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Showing papers by "Case Western Reserve University published in 1993"


Journal ArticleDOI
TL;DR: Current research in this area is directed toward the identification and structural characterization of nucleotide or P2-purinergic receptors that are activated when ATP or other nucleotides are bound.
Abstract: Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.

1,271 citations


Journal ArticleDOI
01 Nov 1993-Nature
TL;DR: It is reported here that a T-helper-1 response to glutamate decarboxylase develops in NOD mice at the same time as the onset of insulitis, and it is suggested that spontaneous autoimmune disease can be prevented by tolerization to the initiating target antigen.
Abstract: INSULIN-DEPENDENT diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice results from the T-lymphocyte-mediated destruction of the insulin-producing pancreatic β-cells and serves as a model for human IDDM1. Whereas a number of autoantibodies are associated with IDDM2, it is unclear when and to what β-cell antigens pathogenic T cells become activated during the disease process. We report here that a T-helper-1 (Thl) response to glutamate decarboxylase develops in NOD mice at the same time as the onset of insulitis. This response is initially limited to a confined region of glutamate decarboxylase, but later spreads intramolecularly to additional determinants. Subsequently, T-cell reactivity arises to other β-cell antigens, consistent with intermolecular diversification of the response. Prevention of the spontaneous anti-glutamate decarboxylase response, by tolerization of glutamate decarboxylase-reactive T cells, blocks the development of T-cell autoimmunity to other β-cell antigens, as well as insulitis and diabetes. Our data suggest that (1) glutamate decarboxylase is a key target antigen in the induction of murine IDDM; (2) autoimmunity to glutamate decarboxylase triggers T-cell responses to other β-cell antigens, and (3) spontaneous autoimmune disease can be prevented by tolerization to the initiating target antigen.

1,106 citations


Journal ArticleDOI
22 Jan 1993-Science
TL;DR: Human neuroblastoma cells transfected with constructs expressing wild-type beta APP or a mutant, beta APP delta NL, recently linked to familial AD were compared and this mutant beta APP may cause AD because its processing is altered in a way that releases increased amounts of A beta.
Abstract: The 4-kilodalton amyloid beta protein (A beta), which forms fibrillar deposits in Alzheimer's disease (AD), is derived from a large protein referred to as the amyloid beta protein precursor (beta APP). Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or a mutant, beta APP delta NL, recently linked to familial AD were compared. After continuous metabolic labeling for 8 hours, cells expressing beta APP delta NL had five times more of an A beta-bearing, carboxyl terminal, beta APP derivative than cells expressing wild-type beta APP and they released six times more A beta into the medium. Thus this mutant beta APP may cause AD because its processing is altered in a way that releases increased amounts of A beta.

971 citations


Journal ArticleDOI
TL;DR: The risk for bleeding can be estimated in an individual patient, giving the primary physician a quantitative basis for weighing the risks and benefits of therapy and for optimizing patient management.

888 citations


Journal ArticleDOI
TL;DR: The body is a biological, material entity, while embodiment can be understood as an indeterminate methodological field defined by perceptual experience and the mode of presence and engagement in the world as mentioned in this paper.
Abstract: Embodiment as a paradigm or methodological orientation requires that the body be understood as the existential ground of culture—not as an object that is “good to think,” but as a subject that is “necessary to be.” To argue by analogy, a phenomenological paradigm of embodiment can be offered as an equivalent, and complement, to the semiotic paradigm of culture as text. Much as Roland Barthes (1986) draws a distinction between the work and the text, a distinction can be drawn between the body and embodiment. For Barthes, the work is a fragment of substance, the material object that occupies the space of a bookstore or a library shelf. The text, in contrast, is an indeterminate methodological field that exists only when caught up in a discourse, and that is experienced only as activity and production (1986:57–68). In parallel fashion, the body is a biological, material entity, while embodiment can be understood as an indeterminate methodological field defined by perceptual experience and the mode of presence and engagement in the world. As applied to anthropology, the model of the text means that cultures can be understood, for purposes of internal and comparative analysis, to have properties similar to texts (Ricoeur 1979). In contrast, the paradigm of embodiment means not that cultures have the same structure as bodily experience, but that embodied experience is the starting point for analyzing human participation in a cultural world.

