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James A Thomas

Publications and source records attributed to James A Thomas.

18 recordsLinked to original sources
PubMed2006

Characterization of human immunodeficiency virus type 1 (HIV-1) containing mutations in the nucleocapsid protein at a putative HIV-1 protease cleavage site.

The HIV-1 nucleocapsid protein (NC) has been hypothesized to be cleaved by the viral protease (PR) during early infection. Characterization of viruses, with amino-acid substitutions that modulate PR cleavage of NC in vitro, was performed in cell culture. Two of the NC mutants, NCN17F and NCN17G, had decreased infectivity and exhibited severe H9 replication defects. Examination of viral DNA after infections revealed defects in reverse transcription and integration, although integration defects were cell-type dependent. However, while the defects in reverse transcription and integration correlate with lowered infectivity in a single-round of infection, they did not approach the magnitude of the replication defect measured in H9 cells over multiple rounds. Importantly, we fail to see evidence that H9 cells are re-infected with the NCN17G and NCN17F viruses 24 h after the initial infection, which suggests that the principal defect caused by these NC mutations occurs during late events of viral replication.

Cell Line↗
PubMed2006

Human immunodeficiency virus type 1 nucleocapsid zinc-finger mutations cause defects in reverse transcription and integration.

The nucleocapsid (NC) protein from HIV-1 contains two zinc-fingers, both of which are necessary for virus replication. This is the first in-depth study that presents the effects of nucleocapsid zinc-finger substitutions on the kinetics of reverse transcription and integration. Over a 72-h time-course of infection, the quantities of viral DNA (vDNA) observed with viruses containing either the nucleocapsid His23Cys or His44Cys mutations were significantly lower than those observed in infections with virus containing wild-type NC. In addition, the kinetics of vDNA formation and loss were significantly different from wild-type. The kinetic profiles observed indicated reduced vDNA stability, as well as defects in reverse transcription and integration. Overall, the defect in integration was much more pronounced than the reverse transcription defects. This suggests that the principal reason for the replication defectiveness of these mutant viruses is impairment of integration, and thus demonstrates the critical importance of NC in HIV-1 infection.

HIV Long Terminal Repeat↗
PubMed2005

Multiple toll-like receptor agonists act as potent adjuvants in the induction of autoimmunity.

Infections can trigger or exacerbate the course of Multiple Sclerosis, and both bacterial and viral agents have been implicated. These agents are recognized by host cells via pathogen-associated molecular patterns activating TLRs. We investigated the role that PAMPs play in the animal model Experimental Autoimmune Encephalomyelitis, and found various MyD88-dependent PAMPs can participate as the adjuvant to induce EAE. Studies with IRAK1-deficient mice suggest that signaling through TLRs is not required in the target organ to develop disease. This suggests that PAMPs play an important role in priming of autoreactive T cells in EAE and potentially MS.

Adaptor Proteins, Signal Transducing↗
PubMed2005

RuII complexes incorporating tetrathiamacrocycles: synthesis and conformational analysis.

The synthesis of a series of RuII complexes incorporating thiacrown ligands ([12]ane-S4, [14]ane-S4, [16]ane-S4), as well as 2,2'-bipyridine (bpy) or pyridine, is reported. Structural studies on these complexes have been carried out using a variety of techniques. Detailed 1H NMR spectroscopic studies on the previously reported [Ru([12]ane-S4)(bpy)]2+ (1) reveal that-contrary to earlier reports-the observed fluxional 1H NMR behavior is not due to chemical exchange involving cleavage of the bpy Ru--N bond but is, in fact, due to lone-pair inversion of coordinated macrocyclic sulfur donor atoms. This phenomenon is also observed for the [14]ane-S4 and [16]ane-S4 analogues of 1. For the first time, using a combination of X-ray crystallography, more detailed 1H NMR experiments, and computational methods, an in-depth study on the energetics and dynamics of invertomer formation and conversion for macrocyclic coordination complexes has been carried out. These studies reveal that the steric constraints of assembling each sulfur macrocycle and bpy ligand around the octahedral Ru(II) center lead to close intramolecular contacts. These contacts are largely dependent on the orientation of the electron lone pairs of equatorial sulfur donor atoms and correlate with the comparative stability of the different invertomeric forms. Thus, the conformational preferences of the three macrocyles in [Ru([n]ane-S4)(bpy)]2+ complexes are determined by steric rather than electronic effects.

