001). Superoxide generation at the beginning of reperfusion was higher than in controls with similar values observed at the end of 30, 45 and 60 min of ischemia. During reperfusion, production of superoxide continued. Bile production was significantly lower at the end of 30 min (0.044 ± 0.026 μl/min/gm), 45 min (0.029 ± 0.0022 μ/min/gm) and 60 min of ischemia (0.022 ± 0.008 μ/min/gm) when compared with bile production by control livers during the corresponding time (0.680 ± 0.195, 0.562 ± 0.133 and 0.480 ± 0.100
- Published
- 1990
4. Inhibition of free radical generation and improved survival by protection of the hepatic microvascular endothelium by targeted erythrocytes in orthotopic rat liver transplantation
- Author
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Rao, PN, Walsh, TR, Makowka, L, Liu, T, Demetris, AJ, Rubin, RS, Snyder, JT, Mischinger, HJ, Starzl, TE, Rao, PN, Walsh, TR, Makowka, L, Liu, T, Demetris, AJ, Rubin, RS, Snyder, JT, Mischinger, HJ, and Starzl, TE
- Abstract
The capacity of specifically targeted erythrocytes to inhibit free radical—mediated injury to the endothelial cell after cold preservation, and improve liver function was studied in two experimental models: An isolated perfused rat liver (IPRL) system and syngeneic orthotopic rat liver transplantation. In the IPRL model, livers were preserved in University of Wisconsin solution for 24 h at 4°C. At the end of the preservation period, livers were flushed with lactated Ringer’s (control), immu- noerythrocytes (IES), or blank intact erythrocytes prior to warm reperfusion for 2 h using an assanguinous Krebs-Henseleit buffer. Production of superoxide (O2-) anion during warm reperfusion in the IES-treated liver was reduced by 65% as compared with controls (P<0.001) and by 74% (P<0.001) when compared with blank erythrocyte—treated livers. Endothelial cell preservation, as assessed by levels of purine nucleoside phos- phorylase (PNP), was much better in the IES-treated group (P<0.001) when compared with untreated livers. Hepatocellular preservation was markedly improved in the IES-treated livers. In the syngeneic liver transplantation model, livers were preserved in UW solution for 24 h at 4°C. Prior to implantation, livers were flushed with 5 ml of cold lactated Ringer’s or immunoerythrocytes. Survival after three weeks was 60% in the IES-treated group and 30% in the untreated group. Survival in the IES-treated group was not significantly different from a control (no preservation) group. IES-treated livers in both models demonstrated better endothelial cell integrity and ultimate liver function. IES treatment therefore appears to protect the hepatic microvascular endothelial cell from reperfusion injury and could prove to be an easy reproducible method of donor organ preparation after cold preservation. © 1990 by Williams & Wilkins.
- Published
- 1990
5. Endoplasmic Reticulum Stress Induced Proliferation Remains Intact in Aging Mouse β-Cells.
- Author
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Snyder JT, Darko C, Sharma RB, and Alonso LC
- Subjects
- Aging metabolism, Animals, Cells, Cultured, Endoplasmic Reticulum metabolism, Female, Insulin-Secreting Cells metabolism, Male, Mice, Mice, Inbred C57BL, Signal Transduction physiology, Aging physiology, Cell Proliferation, Endoplasmic Reticulum Stress physiology, Insulin-Secreting Cells physiology
- Abstract
Aging is associated with loss of proliferation of the insulin-secreting β-cell, a possible contributing factor to the increased prevalence of type 2 diabetes in the elderly. Our group previously discovered that moderate endoplasmic reticulum (ER) stress occurring during glucose exposure increases the adaptive β-cell proliferation response. Specifically, the ATF6α arm of the tripartite Unfolded Protein Response (UPR) promotes β-cell replication in glucose excess conditions. We hypothesized that β-cells from older mice have reduced proliferation due to aberrant UPR signaling or an impaired proliferative response to ER stress or ATF6α activation. To investigate, young and old mouse islet cells were exposed to high glucose with low-dose thapsigargin or activation of overexpressed ATF6α, and β-cell proliferation was quantified by BrdU incorporation. UPR pathway activation was compared by qPCR of target genes and semi-quantitative Xbp1 splicing assay. Intriguingly, although old β-cells had reduced proliferation in high glucose compared to young β-cells, UPR activation and induction of proliferation in response to low-dose thapsigargin or ATF6α activation in high glucose were largely similar between young and old. These results suggest that loss of UPR-led adaptive proliferation does not explain the reduced cell cycle entry in old β-cells, and raise the exciting possibility that future therapies that engage adaptive UPR could increase β-cell number through proliferation even in older individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Snyder, Darko, Sharma and Alonso.)
- Published
- 2021
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6. Atf6α impacts cell number by influencing survival, death and proliferation.
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Sharma RB, Snyder JT, and Alonso LC
- Subjects
- Animals, Cell Count, Cell Survival, Humans, Activating Transcription Factor 6 metabolism, Cell Death, Cell Proliferation
- Abstract
Background: A growing body of literature suggests the cell-intrinsic activity of Atf6α during ER stress responses has implications for tissue cell number during growth and development, as well as in adult biology and tumorigenesis [1]. This concept is important, linking the cellular processes of secretory protein synthesis and endoplasmic reticulum stress response with functional tissue capacity and organ size. However, the field contains conflicting observations, especially notable in secretory cell types like the pancreatic beta cell., Scope of Review: Here we summarize current knowledge of the basic biology of Atf6α, along with the pleiotropic roles Atf6α plays in cell life and death decisions and possible explanations for conflicting observations. We include studies investigating the roles of Atf6α in cell survival, death and proliferation using well-controlled methodology and specific validated outcome measures, with a focus on endocrine and metabolic tissues when information was available., Major Conclusions: The net outcome of Atf6α on cell survival and cell death depends on cell type and growth conditions, the presence and degree of ER stress, and the duration and intensity of Atf6α activation. It is unquestioned that Atf6α activity influences the cell fate decision between survival and death, although opposite directions of this outcome are reported in different contexts. Atf6α can also trigger cell cycle activity to expand tissue cell number through proliferation. Much work remains to be done to clarify the many gaps in understanding in this important emerging field., (Copyright © 2019. Published by Elsevier GmbH.)
- Published
- 2019
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7. Citizen science observations reveal rapid, multi-decadal ecosystem changes in eastern Long Island Sound.
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Snyder JT, Whitney MM, Dam HG, Jacobs MW, and Baumann H
- Subjects
- Animals, Biodiversity, Brachyura, Citizen Science, Fishes, Flounder, Nephropidae, New England, Temperature, Climate Change statistics & numerical data, Ecological Parameter Monitoring, Estuaries
- Abstract
Long-term environmental records are among the most valuable assets for understanding the trajectory and consequences of climate change. Here we report on a newly recovered time-series from Project Oceanology, a non-profit ocean science organization serving New England schools (USA) since 1972. As part of its educational mission, Project Oceanology has routinely and consistently recorded water temperature, pH, and oxygen as well as invertebrate and fish abundance in nearshore waters of the Thames River estuary in eastern Long Island Sound (LIS). We digitized these long-term records to test for decadal trends in abiotic and biotic variables including shifts in species abundance, richness, and diversity. Consistent with previous studies, the data revealed an above-average warming rate of eastern LIS waters over the past four decades (+0.45 °C decade
-1 ), a non-linear acidification trend twice the global average (-0.04 pH units decade-1 ), and a notable decline in whole water-column dissolved oxygen concentrations (-0.29 mg L-1 decade-1 ). Trawl catches between 1997 and 2016 suggested a significant decrease in overall species diversity and richness, declines in cold-water adapted species such as American lobster (Homarus americanus), rock crab (Cancer irroratus), and winter flounder (Pseudopleuronectes americanus), but concurrent increases in the warm-water decapod Libinia emarginata (spider crab). Our study confirmed that Long Island Sound is a rapidly changing urban estuary, while demonstrating the value of long-term observations made by citizen-scientists, educators, and other stakeholders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2019
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8. Metabolism of an Oxime-Linked Antibody Drug Conjugate, AGS62P1, and Characterization of Its Identified Metabolite.