789 citations


Journal ArticleDOI
TL;DR: It is concluded that rMuIL-12 prevents deleterious Th2 T cell responses and promotes curative Th1 responses in an IFN-gamma- dependent fashion during murine leishmaniasis.
Abstract: Resistant C57BL/6 mice infected with Leishmania major are self-healing, whereas susceptible BALB/c mice fail to contain cutaneous infection and subsequently undergo fatal visceral dissemination. These disparate outcomes are mediated by dissimilar expansions of T helper type 1 (Th1) and Th2 CD4+ T lymphocyte subsets in vivo during cure and progression of disease. Because interleukin 12 (IL-12) has potent T cell growth and interferon gamma (IFN-gamma) stimulatory effects, we studied its effect on CD4+ T cell differentiation during murine leishmaniasis. Treatment with recombinant murine (rMu)IL-12 during the first week of infection cured 89% of normally susceptible BALB/c mice, as defined by decreased size of infected footpads and 1,000-10,000-fold reduced parasite burdens, and provided durable resistance against reinfection. Cure was associated with markedly depressed production of IL-4 by lymph node cells cultured with antigen or mitogen, but preserved or increased production of IFN-gamma relative to untreated mice. IL-4 and IFN-gamma mRNA associated with CD4+ T lymphocytes isolated from infected lymph nodes showed similar reciprocal changes in response to rMuIL-12 therapy. A single injection of anti-IFN-gamma monoclonal antibody abrogated the protective effect of rMuIL-12 therapy and restored Th2 cytokine responses. We conclude that rMuIL-12 prevents deleterious Th2 T cell responses and promotes curative Th1 responses in an IFN-gamma-dependent fashion during murine leishmaniasis. Since BALB/c leishmaniasis cannot be cured with rMuIFN-gamma alone, additional direct effects of IL-12 during T cell subset selection are suggested. Because rMuIL-12 is uniquely protective in this well-characterized model of chronic parasitism, differences in IL-12 production may underlie heterogenous host responses to L. major and other intracellular pathogens.

764 citations


Journal ArticleDOI
TL;DR: The tendency for people with high self-esteem to make inflated assessments and predictions about themselves carries the risk of making commitments that exceed capabilities, thus leading to failure, and the danger of letting egotistical illusions interfere with self-regulation processes is indicated.
Abstract: The tendency for people with high self-esteem to make inflated assessments and predictions about themselves carries the risk of making commitments that exceed capabilities, thus leading to failure. Ss chose their performance contingencies in a framework where larger rewards were linked to a greater risk of failure. In the absence of ego threat, Ss with high self-esteem showed superior self-regulation: They set appropriate goals and performed effectively. Ego threat, however, caused Ss with high self-esteem to set inappropriate, risky goals that were beyond their performance capabilities so they ended up with smaller rewards than Ss with low self-esteem. The results indicate the danger of letting egotistical illusions interfere with self-regulation processes. (PsycINFO Database Record (c) 2012 APA, all rights reserved) KW: partner violence

540 citations


Journal ArticleDOI
01 Dec 1993-Neuron
TL;DR: Analysis of the mutant in the olfactory bulb suggests that the mutant phenotype involves a defect in cell migration, possibly through specific loss of the polysialylated form of N-CAM-180, which is expressed in the migration pathway.

487 citations


Journal ArticleDOI
TL;DR: In this article, a dynamic model of auto-immunity with T-cell recruitment and selection leading to changes in the specificity of the anti-self response during the course of disease is proposed.

444 citations


Journal ArticleDOI
TL;DR: Results indicated that the use of transverse field steering current improved selectivity in cats to study selective activation of medial gastrocnemius, soleus, tibialis anterior, and extensor digitorium longus with a cuff electrode.
Abstract: Acute experiments were performed on adult cats to study selective activation of medial gastrocnemius, soleus, tibialis anterior, and extensor digitorium longus with a cuff electrode. A spiral nerve cuff containing twelve dot electrodes was implanted around the sciatic nerve, and evoked muscle twitch forces were recorded in six experiments. Spatially isolated dot electrodes in four geometries (monopolar, longitudinal tripolar, tripolar with four common anodes, and two parallel tripoles) were combined with transverse field steering current(s) from an anode(s) located 180 degrees around from the cathode(s) to activate different regions of the nerve trunk. A selectivity index was used to construct recruitment curves for a muscle with the optimal degree of selectivity. Physiological responses were correlated with the anatomical structure of the sciatic nerve by identifying the nerve fascicles innervating the four muscles, and by determining the relative positions of the electrodes and the nerve fascicles. The results indicated that the use of transverse field steering current improved selectivity. The relative performance of the various electrode arrangements is discussed. >

