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Biomedical subjects

C Carter

Publications and source records attributed to C Carter.

At least 37 records · Page 2Linked to original sources
PubMed2001

Structural consequences of cyclophilin A binding on maturational refolding in human immunodeficiency virus type 1 capsid protein.

While several cellular proteins are incorporated in the human immunodeficiency virus type 1 virion, cyclophilin (CyP) A is the only one whose absence has been demonstrated to impair infectivity. Incorporation of the cytosolic protein results from interaction with a highly exposed Pro-rich loop in the N-terminal region of the capsid (CA) domain of the precursor polyprotein, Pr55(Gag). Even when prevented from interacting with CyP A, Pr55Gag still forms particles that proceed to mature into morphologically wild-type virions, suggesting that CyP A influences a postassembly event. The nature of this CyP A influence has yet to be elucidated. Here, we show that while CyP A binds both Gag and mature CA proteins, the two binding interactions are actually different. Tryptophan 121 (W121) in CyP A distinguished the two proteins: a phenylalanine substitution (W121F) impaired binding of mature CA protein but not of Gag. This indicates the occurrence of a maturation-dependent switch in the conformation of the Pro-rich loop. A structural consequence of Gag binding to CyP A was to block this maturational refolding, resulting in a 24-kDa CA protein retaining the immature Pro-rich loop conformation. Using trypsin as a structure probe, we demonstrate that the conformation of the C-terminal region in mature CA is also a product of maturational refolding. Binding to wild-type CyP A altered this conformation, as indicated by a reduction in the accessibility of Cys residue(s) in the region to chemical modification. Hence, the end result of binding to CyP A, whether the Pro-rich loop is in the context of Gag or mature CA protein, is a structurally modified mature CA protein. The postassembly role of CyP A may be mediated through these modified mature CA proteins.

Animals↗
PubMed2001

Arthroscopic shoulder reconstruction: fast-track recovery and outpatient treatment.

Arthroscopic shoulder reconstructive surgery has been handled in many different ways. However, there currently is significant evidence and experience to show that doing this surgery on an outpatient basis is not only cost-effective and efficient, but safe and beneficial to patients. New arthroscopic surgical techniques and the use of regional interscalene anesthesia have been shown to provide effective and comfortable intraoperative conditions, while allowing for prolonged analgesia and quicker recovery with minimal side effects. The authors will discuss their approach to surgery, anesthesia, and recovery for outpatient shoulder reconstruction.

Ambulatory Surgical Procedures↗
PubMed2001

Identification of a novel membrane protein, HP59, with therapeutic potential as a target of tumor angiogenesis.

CM101, a polysaccharide isolated from the culture medium of Group B streptococcus, a neonatal pathogen, targets pathological angiogenesis and inhibits tumor growth in mice and humans. CM101 also targets neonatal lung and adult sheep lung endothelial cells. A gene encoding a transmembrane protein that interacts with CM101 was isolated from a sheep lung endothelial cell cDNA library. The gene, termed sp55, encodes a 495-amino acid polypeptide. COS-7 cells transfected with a vector containing sp55 express the SP55 protein-bound CM101 in a concentration-dependent manner. Stably transfected CHO cells also bound CM101. The corresponding human gene, hp59, was isolated from a human fetal lung cDNA library and had a predicted identity to SP55 of 86% over 495 amino acids. HP59 protein was shown by immunohistochemistry to be present in the pathological tumor vasculature of the lung, breast, colon, and ovary, but not in the normal vasculature, suggesting that the protein may be critical to pathological angiogenesis. The hp59 gene and/or the HP59 protein was not expressed in a variety of normal tissues, but was significantly expressed in human fetal lung, consistent with the pathophysiology of Group B streptococcus infections in neonates. Mice immunized with HP59 and SP55 peptides showed significant attenuation of tumor growth. Immunization effectively inhibited both the tumor angiogenesis and vasculogenesis processes, as evidenced by lack of both HP59- and CD34-positive vessels. These results and the immunohistochemistry data suggest a therapeutic potential for the CM101 target protein HP59 both as a drug target and as a vaccine against pathoangiogenesis.