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Snyder JT, Malinao MC, Dugal-Tessier J, Atkinson JE, Anand BS, Okada A, and Mendelsohn BA
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- Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents chemistry, Cell Line, Tumor, ERG1 Potassium Channel metabolism, Humans, Immunoconjugates administration & dosage, Immunoconjugates chemistry, Leukemia, Myeloid, Acute pathology, Macaca fascicularis, Mice, Mice, SCID, Oximes chemistry, Rats, Xenograft Model Antitumor Assays, Antineoplastic Agents metabolism, Immunoconjugates metabolism, Leukemia, Myeloid, Acute drug therapy, fms-Like Tyrosine Kinase 3 antagonists & inhibitors
- Abstract
AGS62P1 is an antibody drug conjugate (ADC) composed of a human IgG1κ monoclonal antibody against FLT3 (FMS-like tyrosine kinase 3) with a p-acetyl phenylalanine (pAF) residue inserted at position 124 of each heavy chain linked to the proprietary microtubule disrupting agent AGL-0182-30 via an alkoxyamine linker that forms an oxime upon conjugation to the antibody. AGS62P1 is currently in Phase I human clinical trials for acute myelogenous leukemia (AML). The identified primary metabolite of an oxime-linked ADC is presented for the first time. AGS62P1 metabolism was assessed in xenograft tumor-bearing mice and rats treated with the ADC using liquid chromatography and mass spectrometry-based methods described herein. In this study, we identified the metabolite of AGS62P1 as pAF-AGL-0185-30, which contains a fragment resulting from the catabolism of the antibody component of the ADC and hydrolysis of the terminal amide portion of the linker-payload. We demonstrated that the metabolite of AGS62P1 is tolerated in rats above 1.5 mg/kg and above 0.334 mg/kg in cynomolgus monkeys when given as a single dose. Furthermore, we established in vitro that pAF-AGL-0185-30 does not significantly inhibit hERG or cytochrome P450 family enzymes (CYPs).
- Published
- 2018
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9. Investigation of Hydrophilic Auristatin Derivatives for Use in Antibody Drug Conjugates.
- Author
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Mendelsohn BA, Barnscher SD, Snyder JT, An Z, Dodd JM, and Dugal-Tessier J
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- Amides chemistry, Animals, Cell Line, Tumor, Humans, Immunoconjugates pharmacology, Mice, Protein Multimerization drug effects, Protein Structure, Quaternary, Tubulin chemistry, Xenograft Model Antitumor Assays, Aminobenzoates chemistry, Hydrophobic and Hydrophilic Interactions, Immunoconjugates chemistry, Oligopeptides chemistry
- Abstract
Antibody drug conjugates offer a targeted cancer treatment for the delivery of potent cytotoxic drugs. Derivatives of the natural product dolastatin 10 containing pyridines and other basic amines were examined with the objective of determining if a more hydrophilic auristatin derivative would be potent enough for use as part of an ADC. This may be advantageous if a less hydrophobic drug makes a better ADC. A pyridine derivative, monomethyl auristatin PYE, showed the greatest potency when tested in vivo. While only a modest tumor growth inhibition was observed when the HCC1954 human breast cancer xenografts were treated with"non-cleavable" linker ADCs, tumor regression was seen when treated with an enzymatically degradable "cleavable" linker ADC when conjugated to trastuzumab. Based on these studies, monomethyl auristatin PYE shows promise for use as an ADC payload.
- Published
- 2017
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10. Development of ASG-15ME, a Novel Antibody-Drug Conjugate Targeting SLITRK6, a New Urothelial Cancer Biomarker.
- Author
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Morrison K, Challita-Eid PM, Raitano A, An Z, Yang P, Abad JD, Liu W, Lortie DR, Snyder JT, Capo L, Verlinsky A, Aviña H, Doñate F, Joseph IB, Pereira DS, Morrison K, and Stover DR
- Subjects
- Animals, Antibodies, Monoclonal administration & dosage, Antibodies, Monoclonal pharmacology, Antibodies, Monoclonal, Humanized, Cell Line, Tumor, Cell Proliferation drug effects, Cell Survival drug effects, Drug Discovery, Gene Expression Regulation, Neoplastic drug effects, Humans, Immunoconjugates chemistry, Immunoconjugates pharmacology, Kidney Neoplasms metabolism, Mice, Oligopeptides administration & dosage, Oligopeptides pharmacology, Up-Regulation, Urothelium metabolism, Urothelium pathology, Xenograft Model Antitumor Assays, Antibodies, Monoclonal chemistry, Immunoconjugates administration & dosage, Kidney Neoplasms drug therapy, Membrane Proteins antagonists & inhibitors, Oligopeptides chemistry
- Abstract
SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody-drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301-10. ©2016 AACR., (©2016 American Association for Cancer Research.)
- Published
- 2016
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11. Human CD14hi monocytes and myeloid dendritic cells provide a cell contact-dependent costimulatory signal for early CD40 ligand expression.
- Author
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Chakrabarty S, Snyder JT, Shen J, Azmi H, Hu PQ, Chen Q, and Ragheb JA
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- Antigen-Presenting Cells, Blotting, Northern, Blotting, Western, CD4-Positive T-Lymphocytes, CD40 Ligand genetics, CD58 Antigens genetics, CD58 Antigens metabolism, Cell Communication, Cells, Cultured, Flow Cytometry, Humans, Intercellular Adhesion Molecule-1 genetics, Intercellular Adhesion Molecule-1 metabolism, Lymphocyte Activation, Monocytes cytology, RNA, Messenger genetics, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, CD40 Antigens metabolism, CD40 Ligand metabolism, Cell Adhesion, Dendritic Cells metabolism, Lipopolysaccharide Receptors metabolism, Monocytes metabolism, Myeloid Cells metabolism
- Abstract
CD40L on CD4(+) T cells plays a vital role in the activation of antigen-presenting cells, thus catalyzing a positive feedback loop for T-cell activation. Despite the pivotal juxtaposition of CD40L between antigen-presenting cells and T-cell activation, only a T-cell receptor stimulus is thought to be required for early CD40L surface expression. We show, for the first time, that CD40L expression on peripheral blood CD4(+) T cells is highly dependent on a cell-cell interaction with CD14(hi)CD16(-) monocytes. Interactions with ICAM-1, LFA-3, and to a lesser extent CD80/CD86 contribute to this enhancement of CD40L expression but are not themselves sufficient. The contact-mediated increase in CD40L expression is dependent on new mRNA and protein synthesis. Circulating myeloid dendritic cells also possess this costimulatory activity. By contrast, CD14(lo)CD16(+) monocytes, plasmacytoid dendritic cells, B-cell lymphoma lines, and resting, activated, and Epstein-Barr virus-immortalized primary B cells all lack the capacity to up-regulate early CD40L. The latter indicates that a human B cell cannot activate its cognate T cell to deliver CD40L-mediated help. This finding has functional implications for the role of biphasic CD40L expression, suggesting that the early phase is associated with antigen-presenting cell activation, whereas the late phase is related to B-cell activation.
- Published
- 2011
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12. Structural determinants underlying the temperature-sensitive nature of a Galpha mutant in asymmetric cell division of Caenorhabditis elegans.
- Author
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Johnston CA, Afshar K, Snyder JT, Tall GG, Gönczy P, Siderovski DP, and Willard FS
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- Animals, Cell Division, Circular Dichroism, Crystallography, X-Ray methods, Guanosine Triphosphate chemistry, Humans, Models, Biological, Mutation, Point Mutation, Protein Conformation, Surface Plasmon Resonance, Temperature, Caenorhabditis elegans metabolism, GTP-Binding Protein alpha Subunits, Gi-Go genetics, Gene Expression Regulation
- Abstract
Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Galpha(o)) and GPA-16 (Galpha(i)) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Galpha(i1)(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Galpha. At the permissive temperature, the mutant Galpha was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Galpha bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Galpha subunit function in ACD.
- Published
- 2008
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13. Crystal structure of the multifunctional Gbeta5-RGS9 complex.
- Author
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Cheever ML, Snyder JT, Gershburg S, Siderovski DP, Harden TK, and Sondek J
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- Binding Sites, Crystallography, X-Ray, Dimerization, Humans, Models, Molecular, Protein Binding, Protein Structure, Quaternary, Protein Structure, Tertiary, GTP-Binding Protein beta Subunits chemistry, RGS Proteins chemistry
- Abstract
Regulators of G-protein signaling (RGS) proteins enhance the intrinsic GTPase activity of G protein alpha (Galpha) subunits and are vital for proper signaling kinetics downstream of G protein-coupled receptors (GPCRs). R7 subfamily RGS proteins specifically and obligately dimerize with the atypical G protein beta5 (Gbeta5) subunit through an internal G protein gamma (Ggamma)-subunit-like (GGL) domain. Here we present the 1.95-A crystal structure of the Gbeta5-RGS9 complex, which is essential for normal visual and neuronal signal transduction. This structure reveals a canonical RGS domain that is functionally integrated within a molecular complex that is poised for integration of multiple steps during G-protein activation and deactivation.