Crystallography, X-Ray↗
PubMed2005

Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins.

The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2'-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.

Cell Line↗
PubMed2005

Monitoring liver function during methotrexate therapy for psoriasis: are routine biopsies really necessary?

Methotrexate is a widely used and efficacious treatment for psoriasis. Current recommendations advise liver biopsy after each cumulative 1-1.5g dosage of methotrexate. Although studies of methotrexate hepatotoxicity in psoriasis are of variable quality, the evidence indicates that in patients with no risk factors for liver disease on long-term, low-dosage (<20mg), once-weekly methotrexate, liver biopsy is justified after an initial cumulative dosage of 4g. Advances in the non-invasive assessment of liver fibrogenesis may eventually further reduce the need for biopsy.

Biopsy↗
PubMed2004

Differential induction of apoptosis by LPS and taxol in monocytic cells.

Numerous microbial as well as other stimulants including lipopolysaccharide and taxol can activate TLR4, and elicit diverse downstream signaling events including cytokine gene expression and cell growth regulation. With a mechanism not completely understood, different TLR4 stimulants induce distinct cellular responses. Our present studies showed that taxol, not LPS, induced cell apoptosis in human monocytic THP-1 cells, as indicated by PARP cleavage, as well as bcl-2 phosphorylation. Pretreatment of cells with LPS abolished subsequent taxol effect, suggesting that certain signaling components involved in taxol-mediated apoptosis were disrupted by LPS pretreatment. Since the decrease in IRAK-1 level closely accompanies prolonged LPS treatment in monocytic cells, we investigated the IRAK-1 status upon various taxol and LPS challenges. We observed that only LPS, not taxol, caused dramatic decrease in IRAK-1 protein levels. Using splenic macrophages harvested from IRAK-1 knockout and control mice, we further demonstrated that the presence of IRAK-1 is required for taxol-induced PARP cleavage.

Animals↗
PubMed2004

Bone marrow-derived cells contribute to contractile dysfunction in endotoxic shock.

How infection precipitates depressed contractility is incompletely understood but may involve the immune, nervous, and endocrine systems as well as the heart itself. In this study, we examined the role of Toll-like receptor 4 (TLR4) in LPS-induced myocardial contractile depression. Eighteen hours following endotoxin challenge, we compared contractile responses in hearts from wild-type (WT) and TLR4-deficient mice using modified Langendorff preparations. Unlike hearts from WT mice, TLR4-deficient hearts did not reveal significant contractile dysfunction following LPS administration, as measured by decreased responses in maximal left ventricular pressure, +dP/dtmax, and -dP/dtmax in ex vivo Langendorff preparations. These findings indicate a requirement for TLR4 in LPS-induced contractile depression. To determine the contribution of bone marrow-derived TLR4 function to LPS-induced myocardial dysfunction, we generated TLR4 chimeras using adoptive transfer between histocompatible mouse strains: either TLR4-deficient mice with TLR4+/+ bone marrow-derived cells or TLR4+/+ animals lacking TLR4 in their hematopoietic cells. We then compared the contractile responses of engrafted animals after LPS challenges. Engraftment of TLR4-deficient mice with WT marrow restored sensitivity to the myocardial depressant effects of LPS in TLR4-deficient hearts (P < 0.05). Inactivation of bone marrow-derived TLR4 function, via transplantation of WT mice with TLR4-/- marrow, however, did not protect against the depressant effect of endotoxin. These findings indicate that bone marrow-derived TLR4 activity is sufficient to confer sensitivity to mice lacking TLR4 in all other tissues. However, because inactivation of marrow-derived TLR4 function alone does not protect against endotoxin-triggered contractile dysfunction, TLR4 function in other tissues may also contribute to this response.