Amino Acid Sequence↗
PubMed2001

Magnesium: its proven and potential clinical significance.

Magnesium is the fourth most abundant cation in the body and is present in more than 300 enzymatic systems, where it is crucial for adenosine triphosphate (ATP) metabolism. Deficiency states result in increased insulin resistance, as well as increased smooth muscle and platelet reactivity. Magnesium deficiency has been shown to correlate with a number of chronic cardiovascular diseases, including hypertension, diabetes mellitus, and hyperlipidemia. Intravenous magnesium has been used therapeutically in critical situations such as status asthmaticus, torsades de pointes, and preeclampsia. Few controlled studies exist regarding the therapeutic uses of oral magnesium supplementation in chronic cardiovascular diseases. Randomized clinical trials are urgently needed to determine whether magnesium supplementation will alter the natural history of these disease states.

Arrhythmias, Cardiac↗
PubMed2000

Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defensE of floral reproductive tissues.

Nectarin I, a protein that accumulates in the nectar of Nicotiana sp. , was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 degrees C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H(2)O(2) or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn(2+) addition. Addition of Fe(2+), Cu(2+), Zn(2+), and Cu(2+)/Zn(2+) did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H(2)O(2). Putative nectarin I homologues were found in the nectars of several other plant species.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Binding of equine infectious anemia virus matrix protein to membrane bilayers involves multiple interactions.

Human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) are closely related lentiviruses that infect immune cells, but their pathogenesis differ. Localization to the cytosolic leaflet of the plasma membrane is critical for replication of both viruses. This localization is accomplished through the matrix (MA) domain of the Gag precursor protein. In HIV-1, association of MA to anionic membranes appears to be primarily driven by a linear cluster of basic residues in the MA domain and an N-myristoylation signal. Interestingly, the MA protein of EIAV does not contain either of these signals. To understand which factors could promote EIAV assembly we characterized the membrane binding properties of its MA protein using fluorescence and biochemical methods. We find that EIAV MA exists as a multimer in solution whose protein-protein interactions are destabilized by membrane binding. EIAV MA binds strongly to electrically neutral membranes as well as to negatively charged membranes. Fluorescence quenching and chemical modification techniques, as well as trypsin proteolysis, indicate a different exposure of the EIAV MA Trp residues when bound to the two types of membranes, and EIAV MA proteolysis by trypsin differs when bound to the two types of membranes. Based on these data and the known structures of closely related matrix proteins, we constructed a structural model. This model predicts that EIAV MA binds to negatively charged membranes, but EIAV MA has an additional membrane binding region rich in residues that partition favorably into the membrane headgroup region. This secondary site may play a role in early events of viral infection.

Amino Acid Sequence↗
PubMed2000

Transgenic expression of cyclin D1 in thymic epithelial precursors promotes epithelial and T cell development.

We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5- TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3epsilon transgenic and hCD3epsilon/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.

Animals↗
PubMed2000

Fractalkine modulates TNF-alpha secretion and neurotoxicity induced by microglial activation.

Among the chemokine family, fractalkine shows unusual properties: it exists as a membrane-bound and soluble protein, and both fractalkine and its receptor CX(3)CR1 are expressed predominantly in the central nervous system. In rat cell culture models, the chemokine fractalkine was expressed in neurons and microglia, but not in astrocytes and its receptor exclusively localized to microglial cells, where its expression was downregulated by treatment with the bacterial endotoxin (LPS). In microglial cultures, LPS (10 ng/ml) induced a marked increase in the release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). The effects of LPS on TNF-alpha secretion were partially blocked (30%) by fractalkine and the effects of fractalkine were reversed by a polyclonal anti-fractalkine antibody. When microglial-associated fractalkine was neutralized by anti-fractalkine antibody, the LPS response was increased by 80%, suggesting tonic activation of microglial fractalkine receptors by endogenous fractalkine. The effects of the antibody were antagonized by the addition of fractalkine. LPS-activated microglia were neurotoxic when added to neuronal hippocampal culture, producing 20% neuronal death, as measured by NeuN-positive cell counting. An anti-fractalkine antibody produced neurotoxic effects of similar magnitude in this co-culture system and also markedly potentiated the neurotoxic effects of LPS-activated microglia (40% neuronal death). These results suggest that endogenous fractalkine might act tonically as an anti-inflammatory chemokine in cerebral tissue through its ability to control and suppress certain aspects of microglial activation. These data may have relevance to degenerative conditions such as multiple sclerosis, in which cerebral inflammatory processes may be activated.