- Published
- 2008
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14. Effect of uncovertebral joint excision on the motion response of the cervical spine after total disc replacement.
- Author
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Snyder JT, Tzermiadianos MN, Ghanayem AJ, Voronov LI, Rinella A, Dooris A, Carandang G, Renner SM, Havey RM, and Patwardhan AG
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- Aged, Biomechanical Phenomena instrumentation, Biomechanical Phenomena methods, Cervical Vertebrae pathology, Humans, Intervertebral Disc pathology, Intervertebral Disc physiology, Intervertebral Disc surgery, Intervertebral Disc Displacement pathology, Intervertebral Disc Displacement surgery, Middle Aged, Prosthesis Implantation instrumentation, Cervical Vertebrae physiology, Cervical Vertebrae surgery, Prosthesis Implantation methods, Range of Motion, Articular physiology
- Abstract
Study Design: In vitro biomechanical study., Objective: To quantify the effects of uncinatectomy on cervical motion after total disc replacement (TDR)., Summary of Background Data: The effect of uncinatectomy on TDR motion is unknown. Partial uncinatectomy may be required to decompress the foramen; however, the residual uncinates can potentially limit TDR motion and serve as a source of progressive spondylosis. Complete resection of the uncinates may decrease this risk yet endanger destabilizing the segment., Methods: Seven human cervical spines (C3-C7) (age, 63.4 +/- 6.9 years) were tested first intact and then after implantation of a metal-on-polyethylene ball-and-socket semiconstrained prosthesis at C5-C6. Following this, gradually increased uncinatectomy was performed in the following order: 1) right partial-posteromedial (two thirds), 2) right complete, and 3) bilateral complete resection. Specimens were tested in flexion-extension, lateral bending, and axial rotation (+/-1.5 Nm). Flexion-extension was tested under 150 N follower preload., Results: TDR without uncinatectomy increased C5-C6 flexion-extension range of motion from 8.4 degrees +/- 3.5 degrees to 11.6 degrees +/- 3.4 degrees, but statistical significance was not reached (P > 0.05). Lateral bending decreased from 6.2 degrees +/- 2.2 degrees to 3.1 degrees +/- 1.4 degrees, with a trend for statistical significance (P = 0.07). Axial rotation decreased from 5.5 degrees +/- 2.4 degrees to 4.3 degrees +/- 1.4 degrees after the implantation (P > 0.05). Both right partial and right complete uncinatectomy resulted in nearly symmetrical restoration of lateral bending to intact values and significantly increased flexion-extension compared with intact (P < or = 0.05); however, axial rotation still did not differ from intact (P > 0.05). Complete bilateral resection also restored lateral bending to intact values (7.3 degrees +/- 2.7 degrees, P > 0.05); however, it resulted in significant increase in range of motion in flexion-extension (14.1 degrees +/- 3.0 degrees, P < or = 0.05) and axial rotation (8.7 degrees +/- 2.4 degrees, P < or = 0.05)., Conclusion: Unilateral complete or even partial uncinatectomy can normalize lateral bending after TDR. Bilateral complete uncinatectomy is not necessary to restore lateral bending and may result in significantly increased range of motion in flexion-extension and axial rotation compared with intact values.
- Published
- 2007
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15. Direct inhibition of CD40L expression can contribute to the clinical efficacy of daclizumab independently of its effects on cell division and Th1/Th2 cytokine production.
- Author
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Snyder JT, Shen J, Azmi H, Hou J, Fowler DH, and Ragheb JA
- Subjects
- Animals, Antibodies, Monoclonal, Humanized, CD28 Antigens physiology, CD40 Ligand genetics, Cell Division drug effects, Cytokines biosynthesis, Daclizumab, Humans, Immunologic Factors, Interleukin-2 physiology, Mice, T-Lymphocytes, Regulatory drug effects, Th1 Cells immunology, Th2 Cells immunology, Treatment Outcome, Antibodies, Monoclonal pharmacology, CD40 Ligand antagonists & inhibitors, Immunity, Cellular drug effects, Immunoglobulin G pharmacology
- Abstract
Humanized anti-CD25 antibodies (eg, daclizumab) have been successfully used to treat several autoimmune diseases. Paradoxically, IL-2 blockade in mice can induce autoimmunity. An interspecies difference in the relative contribution of IL-2 to CD25(+) T regulatory cell (CD25(+)Treg) versus CD25(+) effector cell function might explain this conundrum. Consistent with this are reports that daclizumab inhibits human CD25(+) effector cell cytokine production by blocking the expression of CD40L. However, in mice, IL-4 and IL-12 regulate CD40L expression. As human Th1/Th2 cytokine production is also dependent on IL-2, daclizumab's inhibition of CD40L expression could be due to an indirect, rather than a direct, effect of IL-2. Here, we clarify the mechanisms underlying CD40L expression. In contrast to the mouse, human CD40L is regulated by CD28 signaling and IL-2, not the principal Th1/Th2-polarizing cytokines. We find that CD40L is expressed on naive and memory cells and inhibited by daclizumab independently of cell division. Collectively, our results indicate that daclizumab could inhibit CD25(+) effector T-cell function in vivo by directly blocking CD40L expression. This difference between mice and human may help explain the paradoxical effects of IL-2R blockade in the 2 species.
- Published
- 2007
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16. Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif.
- Author
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Yohe ME, Rossman KL, Gardner OS, Karnoub AE, Snyder JT, Gershburg S, Graves LM, Der CJ, and Sondek J
- Subjects
- Amino Acid Motifs genetics, Animals, Catalysis, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Enzyme Activation genetics, Guanine Nucleotide Exchange Factors genetics, Guanine Nucleotide Exchange Factors metabolism, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Mice, Mutation, Protein Structure, Tertiary genetics, Proto-Oncogene Proteins c-vav chemistry, Proto-Oncogene Proteins c-vav genetics, Proto-Oncogene Proteins c-vav metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Cell Cycle Proteins chemistry, Guanine Nucleotide Exchange Factors chemistry, Intracellular Signaling Peptides and Proteins chemistry
- Abstract
Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood. Here we show that the Dbl-family protein, Tim, is auto-inhibited by a short, helical motif immediately N-terminal to its DH domain, which directly occludes the catalytic surface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition of Tim is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. A peptide comprising the helical motif inhibits the exchange activity of Tim in vitro. Furthermore, substitutions within the most highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also activate the exchange process.
- Published
- 2007
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17. Crystal structure of Rac1 bound to its effector phospholipase C-beta2.
- Author
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Jezyk MR, Snyder JT, Gershberg S, Worthylake DK, Harden TK, and Sondek J
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- Crystallography, X-Ray, Humans, Isoenzymes genetics, Models, Molecular, Mutation genetics, Phospholipase C beta, Protein Binding, Protein Structure, Quaternary, Static Electricity, Type C Phospholipases genetics, rac1 GTP-Binding Protein genetics, Isoenzymes chemistry, Isoenzymes metabolism, Type C Phospholipases chemistry, Type C Phospholipases metabolism, rac1 GTP-Binding Protein chemistry, rac1 GTP-Binding Protein metabolism
- Abstract
Although diverse signaling cascades require the coordinated regulation of heterotrimeric G proteins and small GTPases, these connections remain poorly understood. We present the crystal structure of the GTPase Rac1 bound to phospholipase C-beta2 (PLC-beta2), a classic effector of heterotrimeric G proteins. Rac1 engages the pleckstrin-homology (PH) domain of PLC-beta2 to optimize its orientation for substrate membranes. Gbetagamma also engages the PH domain to activate PLC-beta2, and these two activation events are compatible, leading to additive stimulation of phospholipase activity. In contrast to PLC-delta, the PH domain of PLC-beta2 cannot bind phosphoinositides, eliminating this mode of regulation. The structure of the Rac1-PLC-beta2 complex reveals determinants that dictate selectivity of PLC-beta isozymes for Rac GTPases over other Rho-family GTPases, and substitutions within PLC-beta2 abrogate its stimulation by Rac1 but not by Gbetagamma, allowing for functional dissection of this integral signaling node.
- Published
- 2006
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18. Regulation of PLCbeta isoforms by Rac.