Animals↗
PubMed2004

Selective decontamination of the digestive tract attenuated the myocardial inflammation and dysfunction that occur with burn injury.

This study examined the effects of oral antibiotics to selectively decontaminate the digestive tract (SDD) on postburn myocardial signaling, inflammation, and function. We hypothesized that antibiotic therapy to eliminate pathogens from the gastrointestinal (GI) tract would reduce myocardial inflammatory responses and improve postburn myocardial performance. Sprague-Dawley rats received polymyxin E (15 mg), tobramycin (6 mg), and 5-flucytosin (100 mg) by oral gavage twice daily for 3 days preburn and 24 h postburn. Experimental groups included 1) sham burn given vehicle (3 ml water), 2) sham plus SDD, 3) burn over 40% total body surface area (TBSA) plus SDD, and 4) burn over 40% TBSA given vehicle. All burns received lactated Ringer solution (4 mg.kg(-1).%burn(-1)); myocardial signaling (PKCepsilon/p38 MAPK/NF-kappaB) was studied 2, 4, and 24 h postburn; and cytokine secretion (systemic and myocyte secreted cytokines, ELISA) and cardiac function were examined 24 h postburn. Vehicle-treated burn injury increased myocardial PKCepsilon/p38 MAPK expression, promoted NF-kappaB nuclear translocation, promoted TNF-alpha, IL-1beta, IL-6, and IL-10 secretion, and impaired myocardial function. SDD attenuated burn-related proinflammatory myocardial signaling, cytokine secretion, and myocardial contractile defects. Our data suggest that burn-related loss of GI barrier function and translocation of microbial products serve as upstream mediators of postburn myocardial inflammatory signaling and dysfunction.

Animals↗
PubMed2004

IL-1 receptor-associated kinase 1 mediates protection against Staphylococcus aureus infection.

The interleukin-1 receptor-associated kinase-1 (IRAK-1) mediates signal transduction from Toll-like/IL-1/IL-18 receptors. Though a critical protective role against Staphylococcus aureus infection has been previously attributed to myeloid differentiation factor 88 (MyD88) and IRAK-4, both also involved in TLR/IL-1/IL-18 signaling, the role of IRAK-1 is unknown. IRAK-1-deficient (IRAK-1-/-) and wild-type mice were inoculated i.v. with 2 x 10(7) or 1 x 10(6) S. aureus per mouse to evaluate the role of IRAK-1 in S. aureus sepsis. Since IRAK-1 transduces IL-1R signals, IL-1R-/- mice were also included in experiments. IRAK-1-/- mice are susceptible to a high dose of S. aureus compared to wild-type controls. In contrast to the high mortality and extensive weight loss seen in IL-1R-deficient mice in response to 1 x 10(6) S. aureus, IRAK-1-/- mice are resistant to this low dose of S. aureus. Thus IRAK-1 plays an important role in the host response to staphylococcal sepsis.

Animals↗
PubMed2003

IL-1 receptor-associated kinase 1 regulates susceptibility to organ-specific autoimmunity.

Infections often precede the development of autoimmunity. Correlation between infection with a specific pathogen and a particular autoimmune disease ranges from moderately strong to quite weak. This lack of correspondence suggests that autoimmunity may result from microbial activation of a generic, as opposed to pathogen-specific host-defense response. The Toll-like receptors, essential to host recognition of microbial invasion, signal through a common, highly conserved pathway, activate innate immunity, and control adaptive immune responses. To determine the influence of Toll/IL-1 signaling on the development of autoimmunity, the responses of wild-type (WT) mice and IL-1R-associated kinase 1 (IRAK1)-deficient mice to induction of experimental autoimmune encephalomyelitis were compared. C57BL/6 and B6.IRAK1-deficient mice were immunized with MOG 35-55/CFA or MOG 35-55/CpG DNA/IFA. WT animals developed severe disease, whereas IRAK1-deficient mice were resistant to experimental autoimmune encephalomyelitis, exhibiting little or no CNS inflammation. IRAK1-deficient T cells also displayed impaired Th1 development, particularly during disease induction, despite normal TCR signaling. These results suggest that IRAK1 and the Toll/IL-1 pathway play an essential role in T cell priming, and demonstrate one means through which innate immunity can control subsequent development of autoimmunity. These findings may also help explain the association between antecedent infection and the development or exacerbations of some autoimmune diseases.