Animals↗
PubMed2000

A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo.

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.

Animals↗
PubMed2000

Immunofluorescent microscopic investigation of the distal arrector pili: a demonstration of the spatial relationship between alpha5beta1 integrin and fibronectin.

Currently there is limited knowledge regarding the anatomy of the distal arrector pili (AP) muscle. A previous study implicated fibronectin and alpha5beta1 integrin binding as the anchor between the AP and the extracellular matrix (ECM). The purpose of this study was to strengthen this hypothesis. Serial frozen sections of human scalp skin were double-labeled via immunofluorescent staining for alpha5beta1 with fluorescein and fibronectin with rhodamine, followed by fluorescent microscopy. Granular staining for alpha5beta1 with fluorescein and smooth staining for fibronectin with rhodamine were seen at the periphery of the AP muscle bundles and along the distal fibers. Precise co-localization of alpha5beta1 and fibronectin was observed at the AP-ECM interface by means of a dual filter. Analysis of variance was used on the relative density of staining for each epitope. Staining for both epitopes was significantly brighter at the distal fibers than at the middle or proximal portions of the muscle. A computerized three-dimensional reconstruction provides a detailed picture of the microanatomy of the distal AP, which allows mathematical evaluation of the forces of contraction. The anatomic co-localization between alpha5beta1 and fibronectin strengthens our hypothesis that interaction of these epitopes mediates the attachment of the distal AP to the ECM.

Fibronectins↗
PubMed2000

Epidural cooling for spinal cord protection during thoracoabdominal aneurysm repair: A five-year experience.

PURPOSE: We developed and applied a method for providing regional spinal cord hypothermia with epidural cooling (EC) during thoracoabdominal aneurysm (TAA) repair. Preliminary results indicated significant reduction in spinal cord ischemic complications (SCI), compared with historical controls, and a 5-year experience with EC was reviewed. METHODS: From July 1993 to September 1998, 170 patients with thoracic aneurysms (n = 14; 8.2%) or TAAs (types I and II, n = 83 [49%]; type III, n = 66 [39%]; type IV, n = 7 [4.1%]) were treated with EC. An earlier aneurysm resection was noted in 44% of patients, an emergent operation was noted in 20% of patients, and an aortic dissection was noted in 16% of patients. The EC was successful (mean cerebrospinal fluid [CSF] temperature at cross-clamp, 26.4 +/- 3 degrees C) in 97% of cases, with all 170 patients included in an intention-to-treat analysis. The operation was performed with a clamp/sew technique (98% patients) and selective (T(9) to L(1) region) reimplantation of intercostal vessels. Clinical and EC variables were examined for association with operative mortality and SCI by means of the Fischer exact test, and those variables with a P value less than.1 were included in multivariate logistic regression analysis. RESULTS: The operative mortality rate was 9.5% and was weakly associated (P =.07) with SCI; postoperative cardiac complications (odds ratio [OR], 35. 3; 95% CI, 5.3 to 233; P <.001) and renal failure (OR, 32.2; 95% CI, 6.6 to 157; P <.001) were the only independent predictors of postoperative death. SCI of any severity occurred in 7% of cases (type I/II, 10 of 83 [12%]; all other types, 2 of 87 [2.3%]), versus a predicted (Acher model) incidence of 18.5% for this cohort (P =. 003). Half the deficits were minor, with good functional recovery, and devastating paraplegia occurred in three patients (2.0%). Independent correlates of SCI included types I and II TAA (OR, 8.0; 95% CI, 1.4 to 46.3; P =.021), nonelective operation (OR, 8.3, 95% CI, 1.8 to 37.7; P =.006), oversewn T(9) to L(2) intercostal vessels (OR, 6.1; 95% CI, 1.3 to 28.8; P =.023), and postoperative renal failure (OR, 23.6; 95% CI, 4.4 to 126; P <.001). These same clinical variables of nonelective operations (OR, 7.7; 95% CI, 1.4 to 41.4; P =.017), oversewn T(9) to L(2) intercostal arteries (OR, 9.7; 95% CI, 1.5 to 61.2; P =.016), and postoperative renal failure (OR, 20.8; 95% CI, 3.0 to 142.1; P =.002) were independent predictors of SCI in the subgroup analysis of high-risk patients, ie, patients with type I/II TAA. CONCLUSION: EC has been effective in reducing immediate, devastating, total paraplegia after TAA repair. A strategy that combines the neuroprotective effect of regional cord hypothermia, avoiding the sacrifice of potential spinal cord blood supply, and postoperative adjuncts (eg, avoidance of hypotension, CSF drainage) appears necessary to minimize SCI after TAA repair.