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Snyder JT, Jezyk MR, Gershburg S, Harden TK, and Sondek J
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- Animals, Humans, Phospholipase C beta, Spodoptera, Surface Plasmon Resonance methods, Isoenzymes metabolism, Type C Phospholipases metabolism, rac GTP-Binding Proteins physiology
- Abstract
Small GTPases function as molecular switches, which transduce cellular signals from upstream regulators to downstream effectors in a guanine nucleotide-dependent manner. Direct binding partners of small GTPases fall into four classes of both regulators and effectors that can be differentiated on the basis of the state of nucleotide required for binding. Here we describe a procedure for the rapid screening and quantitative assessment of direct interactions of the Rho family of small GTPases with effector molecules of the phospholipase Cbeta class of enzymes using surface plasmon resonance technology. The experimental format described is also readily adaptable toward characterizing guanine nucleotide-dependent binding events of both small and heterotrimeric G proteins with various classes of GTPase regulatory proteins.
- Published
- 2006
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19. Direct activation of purified phospholipase C epsilon by RhoA studied in reconstituted phospholipid vesicles.
- Author
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Seifert JP, Snyder JT, Sondek J, and Harden TK
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- Animals, Cells, Cultured, Chromatography, Affinity, Chromatography, Gel, Enzyme Activation, Humans, Liposomes, Phosphoinositide Phospholipase C, Protein Prenylation, Type C Phospholipases isolation & purification, Type C Phospholipases metabolism, rhoA GTP-Binding Protein physiology
- Abstract
Phospholipase C-epsilon (PLC-epsilon) was shown recently to be a downstream effector of Rho GTPases, and we have used an in vitro phospholipid vesicle reconstitution system with purified proteins to show this regulation to be direct. This chapter describes high-level expression of a hexahistidine-tagged fragment of PLC-epsilon encompassing the catalytic core of the enzyme through the tandem RA domains by use of a recombinant baculovirus and High Five insect cells. The recombinant protein is purified to homogeneity using metal chelate affinity and size exclusion chromatography. The small GTPase RhoA also is expressed to high levels in a lipidated form after baculovirus expression in High Five cells and is purified to near homogeneity after detergent extraction and metal chelate affinity chromatography. The capacity of GTPgammaS-bound RhoA to stimulate the phospholipase activity of PLC-epsilon is assessed by reconstitution of the RhoA in mixed-detergent phospholipid micelles containing PtdIns(4,5)P2 substrate.
- Published
- 2006
- Full Text
- View/download PDF
20. A Cdc42 mutant specifically activated by intersectin.
- Author
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Smith WJ, Hamel B, Yohe ME, Sondek J, Cerione RA, and Snyder JT
- Subjects
- Adaptor Proteins, Vesicular Transport metabolism, Amino Acid Sequence, Animals, Catalysis, Crystallography, X-Ray, Cytoskeleton metabolism, DNA, Complementary metabolism, Green Fluorescent Proteins metabolism, Guanine chemistry, Guanine Nucleotide Exchange Factors chemistry, Guanosine Diphosphate chemistry, Guanosine Triphosphate chemistry, HeLa Cells, Humans, Kinetics, Mice, Microscopy, Fluorescence, Models, Molecular, Molecular Sequence Data, Plasmids metabolism, Point Mutation, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Proto-Oncogene Proteins c-vav chemistry, Rho Guanine Nucleotide Exchange Factors, Signal Transduction, Surface Plasmon Resonance, Time Factors, Tyrosine chemistry, rho GTP-Binding Proteins chemistry, Adaptor Proteins, Vesicular Transport chemistry, Mutation, cdc42 GTP-Binding Protein genetics
- Abstract
The Rho family GTPase Cdc42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate Cdc42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of Cdc42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of Cdc42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of Cdc42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of Cdc42. Moreover, Cdc42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other Cdc42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of Cdc42(Y32F), have been defined. Cdc42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, Cdc42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on Cdc42-mediated signaling phenomena.
- Published
- 2005
- Full Text
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21. RhoA activates purified phospholipase C-epsilon by a guanine nucleotide-dependent mechanism.
- Author
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Seifert JP, Wing MR, Snyder JT, Gershburg S, Sondek J, and Harden TK
- Subjects
- Animals, Calcium metabolism, Cell Line, Enzyme Activation, Guanosine 5'-O-(3-Thiotriphosphate) metabolism, Isoenzymes genetics, Isoenzymes isolation & purification, Peptide Fragments genetics, Peptide Fragments isolation & purification, Phosphatidylinositol 4,5-Diphosphate metabolism, Phosphatidylinositol Phosphates metabolism, Phosphoinositide Phospholipase C, Rats, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Substrate Specificity, Type C Phospholipases genetics, Type C Phospholipases isolation & purification, rac1 GTP-Binding Protein metabolism, Guanine Nucleotides metabolism, Isoenzymes metabolism, Peptide Fragments metabolism, Type C Phospholipases metabolism, rhoA GTP-Binding Protein metabolism
- Abstract
Phospholipase C-epsilon (PLC-epsilon) is a recently identified PLC isoform activated by subunits of heterotrimeric G proteins (Galpha(12), Galpha(13), and Gbetagamma) as well as by the low molecular weight GTPases, Rho and Ras. To define the enzymatic activity and substrate specificity of PLC-epsilon as well as its potential direct activation by Rho family GTPases, a major fragment of PLC-epsilon encompassing the catalytic core (EF-hand repeats through the tandem Ras-associating domains; approximately 118 kDa) was purified to near homogeneity and assayed after reconstitution under various conditions. Similar to the enzymatic profiles of previously purified PLC-beta isozymes, the purified fragment of PLC-epsilon maximally hydrolyzed phosphatidylinositol 4-phosphate at a rate of approximately 10 mumol/mg of protein/min, exhibited phospholipase activity dependent on the concentration of free calcium, and favored phosphatidylinositol 4,5-bisphosphate as substrate relative to other phosphoinositides. Furthermore, in mixed detergent phospholipid micelles, RhoA stimulated the phospholipase activity of the PLC-epsilon fragment in both a concentration-dependent and guanosine 5'-O-(3-thiotriphosphate)-dependent manner. This activation was abolished by the deletion of a unique approximately 65 amino acid-insert within the catalytic core of PLC-epsilon. Although Rac1 activated purified PLC-beta2ina guanine nucleotide-dependent manner, Rac1 failed to promote guanine nucleotide-dependent activation of purified PLC-epsilon. These results indicate that PLC-epsilon is a direct downstream effector for RhoA and that RhoA-dependent activation of PLC-epsilon depends on a unique insert within the catalytic core of the phospholipase.
- Published
- 2004
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22. Protection against lethal vaccinia virus challenge in HLA-A2 transgenic mice by immunization with a single CD8+ T-cell peptide epitope of vaccinia and variola viruses.
- Author
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Snyder JT, Belyakov IM, Dzutsev A, Lemonnier F, and Berzofsky JA
- Subjects
- Amino Acid Sequence, Animals, Cell Line, HLA-A2 Antigen genetics, Humans, Immunodominant Epitopes, Mice, Mice, Inbred C57BL, Mice, Transgenic, Peptides chemistry, Smallpox immunology, Smallpox Vaccine administration & dosage, Smallpox Vaccine immunology, Vaccination, Vaccinia virus pathogenicity, CD8-Positive T-Lymphocytes immunology, Epitopes, T-Lymphocyte immunology, Peptides immunology, Smallpox prevention & control, Vaccinia virus immunology, Variola virus immunology
- Abstract
CD8(+) T lymphocytes have been shown to be involved in controlling poxvirus infection, but no protective cytotoxic T-lymphocyte (CTL) epitopes are defined for variola virus, the causative agent of smallpox, or for vaccinia virus. Of several peptides in vaccinia virus predicted to bind HLA-A2.1, three, VETFsm(498-506), A26L(6-14), and HRP2(74-82), were found to bind HLA-A2.1. Splenocytes from HLA-A2.1 transgenic mice immunized with vaccinia virus responded only to HRP2(74-82) at 1 week and to all three epitopes by ex vivo enzyme-linked immunosorbent spot (ELISPOT) assay at 4 weeks postimmunization. To determine if these epitopes could elicit a protective CD8(+) T-cell response, we challenged peptide-immunized HLA-A2.1 transgenic mice intranasally with a lethal dose of the WR strain of vaccinia virus. HRP2(74-82) peptide-immunized mice recovered from infection, while naïve mice died. Depletion of CD8(+) T cells eliminated protection. Protection of HHD-2 mice, lacking mouse class I major histocompatibility complex molecules, implicates CTLs restricted by human HLA-A2.1 as mediators of protection. These results suggest that HRP2(74-82), which is shared between vaccinia and variola viruses, may be a CD8(+) T-cell epitope of vaccinia virus that will provide cross-protection against smallpox in HLA-A2.1-positive individuals, representing almost half the population.