420 citations


Journal ArticleDOI
TL;DR: It is shown that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase, providing a basis for how cal modulin may regulate NO synthesis.
Abstract: Nitric oxide (NO) is synthesized within the immune, vascular, and nervous systems, where it acts as a wide-ranging mediator of mammalian physiology. The NO synthases (EC 1.14.13.39) isolated from neurons or endothelium are calmodulin dependent. Calmodulin binds reversibly to neuronal NO synthase in response to elevated Ca2+, triggering its NO production by an unknown mechanism. Here we show that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase. Calmodulin-triggered electron transfer to heme was independent of substrate binding, caused rapid enzymatic oxidation of NADPH in the presence of O2, and was required for NO synthesis. An NO synthase isolated from cytokine-induced macrophages that contains tightly bound calmodulin catalyzed spontaneous electron transfer to its heme, consistent with bound calmodulin also enabling electron transfer within this isoform. Together, these results provide a basis for how calmodulin may regulate NO synthesis. The ability of calmodulin to trigger electron transfer within an enzyme is unexpected and represents an additional function for calcium-binding proteins in biology.

Journal Article
TL;DR: The presence of major alterations in the tumor-infiltrating lymphocytes with only minor changes in the peripheral blood leukocyte T-cells supports the notion that the defects are induced by exposure to tumor.
Abstract: Cancer patients and mice bearing tumors develop a progressive immunosuppression manifested by a decreased delayed-type hypersensitivity, decreased T-cell lytic activity, diminished production of lymphokines, and a reduced T-cell proliferative response. The mechanisms underlying these changes are incompletely understood. We recently reported the presence of marked alterations in signal transduction in T-cells from mice bearing long-term (28-day) tumors. We hypothesized that a soluble product produced by the tumor or resulting from the immune response to tumor might be responsible for inducing the changes in T-cells. Tumor-infiltrating lymphocytes from patients with renal cell carcinoma tested here showed, in 10 of 11 cases, a marked decrease in the expression of the T-cell receptor ζ chain and in p56 lck tyrosine kinase. The presence of major alterations in the tumor-infiltrating lymphocytes with only minor changes in the peripheral blood leukocyte T-cells supports the notion that the defects are induced by exposure to tumor. These results suggest that tumor-infiltrating lymphocytes may be compromised in their antitumor efficacy in patients with renal cell cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Journal ArticleDOI
TL;DR: The improvement in cognitive function during clozapine treatment could have consequences for capacity to work and social function and there was some evidence for a relationship between improvement in psychopathology and cognitive function.

Journal ArticleDOI
TL;DR: The T2* signal response associated with cortical activation due to finger motion at 1.5 Tesla is measured, consistent with reducing partial volume effects and a simple difference in phase between the intravascular signal and surrounding parenchyma.
Abstract: We have measured the T2* signal response associated with cortical activation due to finger motion at 1.5 Tesla. Both thin slice 2D and 3D images show signal intensity changes which vary from 2% to 32% depending on volunteer, echo time, slice thickness, and in-plane resolution. The largest signal change occurred for the thinnest slices and highest resolution (2 mm3). This is consistent with reducing partial volume effects and a simple difference in phase between the intravascular signal and surrounding parenchyma. No inflow enhancement was seen on the 2D or 3D scans, confirming the nature of the signal difference for this approach was due to local field in-homogeneity effects. Using 3D imaging, multiple effects can be seen simultaneously. With a 3D MRA method, it was possible to locate the vessel that was the source of the T2* behavior; it was in each case a vein on the surface of the cortical parenchyma.