Adolescent↗
PubMed2000

Effect of the factor V Leiden mutation on the clinical expression of severe hemophilia A.

To determine whether the factor V Leiden mutation is associated with decreased bleeding in individuals with severe hemophilia A, factor concentrate utilization, maximum annual number of bleeding episodes, and the prevalence of hemophilic arthropathy between carriers and non-carriers of the factor V Leiden mutation were compared. Heterozygosity for the factor V Leiden mutation was found in 6 of 137 subjects (4.4%). Carriers of the factor V Leiden mutation utilized less factor concentrate (geometric mean: 310 vs. 1185 units/kg/year) and had fewer bleeding episodes than non-carriers (proportion with 10 or fewer bleeding episodes in their worst year: 50 vs. 11%). However, the factor V Leiden mutation was not associated with the absence of arthropathy. The intron 22 inversion mutation of the factor VIII gene was tested for in a subgroup of 80 subjects, but it was not found to be a significant variable for any of the bleeding endpoints. The results of this small study are consistent with the hypothesis that the factor V Leiden mutation imparts a protective effect; however, a larger confirmatory study in which the factor VIII molecular defects can be controlled for is needed. Furthermore, most severe hemophiliacs who used fewer than 200 units/kg/year of factor concentrate or who had experienced 10 or fewer bleeding episodes per year did not carry the factor V Leiden mutation, suggesting that the proportion of severe hemophiliacs whose mild clinical course can be attributed to the factor V Leiden mutation is small.

Adolescent↗
PubMed1999

Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses.

Nonmyeloablative allogeneic stem cell transplantation has recently been explored as a safer alternative to conventional high-dose transplant regimens. Although a high incidence of mixed chimerism after nonmyeloablative procedures has been reported, the exact kinetics of engrafting donor cells in specific cellular lineages has yet to be defined. We investigated lineage-specific chimerism in 15 patients receiving an allogeneic peripheral blood stem cell (PBSC) transplant from an HLA-identical (n = 14) or a 5/6 antigen-matched sibling donor after a preparative regimen of cyclophosphamide and fludarabine. Donor chimerism was assessed weekly in T lymphocytes and myeloid cells by polymerase chain reaction (PCR) of minisatellite regions. Eight patients survived between 121 to 409 days after transplant. Ten of 14 patients surviving more than 30 days (71.4%) had delayed disease regression consistent with a graft-versus-malignancy (GVM) effect. One patient rejected the transplant with subsequent recovery of autologous hematopoiesis. Hematological recovery was rapid (median, 11 days to >/=500 neutrophils/microL) and was initially predominantly recipient in origin. Donor myeloid chimerism gradually supplanted recipient hematopoiesis and became fully donor in all survivors by 200 days after transplantation. In contrast, T-cell engraftment was more rapid, with full chimerism in 7 patients by day 30 and in 6 further patients by day 200 after cyclosporine withdrawal and donor lymphocyte infusion. Full donor T-cell engraftment preceded donor myeloid engraftment, acute graft-versus-host disease, and disease regression, consistent with a requirement for 100% donor T-cell chimerism for full expression of the alloresponse. These results emphasize the importance of lineage-specific chimerism analysis to successfully manipulate engraftment after nonmyeloablative allogeneic PBSC transplantation.