- Published
- 2004
- Full Text
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23. Direct activation of phospholipase C-epsilon by Rho.
- Author
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Wing MR, Snyder JT, Sondek J, and Harden TK
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, COS Cells, Dose-Response Relationship, Drug, Enzyme Activation, Glutathione Transferase metabolism, Inositol Phosphates metabolism, Models, Molecular, Molecular Sequence Data, Open Reading Frames, Phosphoinositide Phospholipase C, Protein Binding, Protein Isoforms, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Software, cdc25 Phosphatases metabolism, rap GTP-Binding Proteins metabolism, ras Proteins metabolism, Type C Phospholipases metabolism, rho GTP-Binding Proteins metabolism
- Abstract
Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.
- Published
- 2003
- Full Text
- View/download PDF
24. Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses.
- Author
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Belyakov IM, Earl P, Dzutsev A, Kuznetsov VA, Lemon M, Wyatt LS, Snyder JT, Ahlers JD, Franchini G, Moss B, and Berzofsky JA
- Subjects
- Animals, CD4 Antigens biosynthesis, CD8 Antigens biosynthesis, Enzyme-Linked Immunosorbent Assay, Female, Humans, Interferon-gamma biosynthesis, Mice, Mice, Inbred BALB C, T-Lymphocytes metabolism, Time Factors, Vaccines, Poxviridae Infections virology, Smallpox prevention & control, Smallpox Vaccine, Vaccinia virus physiology
- Abstract
The concern about bioterrorism with smallpox has raised the possibility of widespread vaccination, but the greater prevalence of immunocompromised individuals today requires a safer vaccine, and the mechanisms of protection are not well understood. Here we show that, at sufficient doses, the protection provided by both modified vaccinia Ankara and NYVAC replication-deficient vaccinia viruses, safe in immunocompromised animals, was equivalent to that of the licensed Wyeth vaccine strain against a pathogenic vaccinia virus intranasal challenge of mice. A similar variety and pattern of immune responses were involved in protection induced by modified vaccinia Ankara and Wyeth viruses. For both, antibody was essential to protect against disease, whereas neither effector CD4+ nor CD8+ T cells were necessary or sufficient. However, in the absence of antibody, T cells were necessary and sufficient for survival and recovery. Also, T cells played a greater role in control of sublethal infection in unimmunized animals. These properties, shared with the existing smallpox vaccine, provide a basis for further evaluation of these replication-deficient vaccinia viruses as safer vaccines against smallpox or against complications from vaccinia virus.
- Published
- 2003
- Full Text
- View/download PDF
25. Molecular mechanisms and biological significance of CTL avidity.
- Author
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Snyder JT, Alexander-Miller MA, Berzofskyl JA, and Belyakov IM
- Subjects
- Dendritic Cells immunology, Genes, MHC Class I, Genes, T-Cell Receptor, HIV Infections immunology, Histocompatibility Antigens Class I immunology, Humans, Lymphocyte Activation, Receptors, Antigen, T-Cell immunology, T-Lymphocytes, Cytotoxic metabolism, Cytotoxicity, Immunologic, Neoplasms immunology, T-Lymphocytes, Cytotoxic immunology, Virus Diseases immunology
- Abstract
CD8 CTLs are a major effector for protection against cancer as well as many infectious diseases, including HIV/AIDS. CD8 CTL recognize antigenic peptides in the context of class I MHC. CTL functional avidity has been shown to be an important determinant of in vivo efficacy. CTL that can recognize peptide/MHC only at high antigen density are termed low avidity CTL, while those that can recognize their cognate antigen at low densities are termed high avidity CTL. Recent studies have demonstrated that high avidity CTLs are essential for the effective clearance of viral infections and for the elimination of tumor cells. At this time, approaches that can target high avidity cells for expansion in vivo are not well defined; however, new insights are beginning to emerge. A recent study has shown that prime-boost immunization may be an effective method to generate high avidity CTLs that recognize HIV antigens. In addition, we recently found that high levels of costimulation (signal 2) can skew the CTL response toward higher avidity cells. Thus, vectors expressing a triad of costimulatory molecules (TRICOM) or dendritic cells expressing higher levels of costimulatory molecules, can be used to induce high avidity CTL. Finally a critical role for CD4+ T cell help in the generation of high avidity cells has recently been identified (Palmer, manuscript submitted). While high avidity CTLs are superior for viral and tumor clearance, they also have a greater sensitivity to antigen induced cell death. In some types of chronic infections, such as HIV and HCV, as well as in cancer, the host may lose, by clonal exhaustion or other apoptotic mechanisms, the effector cells that are most critical to viral or tumor clearance. In this review, we examine the current knowledge concerning CTL avidity. We discuss the factors that may distinguish high avidity CTLs from low avidity CTLs and describe some of the mechanisms these cells use to clear viral infections. In addition, we study possible immunization strategies that may be used to elicit higher avidity CTLs and describe what is known about the factors that render these cells more susceptible to apoptosis than low avidity CTLs. Finally, we will incorporate these various elements into a general discussion of possible approaches for induction and maintenance of an effective immune response that can result in clearance of tumors or chronic viral infections and the relevance to vaccine development.
- Published
- 2003
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26. The pleckstrin homology domain of phospholipase C-beta2 as an effector site for Rac.
- Author
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Snyder JT, Singer AU, Wing MR, Harden TK, and Sondek J
- Subjects
- Animals, Biosensing Techniques, COS Cells, DNA, Complementary, Enzyme Activation, Humans, Isoenzymes metabolism, Phospholipase C beta, Protein Binding, Protein Structure, Tertiary, Type C Phospholipases metabolism, rac GTP-Binding Proteins metabolism, RAC2 GTP-Binding Protein, Blood Proteins genetics, Isoenzymes chemistry, Isoenzymes genetics, Phosphoproteins genetics, Type C Phospholipases chemistry, Type C Phospholipases genetics, rac1 GTP-Binding Protein metabolism
- Abstract
Increasing evidence links the activation of Rho family GTPases to the stimulation of lipid hydrolysis catalyzed by phospholipase C (PLC)-beta isozymes. To better define this relationship, members of a library of recombinant Rho GTPases were screened for their capacity to directly engage various purified PLC-beta isozymes. Of the 17 tested members of the Rho family, only the active isoforms of Rac (Rac1, Rac2, and Rac3) both stimulate PLC-beta activity in vivo and bind PLC-beta2 and PLC-beta3, but not PLC-beta1, in vitro. Furthermore, the recognition site for Rac GTPases was localized to the pleckstrin homology (PH) domain of PLC-beta2, and this PH domain is fully sufficient to selectively interact with the active versions of the Rac GTPases, but not with other similar Rho GTPases. Together, these findings present a quantitative evaluation of the direct interactions between Rac GTPases and PLC-beta isozymes and define a novel role for the PH domain of PLC-beta2 as a putative effector site for Rac GTPases.
- Published
- 2003
- Full Text
- View/download PDF
27. Multifunctional roles for the PH domain of Dbs in regulating Rho GTPase activation.
- Author
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Rossman KL, Cheng L, Mahon GM, Rojas RJ, Snyder JT, Whitehead IP, and Sondek J
- Subjects
- 3T3 Cells, Animals, Blotting, Western, Cell Membrane metabolism, DNA Mutational Analysis, DNA, Complementary metabolism, Enzyme Activation, Enzyme-Linked Immunosorbent Assay, Mice, Models, Molecular, Protein Binding, Protein Structure, Tertiary, Rho Guanine Nucleotide Exchange Factors, Spectrometry, Fluorescence, Subcellular Fractions metabolism, Time Factors, rhoA GTP-Binding Protein metabolism, Guanine Nucleotide Exchange Factors metabolism, Guanine Nucleotide Exchange Factors physiology, rho GTP-Binding Proteins metabolism
- Abstract
Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.