Adjuvants, Immunologic↗
PubMed2003

IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction.

Myocardial contractile dysfunction accompanies both systemic and cardiac insults. Septic shock and burn trauma can lead to reversible contractile deficits, whereas ischemia and direct inflammation of the heart can precipitate transient or permanent impairments in contractility. Many of the insults that trigger contractile dysfunction also activate the innate immune system. Activation of the innate immune response to infection is coordinated by the conserved Toll/interleukin-1 (IL-1) signal transduction pathway. Interestingly, components of this pathway are also expressed in normal and failing hearts, although their function is unknown. The hypotheses that Toll/IL-1 signaling occurs in the heart and that intact pathway function is required for contractile dysfunction after different insults were tested. Results from these experiments demonstrate that lipopolysaccharides (LPS) activate Toll/IL-1 signaling and IL-1 receptor-associated kinase-1 (IRAK1), a critical pathway intermediate in the heart, indicating that the function of this pathway is not limited to immune system tissues. Moreover, hearts lacking IRAK1 exhibit impaired LPS-triggered downstream signal transduction. Hearts from IRAK1-deficient mice also resist acute LPS-induced contractile dysfunction. Finally, IRAK1 inactivation enhances survival of transgenic mice that develop severe myocarditis and lethal heart failure. Thus the Toll/IL-1 pathway is active in myocardial tissue and interference with pathway function, through IRAK1 inactivation, may represent a novel strategy to protect against cardiac contractile dysfunction.

Acute Disease↗
PubMed2002

TLR4 inactivation and rBPI(21) block burn-induced myocardial contractile dysfunction.

Both large burns and severe gram-negative sepsis are associated with acute myocardial contractile dysfunction. Because others have reported that burn injury may be followed by transient endotoxemia, we hypothesized that bacterial endotoxin induces contractile impairment after burn trauma. We tested this hypothesis in two rodent models. In each model, postburn myocardial contractility was assessed using Langendorff preparations of excised hearts. In the first model, mice expressing either a mutant form of or no Toll-like receptor 4 (TLR4), a critical element of the mammalian endotoxin receptor, were resistant to postburn myocardial contractile dysfunction. In the second model, starting 30 min or 4 h after burn injury, rats were infused with recombinant bactericidal/permeability-increasing protein (rBPI(21)), a protein that binds and neutralizes endotoxin. Hearts from rBPI(21)-treated animals were completely protected from postburn contractile impairment. Because burn-induced contractile dysfunction can be prevented either by blocking signaling through the endotoxin receptor or by neutralizing circulating LPS, bacterial endotoxin may contribute to impaired myocardial contractility after burn injury.

Animals↗
PubMed2002

IRAK contributes to burn-triggered myocardial contractile dysfunction.

Major burn injury causes myocardial contractile dysfunction, but the molecular basis of this physiological response is incompletely understood. Previous studies demonstrated a role for the interleukin-1 receptor-associated kinase (IRAK) in the cardiac response to acute lipopolysaccharide administration as well as congestive heart failure. In this study, we examined the contribution of IRAK to burn-mediated cardiac responses. After burn injury, hearts from wild-type and IRAK-deficient mice were compared for intracellular signaling pathway activation and contractile function. IRAK-deficient hearts showed impaired activation of kinases that function downstream of IRAK and were partially protected against burn-induced contractile dysfunction. The findings demonstrate that IRAK and the Toll/interleukin-1 pathways participate in the response to large body surface area burns that leads to impaired cardiac contractility.

Animals↗
PubMedDate not supplied

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats.

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.

Aging↗