Journal ArticleDOI
TL;DR: This article explored unreciprocated romantic attraction by comparing narrative accounts and found that would-be lovers believed that the attraction had been mutual, that they had been led on, and that the rejection had never been communicated definitely.
Abstract: Unreciprocated romantic attraction was explored by comparing narrative accounts. Unrequited love emerged as a bilaterally distressing experience marked by mutual incomprehension and emotional interdependence. Would-be lovers looked back with both positive and intensely negative emotions, whereas rejectors were more uniformly negative in their accounts. Unlike rejectors, would-be lovers believed that the attraction had been mutual, that they had been led on, and that the rejection had never been communicated definitely. Rejectors depicted themselves as morally innocent but still felt guilty about hurting someone; many rejectors depicted the would-be lover's persistent efforts as intrusive and annoying

Journal ArticleDOI
TL;DR: There is currently no uniformly accepted or proven approach for prevention of fungal infections in neutropenic patients with acute leukemia and fluconazole is a new triazole antifungal agent with activity against many common fungal pathogens causing infection in patients with severe liver disease.
Abstract: OBJECTIVE To evaluate the efficacy and safety of fluconazole for prevention of fungal infections. DESIGN A randomized, placebo-controlled, double-blind, multicenter trial. PATIENTS Adults (257) undergoing chemotherapy for acute leukemia. INTERVENTION Patients were randomly assigned to receive either fluconazole (400 mg orally once daily or 200 mg intravenously every 12 hours) or placebo. The study drug was started at initiation of chemotherapy and continued until recovery of neutrophil count, development of proven or suspected invasive fungal infection, or the occurrence of a drug-related toxicity. MEASUREMENTS Fungal colonization, proven superficial or invasive fungal infection, empiric antifungal therapy with amphotericin B, drug-related side effects, and mortality. MAIN RESULTS Fluconazole decreased fungal colonization (83 of 122 [68%] placebo patients compared with 34 of 119 [29%] fluconazole patients colonized at end of prophylaxis, P < 0.001) and proven fungal infections (27 of 132 [21%] placebo patients compared with 11 of 123 [9%] fluconazole patients infected, P = 0.02). Superficial fungal infections occurred in 20 of 132 (15%) placebo patients but in only 7 of 123 (6%) fluconazole patients (P = 0.01), whereas invasive fungal infections developed in 10 of 132 (8%) placebo patients and in 5 of 123 (4%) fluconazole patients (P = 0.3). Fluconazole was especially effective in eliminating colonization and infection by Candida species other than Candida krusei (66 of 122 [64%] placebo patients colonized at end of prophylaxis compared with 11 of 119 [9%] fluconazole patients, P < 0.001; 22 of 132 [17%] placebo patients infected compared with 7 of 123 [6%] fluconazole patients, P = 0.005). Aspergillus infections were infrequent in both fluconazole (3 cases) and placebo groups (3 cases). The use of amphotericin B, the incidence of drug-related side effects, and overall mortality were similar in both study groups. CONCLUSION Prophylactic fluconazole prevents colonization and superficial infections by Candida species other than Candida krusei in patients undergoing chemotherapy for acute leukemia and is well tolerated. Fluconazole could not be clearly shown to be effective for preventing invasive fungal infections, reducing the use of amphotericin B, or decreasing the number of deaths.

Journal ArticleDOI
TL;DR: It is concluded that FDG PET may give useful information on breast masses and axillary node status prior to surgery.
Abstract: Twenty-eight patients with a total of 35 suspect breast masses underwent positron emission tomography (PET) with [fluorine-18] 2-deoxy-2-fluoro-D-glucose (FDG) in order to study the utility of this technique in the evaluation of breast cancer. FDG PET allowed discrimination between eight benign and 27 malignant breast masses, with a sensitivity of 96% and specificity of 100%. Among the malignancies, there was a significant correlation between normalized FDG uptake and nuclear grade (P = .006). In addition, the results of PET imaging were compared with results of axillary node dissection in 20 cases of breast cancer. PET allowed correct categorization of 10 of 10 axillae as negative (specificity = 100%). PET results were equivocal in one axilla and positive in the remaining nine of 10 axillae with positive dissection results (sensitivity = 90%). The authors conclude that FDG PET may give useful information on breast masses and axillary node status prior to surgery.