- Published
- 2003
- Full Text
- View/download PDF
28. Functional analysis of cdc42 residues required for Guanine nucleotide exchange.
- Author
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Rossman KL, Worthylake DK, Snyder JT, Cheng L, Whitehead IP, and Sondek J
- Subjects
- Cloning, Molecular, Edetic Acid pharmacology, Kinetics, Models, Molecular, Peptide Fragments chemistry, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins metabolism, cdc42 GTP-Binding Protein chemistry, rhoA GTP-Binding Protein metabolism, Guanosine Diphosphate metabolism, Guanosine Triphosphate metabolism, Peptide Fragments metabolism, cdc42 GTP-Binding Protein metabolism
- Abstract
Guanine nucleotide exchange factors (GEFs) directly engage small GTPases to facilitate the exchange of bound GDP for GTP, leading to GTPase activation. Several recent crystal structures of GEFs in complex with Rho family GTPases highlight the conserved interactions and conformational alterations necessary for catalyzing exchange. In the present study, functional roles were defined for specific residues within Cdc42 implicated by the crystal structures as important for physiological exchange of guanine nucleotides within Rho GTPases. In particular, this study highlights the paramount importance of the phosphate-binding loop and interactions with the magnesium co-factor as critical for proper regulation of RhoGEF-catalyzed exchange. Other conformational alterations of the GTPases affecting interactions with the sugar and base of guanine nucleotides are also important but are secondary. Of particular note, substitution of alanine for cysteine at position 18 of Cdc42 leads to a fast cycling phenotype for Cdc42 with heightened affinity for RhoGEFs and produces a dominant negative form of Cdc42 capable of inhibiting RhoGEFs both in vitro and in vivo.
- Published
- 2002
- Full Text
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29. Structural basis for the selective activation of Rho GTPases by Dbl exchange factors.
- Author
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Snyder JT, Worthylake DK, Rossman KL, Betts L, Pruitt WM, Siderovski DP, Der CJ, and Sondek J
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Enzyme Activation, Guanosine Triphosphate metabolism, Humans, Models, Molecular, Molecular Sequence Data, Protein Structure, Secondary, Protein Structure, Tertiary, Proteins chemistry, Proteins metabolism, Sequence Alignment, Structure-Activity Relationship, Substrate Specificity, T-Lymphoma Invasion and Metastasis-inducing Protein 1, cdc42 GTP-Binding Protein chemistry, cdc42 GTP-Binding Protein metabolism, rac1 GTP-Binding Protein chemistry, rac1 GTP-Binding Protein metabolism, rhoA GTP-Binding Protein chemistry, rhoA GTP-Binding Protein metabolism, Guanine Nucleotide Exchange Factors chemistry, Guanine Nucleotide Exchange Factors metabolism, Proto-Oncogene Proteins chemistry, rho GTP-Binding Proteins chemistry, rho GTP-Binding Proteins metabolism
- Abstract
Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively. Structure-based mutagenesis of intersectin and Dbs reveals the key determinants responsible for promoting exchange activity in Cdc42, Rac1 and RhoA. These findings provide critical insight into the structural features necessary for the proper pairing of Dbl-exchange factors with Rho GTPases and now allow for the detailed manipulation of signaling pathways mediated by these oncoproteins in vivo.
- Published
- 2002
- Full Text
- View/download PDF
30. A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange.
- Author
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Rossman KL, Worthylake DK, Snyder JT, Siderovski DP, Campbell SL, and Sondek J
- Subjects
- Amino Acid Sequence, Animals, Crystallography, X-Ray, Guanine Nucleotide Exchange Factors genetics, Mice, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Structure, Tertiary, Rho Guanine Nucleotide Exchange Factors, Sequence Homology, Amino Acid, Guanine Nucleotide Exchange Factors chemistry, cdc42 GTP-Binding Protein chemistry
- Abstract
Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1.Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.
- Published
- 2002
- Full Text
- View/download PDF
31. Quantitative analysis of the effect of phosphoinositide interactions on the function of Dbl family proteins.
- Author
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Snyder JT, Rossman KL, Baumeister MA, Pruitt WM, Siderovski DP, Der CJ, Lemmon MA, and Sondek J
- Subjects
- Immunoblotting, Protein Binding, Rho Guanine Nucleotide Exchange Factors, Surface Plasmon Resonance, T-Lymphoma Invasion and Metastasis-inducing Protein 1, Adaptor Proteins, Vesicular Transport, Carrier Proteins metabolism, Guanine Nucleotide Exchange Factors metabolism, Phosphatidylinositols metabolism, Proteins metabolism
- Abstract
Normally, Rho GTPases are activated by the removal of bound GDP and the concomitant loading of GTP catalyzed by members of the Dbl family of guanine nucleotide exchange factors (GEFs). This family of GEFs invariantly contain a Dbl homology (DH) domain adjacent to a pleckstrin homology (PH) domain, and while the DH domain usually is sufficient to catalyze nucleotide exchange, possible roles for the conserved PH domain remain ambiguous. Here we demonstrate that the conserved PH domains of three distinct Dbl family proteins, intersectin, Dbs, and Tiam1, selectively bind lipid vesicles only when phosphoinositides are present. While the PH domains of intersectin and Dbs promiscuously bind several multiphosphorylated phosphoinositides, Tiam1 selectively interacts with phosphatidylinositol 3-phosphate (K(D) approximately 5-10 microm). In addition, and in contrast to recent reports, catalysis of nucleotide exchange on nonprenylated Rac1 provided by various extended portions of Tiam1 is not influenced by (a) soluble phosphoinositide head groups, (b) dibutyl versions of phosphoinositides, or (c) lipid vesicles containing phosphoinositides. Likewise, GEF activity afforded by DH/PH fragments of intersectin and Dbs are also not altered by phosphoinositide interactions. These results strongly suggest that unless all relevant components are localized to a lipid membrane surface, Dbl family GEFs generally are not intrinsically modulated by binding phosphoinositides.
- Published
- 2001
- Full Text
- View/download PDF
32. Activating CTL precursors to reveal CTL function without skewing the repertoire by in vitro expansion.
- Author
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Belyakov IM, Wang J, Koka R, Ahlers JD, Snyder JT, Tse R, Cox J, Gibbs JS, Margulies DH, and Berzofsky JA
- Subjects
- Animals, Female, HIV Envelope Protein gp160 immunology, HIV-1 immunology, Mice, Mice, Inbred BALB C, Receptors, Antigen, T-Cell, alpha-beta analysis, Hematopoietic Stem Cells immunology, Lymphocyte Activation, T-Lymphocytes, Cytotoxic immunology
- Abstract
Detection of the functional CD8(+) CTL response usually requires in vitro restimulation. The differences between the CD8(+) CTL repertoire in freshly isolated precursor cells and CD8(+) CTL after short-term in vitro expansion have been generally assumed to be minimal, but have never been defined experimentally. Using staining with P18-I10/H-2D(d) tetramers and monoclonal antibodies (mAb) against Vbeta, we show the surprising result that there was significant skewing of the CD8(+) CTL repertoire after just 7 days of stimulation. In contrast, we found that overnight incubation of precursor cells with peptide allows the functional assessment of CD8(+) CTL (which cannot be detected ex vivo from freshly isolated cells) without changing the absolute number of antigen-specific CTL as measured by tetramer staining or the repertoire of TCR analyzed with mAb. This study affords a better understanding of the differences between the ex vivo and in vitro stimulated CTL repertoire, and provides an approach to reveal a more faithful representation of the functional in vivo CTL response without skewing of the repertoire of T cells detected.
- Published
- 2001
- Full Text
- View/download PDF
33. An abrupt and concordant initiation of apoptosis: antigen-dependent death of CD8+ CTL.
- Author
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Derby MA, Snyder JT, Tse R, Alexander-Miller MA, and Berzofsky JA
- Subjects
- Animals, Annexin A5 analysis, Antigens, CD physiology, Caspase Inhibitors, Caspases physiology, Down-Regulation, Mice, Mice, Inbred BALB C, Proteins analysis, Proto-Oncogene Proteins c-bcl-2 analysis, Receptors, Antigen, T-Cell physiology, Receptors, Tumor Necrosis Factor physiology, Receptors, Tumor Necrosis Factor, Type II, TNF Receptor-Associated Factor 2, Tumor Necrosis Factor-alpha physiology, Antigens immunology, Apoptosis, CD8-Positive T-Lymphocytes physiology
- Abstract
The ability of CD8+ cytotoxic T lymphocytes (CTL) to clear viral infections may be limited when high avidity CTL encounter supra-optimal antigen density on antigen-presenting cells (APC) and undergo antigen-dependent apoptosis of CTL (ADAC). Previously, we have shown ADAC in CD8+ populations to be Fas independent, TNF-alpha receptor 2 (TNFR2) mediated, caspase dependent, and accompanied by a decrease in Bcl-2. We now employ flow cytometry to follow ADAC within individual CD8+ cells to demonstrate that the intense TCR signal induced in high avidity CTL by supra-optimal antigen density results 8 - 16 h later in a caspase-independent TNFR2 down-modulation that is directly related to the stimulating APC antigen density and concludes in a rapid onset of apoptosis by 18 - 24 h. Individual CTL undergoing apoptosis exhibit a dramatic and concurrent: (1) positive staining with Annexin V and propidium iodide; (2) transformation to a smaller cell size characteristic of apoptosis; and (3) a nearly complete loss of Bcl-2, c-IAP1, and TRAF2. We conclude that the antigen-dependent apoptosis of CD8+ CTL occurs when a tandem TCR/TNFR2 signal initiates an abrupt and concordant onset of multiple apoptotic events.