Adult↗
PubMed1999

A phase I study of a 4-week course of SDZ-RAD (RAD) quiescent cyclosporine-prednisone-treated renal transplant recipients.

BACKGROUND AND METHODS: This phase I, randomized, blinded, placebo-controlled study assessed the safety profile and pharmacokinetics of a 4-week course of once-daily, sequential ascending doses (0.75, 2.5, or 7.5 mg/day) of SDZ-RAD (RAD) capsules in renal transplant recipients on a stable regimen of cyclosporine (CsA; Neoral) and prednisone. RESULTS: RAD displayed a similar spectrum of side effects as observed with rapamycin, namely, an increased incidence of infection associated with the augmented immunosuppression and a dose-related occurrence of thrombocytopenia, hypercholesterolemia, and hypertriglyceridemia, particularly at the 7.5-mg dose. The pharmacokinetic parameters of RAD showed dose proportionality, a good correlation between trough and area under the curve (AUC) concentrations, and a moderate accumulation of 2.5-fold. The drug was absorbed within 2 hr and displayed a 16-19-hr half-life, which is shorter than that of rapamycin. RAD reached steady state at 4 days. Preliminary kinetic-dynamic correlations indicate a correlation between thrombocytopenia (but not hyperlipidemia) and AUC, as well as maximum drug concentrations, and weight-adjusted dose. At the end of a 4-week course of simultaneous dosing, there was no evidence of a pharmacokinetic interaction between CsA and RAD. CONCLUSION: This study suggests that the shorter half-life of RAD compared to rapamycin may confer the benefits of rapid attainment of steady state and dissipation of effects upon drug cessation. Controlled, multicenter trials are being planned to assess the impact of these features on clinical outcomes.

Adult↗
PubMed1999

Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.

To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that the MDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Calcium mobilization in human myeloid cells results in acquisition of individual dendritic cell-like characteristics through discrete signaling pathways.

We have shown previously that calcium ionophore (CI) treatment of various myeloid origin cells results in rapid acquisition of properties associated with mature, activated dendritic cells. These properties include increased CD83 and costimulatory molecule expression, tendencies to form dendritic processes, loss of CD14 expression by monocytes, and typically an enhanced capacity to sensitize T lymphocytes to Ag. We here analyze the intracellular signaling pathways by which CI induces acquisition of such properties. Thapsigargin, which raises intracellular Ca2+ levels by antagonizing its sequestration, induced immunophenotypic and morphologic changes that paralleled CI treatment. CI-induced activation was broadly attenuated by the Ca2+ chelating compound EGTA and by calmodulin antagonists trifluoperazine dimaleate and W-7. However, antagonists of signaling pathways downstream to calmodulin displayed more selective inhibitory effects. Calcineurin antagonists cyclosporin A and the FK-506 analogue, ascomycin, diminished costimulatory molecule and CD83 expression, as well as formation of dendritic processes in CI-treated myeloid cells, and strongly attenuated the T cell allosensitizing capacity of CI-treated HL-60 cells. These calcineurin antagonists displayed minimal effect on CI-induced CD14 down-regulation in monocytes. In contrast, the calmodulin-dependent protein kinase antagonists, K252a and KT5926, while displaying only modest effects on CI-induced costimulatory molecule and CD83 expression, strongly blocked CD14 down-regulation. These results are consistent with a Ca2+-dependent mechanism for CI-induced differentiation of myeloid cells, and indicate that multiple discrete signaling pathways downstream to calcium mobilization and calmodulin activation may be essential in regulating this process.

Antigens, CD↗