Journal ArticleDOI
TL;DR: It is suggested that glucocorticoid‐in‐duced elevations in extracellular glutamate concentrations may contribute to the deleterious effects of stress on hippocampal neurons.
Abstract: Glucocorticoids and stress have deleterious effects on hippocampal cell morphology and survival. It has been hypothesized that these effects are mediated via an excitatory amino acid mechanism. The present study was designed to evaluate the effects of acute stress on the extracellular levels of glutamate in the hippocampus and to determine if adrenalectomy modifies this response. Rats were adrenalectomized or sham-adrenalectomized and implanted with microdialysis probes in the CA3 region of the hippocampus. Three days later rats were subjected to an acute 1-h period of immobilization stress. Stress significantly increased extracellular glutamate levels in the sham-operated rats, which peaked at 20 min following the initiation of stress. Extracellular glutamate levels also increased immediately following the termination of stress. In the adrenalectomized rats there was a 30% decrease in basal extracellular concentrations of glutamate and a marked attenuation (-70%) of the stress-induced increase in extracellular glutamate levels. Extracellular concentrations of taurine were not modified by adrenalectomy and did not change in response to stress. These results suggest that glucocorticoid-induced elevations in extracellular glutamate concentrations may contribute to the deleterious effects of stress on hippocampal neurons.

Journal ArticleDOI
01 Jan 1993-Science
TL;DR: Antisense blocking of IGF-I expression may reverse a phenotype that allows C6 glioma cells to evade the immune system and cause regression of established brain glioblastomas when injected at a point distal to the tumor.
Abstract: Rat C6 glioma cells express insulin-like growth factor I (IGF-I) and form rapidly growing tumors in syngeneic animals. When transfected with an episome-based vector encoding antisense IGF-I complementary DNA, these cells lost tumorigenicity. Subcutaneous injection of IGF-I antisense-transfected C6 cells into rats prevented formation of both subcutaneous tumors and brain tumors induced by nontransfected C6 cells. The antisense-transfected cells also caused regression of established brain glioblastomas when injected at a point distal to the tumor. These antitumor effects result from a glioma-specific immune response involving CD8+ lymphocytes. Antisense blocking of IGF-I expression may reverse a phenotype that allows C6 glioma cells to evade the immune system.

Journal ArticleDOI
TL;DR: In this article, the authors employed polarized epithelial monolayers to neutralize intracellular microbial pathogens, such as viruses, directly within epithelial cells and to bind antigens in the mucosal lamina propria and excrete them through the adjacent epithelium into the lumen.

Journal ArticleDOI
TL;DR: The range of the posterior atlanto-odontoid interval that was associated with poor or no recovery after the operation was observed, and the safe range was identified on the basis of the patients in whom paralysis did not develop.
Abstract: We analyzed the cases of seventy-three patients who were managed over a twenty-year period for rheumatoid involvement of the cervical spine and were followed for a minimum of two years, with an average follow-up of seven years. A neurological deficit did not develop in thirty-one patients (Ranawat et al. Class I) and paralysis developed in the remaining forty-two patients: Class II in eleven and Class III in thirty-one. Of the forty-two patients in whom paralysis developed, thirty-five had operative stabilization. Seven patients were managed with a soft cervical collar because they refused or were medically unable to have the operation; all of the had an increase in the severity of the paralysis. The posterior atlanto-odontoid interval and the diameter of the subaxial sagittal canal measured on the cervical radiographs demonstrated statistically significant correlations with the presence and severity of paralysis. All of the patients who had a Class-III neurological deficit had a posterior atlanto-odontoid interval or diameter of the subaxial canal that was less than fourteen millimeters. In contrast, the anterior atlanto-odontoid interval, which has traditionally been reported, did not correlate with paralysis. The prognosis for neurological recovery following the operation was not affected by the duration of the paralysis but was influenced by the severity of the paralysis at the time of the operation. The most important predictor of the potential for neurological recovery after the operation was the preoperative posterior atlanto-odontoid interval. In patients who had paralysis due to atlanto-axial subluxation, no recovery occurred if the posterior atlanto-odontoid interval was less than ten millimeters, whereas recovery of at least one neurological class always occurred when the posterior atlanto-odontoid interval was at least ten millimeters. If basilar invagination was superimposed, clinically important neurological recovery occurred only when the posterior atlanto-odontoid interval was at least thirteen millimeters. All patients who had paralysis and a posterior atlanto-odontoid interval or diameter of the subaxial canal of fourteen millimeters had complete motor recovery after the operation. In this series, although only patients who had a neurological deficit were operated on, we observed the range of the posterior atlanto-odontoid interval that was associated with poor or no recovery after the operation, and we identified the safe range on the basis of the patients in whom paralysis did not develop.(ABSTRACT TRUNCATED AT 400 WORDS)