- Published
- 2001
- Full Text
- View/download PDF
34. Angioscopic assessment of coronary lesions underlying thrombus.
- Author
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Waxman S, Mittleman MA, Zarich SW, Fitzpatrick PJ, Lewis SM, Leeman DE, Shubrooks SJ Jr, Snyder JT, Muller JE, and Nesto RW
- Subjects
- Coronary Disease etiology, Humans, Risk Factors, Angioscopy, Coronary Disease complications, Coronary Disease diagnosis, Coronary Thrombosis etiology, Coronary Thrombosis pathology
- Abstract
This study examines the characteristics of coronary lesions in which thrombus is found as assessed by angioscopy before percutaneous transluminal coronary angioplasty in patients with various coronary syndromes. Our findings demonstrate that the plaque underlying intracoronary thrombus is usually yellow and/or disrupted, and support in vitro observations that lipid-rich plaques are highly thrombogenic and that disruption of these plaques is associated with in situ thrombosis.
- Published
- 1997
- Full Text
- View/download PDF
35. Hyaluronate levels in donor organ washout effluents: a simple and predictive parameter of graft viability.
- Author
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Rao PN, Bronsther OL, Pinna AD, Snyder JT, Cowan S, Sankey S, Kramer D, Takaya S, and Starzl T
- Subjects
- Adult, Alanine Transaminase blood, Aspartate Aminotransferases blood, Endothelium, Vascular metabolism, False Positive Reactions, Humans, Hyaluronic Acid analysis, Linear Models, Liver blood supply, Perfusion, Postoperative Complications, Graft Survival, Hyaluronic Acid metabolism, Liver metabolism, Liver Transplantation
- Abstract
The principal cause of primary non-function in orthotopic liver transplantation is thought to be preservation injury to the microvasculature. We, therefore, evaluated if effluent levels of hyaluronate, whose uptake is an endothelial cell marker, could predict early graft function and ultimate graft outcome in orthotopic liver transplantation. A total of 102 cases were studied in two phases. In the first phase, we attempted to determine if a correlation existed between effluent hyaluronate levels, early graft function and ultimate graft outcome. This phase of the study was also used to determine hypothetical cut-off values for hyaluronate which could discriminate between good and bad livers. Thirty-two livers orthotopically transplanted to randomly selected primary recipients were studied. After varying periods of static cold storage (4 degrees C) in University of Wisconsin solution, the livers were reinfused with cold (4 degrees C) lactated Ringer's solution. The first 50 ml of the reperfusion effluent was collected from the infrahepatic vena cava. Effluent samples were analyzed for hyaluronate. Linear regression analysis demonstrated a significant correlation between effluent hyaluronate levels and post-operative aspartate and alanine aminotransferase levels (p < 0.001 for both). Logistic regression demonstrated a highly significant correlation (p = 0.0056) between effluent hyaluronate levels and ultimate graft outcome. Generation of Receiver Characteristics Curves indicated that a level between 400 and 430 micrograms.l-1 could possibly discriminate between good livers and those at risk of early graft failure. The authenticity of this hyaluronate cut-off level was further confirmed in the second phase of the study where 70 consecutive primary crossmatch-negative transplants were performed. A highly significant difference was observed in peak aspartate and alanine aminotransferase levels in the first week (p < 0.0006 and p < 0.0005, respectively) between livers with effluent hyaluronate levels < or = 400 micrograms.l-1 and livers with hyaluronate levels higher than 400 micrograms.l-1. Logistic regression revealed a highly significant correlation between effluent hyaluronate levels and graft success (p = 0.0001). Since hyaluronate uptake by the microvascular endothelial cell is significantly greater than production, high hyaluronate effluent levels in failed livers would be due to decreased hyaluronate uptake by the injured microvascular endothelial cell. We therefore conclude that effluent hyaluronate levels may prove to be a reliable preoperative test to assess early graft function and outcome in clinical orthotopic liver transplantation.
- Published
- 1996
- Full Text
- View/download PDF
36. Evidence that small bowel preservation causes primarily basement membrane and endothelial rather than epithelial cell injury.
- Author
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Mueller AR, Nalesnik MA, Langrehr JM, Rao PN, Snyder JT, Hoffman RA, and Schraut WH
- Subjects
- Animals, Basement Membrane injuries, Basement Membrane metabolism, Basement Membrane pathology, Cold Temperature, Endothelium injuries, Endothelium metabolism, Endothelium pathology, Epithelium injuries, Epithelium metabolism, Epithelium pathology, Evaluation Studies as Topic, Glutaminase metabolism, Hyaluronic Acid metabolism, Immunohistochemistry, Intestine, Small pathology, Laminin metabolism, Male, Rats, Rats, Inbred Lew, Transplantation, Isogeneic, Intestine, Small injuries, Intestine, Small transplantation, Organ Preservation adverse effects
- Abstract
The main site of injury induced during small bowel preservation is perceived to be the basement membrane and the endothelium of the highly vascularized mucosa, an aspect evaluated here in further detail. The effects of preservation were studied using a specific basement membrane stain (laminin antibody), an endothelial cell stain (factor 8 antibody) and standard histology. In addition, mucosal glutaminase activity reflecting enterocyte integrity was measured as monitor of the extent of preservation injury. Using a rat model, small bowel grafts were harvested, the vascular bed and bowel lumen were flushed, and the grafts were stored (4 degrees C) for 1, 6, 9, and 12 hr and transplanted into syngeneic hosts. After cold storage prior to transplantation, full-thickness small bowel biopsies were obtained for the various tissue preparations. Histologic evaluation at the end of the preservation period revealed separation of the villous epithelium from the lamina propria that increased with extending preservation time. Tissue staining with the laminin antibody disclosed progressive changes with increasing preservation intervals. Staining with the factor 8 antibody demonstrated also progressive changes, but failed to reflect in a gradual fashion increasing endothelial cell injury. Histologic injury became more pronounced after transplantation and reperfusion, then showing destruction of epithelial cells; the extent of injury correlated with the duration of preservation. Glutaminase activity was maintained after cold storage, indicating that the enterocytes remained intact during this period, but when assayed after reperfusion, glutaminase decreased with increasing preservation intervals and increasing histologic mucosal damage. We conclude that cold ischemic injury involves primarily the endothelium and the basement membrane, which progresses to global mucosal impairment with reperfusion.
- Published
- 1993
- Full Text
- View/download PDF
37. Hyaluronic acid and purine nucleoside phosphorylase in vascular and luminal effluents of small bowel grafts as parameters of preservation injury.
- Author
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Mueller AR, Rao PN, Snyder JT, Hoffman RA, and Schraut WH
- Subjects
- Animals, Cell Membrane Permeability, Intestine, Small chemistry, Male, Rats, Rats, Inbred Lew, Hyaluronic Acid analysis, Intestine, Small transplantation, Purine-Nucleoside Phosphorylase analysis, Tissue Preservation
- Abstract
Reliable parameters reflecting the degree of graft injury after small bowel preservation are currently not established. We investigated hyaluronic acid (HA) and purine nucleoside phosphorylase (PNP) as indicators of preservation injury before small bowel transplantation. In the first part of the study, intestinal grafts were harvested, perfused with saline, and flushed either immediately or after 1, 6, 12, 24, and 48 hr of cold storage (n = 6/group). HA and PNP were assayed in vascular and luminal effluents. In the second part of the study, 24 grafts were transplanted after preservation periods of 1, 6, 9, and 12 hr (n = 6/group) to assess if HA and PNP are predictors of postoperative graft survival. HA levels in vascular effluents and PNP activities in luminal effluents correlated with duration of preservation time and predicted graft survival. Utilizing both parameters significantly increased the predictive accuracy.
- Published
- 1993
- Full Text
- View/download PDF
38. Levels of purine nucleoside phosphorylase (PNP) as a viability marker of nonparenchymal cells in cold preserved livers.
- Author
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Mischinger HJ, Rao PN, Todo S, Snyder JT, Quehenberger F, Murase N, and Starzl TE
- Subjects
- Adenosine Triphosphate metabolism, Animals, Bile metabolism, Biomarkers, Cell Survival, Liver enzymology, Liver physiology, Liver Function Tests, Male, Rats, Rats, Inbred Lew, Time Factors, Liver cytology, Organ Preservation, Purine-Nucleoside Phosphorylase metabolism
- Published
- 1991
39. Survival following severe overdose with mexiletene, nifedipine, and nitroglycerine.
- Author
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Frank SE and Snyder JT
- Subjects
- Hemodynamics drug effects, Humans, Male, Middle Aged, Poisoning diagnosis, Poisoning physiopathology, Poisoning therapy, Suicide, Attempted, Mexiletine poisoning, Nifedipine poisoning, Nitroglycerin poisoning
- Abstract
Survival following attempted suicide in a 50-year-old man by ingestion of 12.4 g of mexiletine, 620 mg of nifedipine and 50 to 100 tablets of sublingual nitroglycerine 1:150 is reported. Initial presentation was that of mental obtundation, vomiting, tonic-clonic seizure, high-degree atrioventricular block, profound vasodilation and cardiovascular collapse. Treatment consisted of intravenous calcium gluconate and aggressive fluid management. Maintenance of cardiovascular stability required continuous infusion phenylephrine, dopamine and epinephrine. The patient made a full recovery and was medically discharged on the fourth hospital day. This case represents the largest overdose of mexiletine to date to end in patient survival.
- Published
- 1991
- Full Text
- View/download PDF
40. Inhibition of free radical generation and improved survival by protection of the hepatic microvascular endothelium by targeted erythrocytes in orthotopic rat liver transplantation.
- Author
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Rao PN, Walsh TR, Makowka L, Liu T, Demetris AJ, Rubin RS, Snyder JT, Mischinger HJ, and Starzl TE
- Subjects
- Animals, Bile metabolism, Erythrocytes immunology, Free Radicals, Graft Survival, Isotonic Solutions, Male, Rats, Reperfusion Injury prevention & control, Ringer's Solution, Tissue Preservation, Erythrocytes metabolism, Liver blood supply, Liver Transplantation immunology, Reperfusion Injury enzymology, Superoxides metabolism
- Abstract
The capacity of specifically targeted erythrocytes to inhibit free radical-mediated injury to the endothelial cell after cold preservation, and improve liver function was studied in two experimental models: An isolated perfused rat liver (IPRL) system and syngeneic orthotopic rat liver transplantation. In the IPRL model, livers were preserved in University of Wisconsin solution for 24 h at 4 degrees C. At the end of the preservation period, livers were flushed with lactated Ringer's (control), immunoerythrocytes (IES), or blank intact erythrocytes prior to warm reperfusion for 2 h using an assanguinous Krebs-Henseleit buffer. Production of superoxide (O2-) anion during warm reperfusion in the IES-treated liver was reduced by 65% as compared with controls (P less than 0.001) and by 74% (P less than 0.001) when compared with blank erythrocyte-treated livers. Endothelial cell preservation, as assessed by levels of purine nucleoside phosphorylase (PNP), was much better in the IES-treated group (P less than 0.001) when compared with untreated livers. Hepatocellular preservation was markedly improved in the IES-treated livers. In the syngeneic liver transplantation model, livers were preserved in UW solution for 24 h at 4 degrees C. Prior to implantation, livers were flushed with 5 ml of cold lactated Ringer's or immunoerythrocytes. Survival after three weeks was 60% in the IES-treated group and 30% in the untreated group. Survival in the IES-treated group was not significantly different from a control (no preservation) group. IES-treated livers in both models demonstrated better endothelial cell integrity and ultimate liver function. IES treatment therefore appears to protect the hepatic microvascular endothelial cell from reperfusion injury and could prove to be an easy reproducible method of donor organ preparation after cold preservation.
- Published
- 1990
- Full Text
- View/download PDF
41. Purine nucleoside phosphorylase: a new marker for free oxygen radical injury to the endothelial cell.
- Author
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Rao PN, Walsh TR, Makowka L, Rubin RS, Weber T, Snyder JT, and Starzl TE
- Subjects
- Animals, Bile metabolism, Free Radicals, Ischemia, Liver blood supply, Liver enzymology, Oxygen toxicity, Rats, Rats, Inbred Lew, Superoxides metabolism, Endothelium, Vascular enzymology, Pentosyltransferases metabolism, Purine-Nucleoside Phosphorylase metabolism, Reperfusion Injury enzymology
- Abstract
The effect of ischemia and reperfusion on purine nucleoside phosphorylase was studied in an isolated perfused rat liver model. This enzyme is localized primarily in the cytoplasm of the endothelial and Kupffer cells; some activity is associated with the parenchymal cells. Levels of this enzyme accurately predicted the extent of ischemia and reperfusion damage to the microvascular endothelial cell of the liver. Livers from Lewis rats were subjected to 30, 45 and 60 min of warm (37 degrees C) no flow ischemia that was followed by a standard reperfusion period lasting 45 min. Purine nucleoside phosphorylase was measured at the end of the no flow ischemia and reperfusion periods as was superoxide generation (O2-). Bile production was monitored throughout the no flow ischemia and reperfusion periods. Control perfusions were carried out for 120 min. A significant rise in purine nucleoside phosphorylase levels as compared with controls was observed at the end of ischemia in all the three groups. The highest level, 203.5 +/- 29.2 mU/ml, was observed after 60 min of ischemia. After the reperfusion period, levels of purine nucleoside phosphorylase decreased in the 30- and 45-min groups 58.17 +/- 9.66 mU/ml and 67.5 +/- 17.1 mU/ml, respectively. These levels were equal to control perfusions. In contrast, after 60 min of ischemia, levels of purine nucleoside phosphorylase decreased early in the reperfusion period and then rose to 127.8 +/- 14.8 mU/ml by the end of reperfusion (p less than 0.0001). Superoxide generation at the beginning of reperfusion was higher than in controls with similar values observed at the end of 30, 45 and 60 min of ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1990
- Full Text
- View/download PDF
42. Characterization of an outbreak of Clostridium perfringens food poisoning by quantitative fecal culture and fecal enterotoxin measurement.
- Author
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Birkhead G, Vogt RL, Heun EM, Snyder JT, and McClane BA
- Subjects
- Aged, Animals, Diarrhea etiology, Enzyme-Linked Immunosorbent Assay, Feces analysis, Humans, Meat, Nursing Homes, Spores, Bacterial isolation & purification, Turkeys, Vermont, Clostridium Infections microbiology, Clostridium perfringens isolation & purification, Disease Outbreaks, Enterotoxins analysis, Feces microbiology, Foodborne Diseases microbiology
- Abstract
Published criteria for implicating Clostridium perfringens as the cause of food-poisoning outbreaks include finding a median fecal C. perfringens spore count of greater than 10(6)/g among specimens from ill persons. We investigated a food-poisoning outbreak with the epidemiologic characteristics of C. perfringens-related disease in a nursing home in which the median fecal spore count for ill patients (2.5 X 10(7)/g) was similar to that for well patients (4.0 X 10(6)/g), making the etiology of the outbreak uncertain. All ill and well patients tested had eaten turkey, the implicated food item. C. perfringens enterotoxin was detected by reverse passive latex agglutination in fecal specimens from six of six ill and none of four well patients who had eaten turkey (P = 0.005), suggesting that this organism had caused the outbreak. This investigation suggests that detection of fecal C. perfringens enterotoxin is a specific way to identify this organism as the causative agent in food-poisoning outbreaks.
- Published
- 1988
- Full Text
- View/download PDF
43. Development and preliminary evaluation of a slide latex agglutination assay for detection of Clostridium perfringens type A enterotoxin.
- Author
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McClane BA and Snyder JT
- Subjects
- Enterotoxins immunology, Enzyme-Linked Immunosorbent Assay, Feces analysis, Foodborne Diseases diagnosis, Humans, Latex Fixation Tests, Clostridium perfringens, Enterotoxins analysis
- Abstract
A slide latex agglutination (SLA) assay was developed for rapid screening for Clostridium perfringens type A enterotoxin (CPE). SLA specifically detected CPE added to buffer or normal feces (sensitivity limit of 1 microgram CPE/g feces). Using clinical fecal samples from C. perfringens food poisoning cases, a strong correlation was shown between SLA results and results from other CPE assays and between SLA results and illness status.
- Published
- 1987
- Full Text
- View/download PDF
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