Journal ArticleDOI
TL;DR: In this paper, the authors identify key buyers' behaviors in high technology markets and identify the key factors that influence buyer behavior in high-technology markets, focusing on specific outcomes (product adoption or rejection) as opposed to buyers' underlying processes.
Abstract: Previous research on buyer behavior in high technology markets has focused on specific outcomes (product adoption or rejection) as opposed to buyers’ underlying processes. The authors identify key ...

Journal ArticleDOI
TL;DR: This chapter is intended as an overview of the inflammatory and infectious responses to implanted devices and its biocompatibility and its success or failure as a device or prosthesis.

Journal ArticleDOI
TL;DR: These findings demonstrate that there is a cytosolic pathway for folding tubulin and actin in vivo that involves the TCP1 complex and could be immunoprecipitated by using an anti-TCP1 antibody.
Abstract: A role in folding newly translated cytoskeletal proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide 1 (TCP1). In this study, we investigated tubulin and actin biogenesis in Chinese hamster ovary (CHO) cells. When extracts of pulse-labeled cells were analyzed by anion-exchange and size-exclusion chromatography, newly synthesized alpha-tubulin, beta-tubulin, and actin were observed to enter a large molecular mass complex (approximately 900 kDa). These proteins were released from this complex capable, in the case of tubulin, of forming heterodimers. The large molecular mass complexes coeluted with TCP1 and could be immunoprecipitated by using an anti-TCP1 antibody. These findings demonstrate that there is a cytosolic pathway for folding tubulin and actin in vivo that involves the TCP1 complex.

Journal ArticleDOI
TL;DR: This review on endothelins in the kidney addressed major topics addressed in this review on endothelin isopeptides: a gene family of regulatory peptides.
Abstract: Major topics addressed in this review on endothelins in the kidney are; 1) endothelin isopeptides: a gene family of regulatory peptides; 2) mapping endothelin receptors in the kidney: sites of action; 3) endothelin gene expression and secretion in the kidney; 4) endothelin regulates renal hemodynamics; 5) effects of endothelin on glomerular filtration rate and glomerular function; 6) endothelin and renal sodium excretion; 7) endothelin and renal water reabsorption

Journal ArticleDOI
TL;DR: Thioltransferase (glutaredoxin) appears to be specific for glutathione-containing mixed disulfides, and in separate experiments, TTase from rat liver displayed analogous selectivity.
Abstract: To study the substrate specificity and mechanism of thioltransferase (TTase) catalysis, we have used 14C- and 35S-radiolabeled mixed disulfides of cysteine and glutathione (GSH) with various cysteine-containing proteins. These protein mixed disulfide substrates were incubated with glutathione, glutathione disulfide (GSSG) reductase, and NADPH in the presence or absence of thioltransferase. Glutathione-dependent reduction of protein mixed disulfides was monitored both by release of trichloroacetic acid soluble radiolabel and by formation of GSSG in an NADPH-linked spectrophotometric assay. GSH-dependent dethiolation of [35S]glutathione-papain mixed disulfide (papain-SSG) and the corresponding bovine serum albumin mixed disulfide (BSA-SSG) were catalyzed by thioltransferase (from human red blood cells) as shown by the radiolabel assay, and equivalent rates were measured by the spectrophotometric assay. Dethiolation of [35S]hemoglobin-glutathione mixed disulfide (Hb-SSG) was also catalyzed by TTase. In contrast, TTase did not catalyze GSH-dependent dethiolation of [14C]papain-SScysteine or [14C]BSA-SScysteine as measured by the radiolabel assay. [14C]Hb-SScysteine and Hb-SScysteamine also did not serve as substrates. In separate experiments, TTase from rat liver displayed analogous selectivity. Thus, thioltransferase (glutaredoxin) appears to be specific for glutathione-containing mixed disulfides. Apparent TTase catalysis of GSSG formation from the papain- and BSA-SScysteine mixed disulfides was observed by the spectrophotometric assay, but a lag phase occurred consistent with preenzymatic formation of GSScysteine which could serve as the actual TTase substrate. Two-substrate kinetic studies of TTase with GSH and GSScysteine gave patterns of parallel lines on double-reciprocal plots (1/V vs 1/[S]), consistent with a simple ping-pong mechanism involving a TTase-SSG intermediate.

Journal ArticleDOI
TL;DR: Not enough is known at present to conclude that IL-6 is a major participant in these changes in man, but an in vivo scenario in the acute phase response in which the hepatocyte receives a complex mixture of humoral or paracrine signals which are integrated by multiple interacting post-receptor and gene regulatory mechanisms to cause finely regulated changes in plasma protein synthesis is suggested.
Abstract: Cytokines appear to function as part of a complex regulatory network, a signaling language in which informational content resides in the combinations, and perhaps sequence, of cytokines and other extracellular messenger molecules received by a cell. The effects of combinations of cytokines are complex and often differ from their in vitro effects or when administered by themselves. (From this perspective, to use a somewhat crude simile, individual cytokines can be thought of as words which bear informational content. Although individual cytokines may, on occasion, communicate a complete message, more commonly the actual messages received by cells probably resemble sentences, in which it is the combination and sequence of words which convey information.) Currently available data suggest an in vivo scenario in the acute phase response in which the hepatocyte receives a complex mixture of humoral or paracrine signals which are integrated by multiple interacting post-receptor and gene regulatory mechanisms to cause finely regulated changes in plasma protein synthesis. Regulation often occurs by transcriptional control, but post-transcriptional mechanisms, including translational regulation, may participate. Both the extracellular and intracellular mechanisms that mediate the response of the hepatocyte to inflammatory stimuli appear to be highly complex and involve multiple overlapping, concurrent, and parallel pathways. Enough is known at present to conclude that IL-6 is a major participant in these changes in man. Regulation of non-hepatocyte acute phase phenomena has not been delineated as thoroughly, but clearly involves a number of cytokines.

Journal ArticleDOI
TL;DR: The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states.
Abstract: The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine N epsilon atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N delta atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5 degrees with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 degrees rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.

Journal ArticleDOI
01 Jan 1993-Vaccine
TL;DR: Oral vaccination with bacterial antigens plus cholera toxin and monoclonal IgA anti-H.

Journal ArticleDOI
TL;DR: The demonstration of impaired IL2RG mRNA expression in males with X-linked SCID and of unique point mutations in SCIDX1 pedigrees constitutes powerful evidence that the SCID X1 gene is IL2 RG, a candidate for the X- linked form of severe combined immunodeficiency at the SC IDX1 locus.
Abstract: The gene encoding the gamma chain of the lymphocyte interleukin-2 receptor has been cloned and shown to be required to associate with the beta chain in order for IL-2 internalization and cell activation to occur (1). We considered this gene, IL2RG, a candidate for the X-linked form of severe combined immunodeficiency at the SCIDX1 locus, in which affected males have impaired lymphocyte development. Using fluorescence in situ hybridization and PCR amplification of somatic cell hybrid DNAs, we mapped IL2RG to human Xq13.1, a location within the SCIDX1 critical region established by linkage analysis. The 4.2 kb IL2RG gene was sequenced, and its genomic organization was elucidated. Seven of 19 transformed B-lymphocyte cell lines with independent SCIDX1 mutations had absent or minimal IL2RG mRNA. Unique point mutations were documented to be specifically associated with the disease and the carrier state in four unrelated affected males and their family members: one in a boy with no detectable IL2RG mRNA, in which the mutation ablated a splice donor site; one causing premature chain termination; and two causing distinct amino acid changes. The demonstration of impaired IL2RG mRNA expression in males with X-linked SCID and of unique point mutations in SCIDX1 pedigrees constitutes powerful evidence that the SCIDX1 gene is IL2RG. Noguchi et al. (2) have independently published IL2RG mapping to Xq13 and discovery of mutations in three affected males. The specific pathogenesis of IL2RG mutations and approaches to gene therapy can now be addressed in the X-linked form of SCID.