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D J Reed

Publications and source records attributed to D J Reed.

At least 19 recordsLinked to original sources
PubMed2000

Primary structure and functional characterization of a soluble, alternatively spliced form of B7-1.

Recent studies have suggested that soluble forms of B7-1 and B7-2 may exist, but transcripts that code for these molecules have not been previously described. In this study, we report the cloning and characterization of an alternatively spliced soluble form of porcine B7-1 (sB7-1) that lacks exons coding for both the transmembrane and cytoplasmic domains. Northern blot analysis of RNA from alveolar macrophages revealed an approximate 3:1 ratio of the transmembrane form of B7-1 mRNA relative to sB7-1 mRNA. Porcine B7-1 was present on the surface of both B and T cells following stimulation with PMA/ionomycin. A histidine-tagged form of porcine sB7-1 (sB7-1-His) interacted with both CD28 and CTLA-4, and effectively blocked IL-2 production from human responder cells stimulated with PHA and either porcine or human stimulator cells. In addition, sB7-1-His inhibited human T cell proliferation in response to porcine or human peripheral blood leukocytes. This study is the first report of an alternatively spliced form of B7 that codes for a soluble protein. Furthermore, these data demonstrate that porcine B7-1 interacts with the human receptors CD28 and CTLA-4, suggesting a potential role for this molecule in pig to human xenotransplantation. Possible physiological functions for the soluble form of B7-1 are discussed.

Abatacept↗
PubMed1999

Role of glutathione and reactive oxygen intermediates in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced immune suppression in C57Bl/6 mice.

Recent developments in basic immunology have revealed the importance of glutathione (GSH) and cellular redox balance in the generation of an immune response. In the liver, it has been shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters cellular GSH and reactive oxygen intermediate (ROI) production. We have tested the hypothesis that TCDD mediates the suppression of the cytotoxic T lymphocyte (CTL) response to alloantigen by increasing oxidative stress. Total cellular GSH, GSSG, and GSH-protein adducts were analyzed by HPLC. Changes in intracellular GSH and ROI were simultaneously measured in isolated hepatocytes and individual subpopulations of spleen cells (CD4+, CD8+, B220+, and Mac-1+) following in vivo exposure to TCDD and antigenic challenge with P815 mastocytoma cells. Monochlorobimane was utilized to measure GSH levels, and two fluorescent probes were used to evaluate ROI levels: dichlorofluoroscein diacetate to monitor peroxides and dihydroethidine to assess superoxide anion. In hepatocytes, in vivo treatment with TCDD resulted in a transient, 2-fold increase in GSH, a 50% decrease in peroxide levels and a small (20-40%) decrease in superoxide anion levels. Although alloantigen challenge resulted in increased GSH and peroxide in spleen cells, in vivo exposure to TCDD had no effect on splenic ROI levels, nor did it consistently alter GSH levels in any subpopulation of spleen cells examined. Moreover, in vivo treatment with the antioxidant N-acetyl cysteine failed to affect the immune suppression caused by TCDD. These results suggest to us that although TCDD perturbs cellular redox balance in the liver, it does not exacerbate or diminish the normal increased GSH and ROI which occur in the spleen in response to antigenic challenge.

Animals↗
PubMed1998

Effect of cocaine, 95% oxygen and ellagic acid on the development and antioxidant status of cultured rat embryos.

Prenatal exposure to cocaine has been associated with adverse developmental effects and current data suggest cocaine induced malformations are caused by ischemic-reperfusion injury. This study was undertaken to assess a new in vitro model which uses a routine rat whole embryo culture system that incorporates a change in oxygen status, and to examine the effects of altered oxygen status and pretreatment with ellagic acid (EA), an anti-oxidant, after cocaine exposure. Embryos were evaluated by determining a developmental score and by measuring tissue reduced glutathione (GSH) levels. Following re-oxygenation with 95% O2 for the last 6 h of culture, embryos treated with cocaine had reduced developmental scores and GSH levels. Embryos treated with cocaine and not re-oxygenated with 95% O2 did not have reduced developmental scores. EA blocked the effects of cocaine on developmental score and GSH level. These data support ischemia-reperfusion injury as the mechanism of cocaine developmental toxicity.

Animals↗
PubMed1998

Biliary secretion of alpha-tocopherol and the role of the mdr2 P-glycoprotein in rats and mice.

The mechanism by which alpha-tocopherol (alpha-T) is secreted into the bile is not known; however, we have previously demonstrated that treatment with piperonyl butoxide (PIP, 1 g/kg) results in increased biliary output of both alpha-T and phosphatidylcholine within 3 h of ip injection in rats and that the biliary output of both substances was prevented by chemicals that disrupt microtubules (Toxicol. Appl. Pharmacol. 139, 411-417 (1996)). The P-glycoprotein (Pgp) encoded by the mdr2 gene has been shown to transport phosphatidylcholine into the bile; therefore, in the current study, we utilized the Pgp inhibitor verapamil to investigate the possible involvement of Pgps in the biliary secretion of alpha-T. When rats were iv injected with verapamil (4 mg/kg) 10 min prior to PIP treatment, verapamil prevented the PIP-induced increases in biliary alpha-T and phosphatidylcholine output and resulted in biliary alpha-T outputs that were significantly less than controls. Also, we determined that the biliary alpha-T levels in mdr2 knockout mice were 25% of those in wildtype mice; furthermore, mdr2 liver, lung, and kidney levels of alpha-T and glutathione differed from those of wildtype. To investigate the fate of biliary alpha-T, we injected 14C-labeled alpha-T into the bile duct cannulae of rats and determined that approximately 60% of the radioactivity was reabsorbed within 1 h. Our results indicate that alpha-T undergoes enterohepatic circulation and that the biliary secretion of alpha-T, basally and following chemical treatment, is dependent on the presence of a functioning mdr2 Pgp in rats and mice.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1998

Evaluation of the Galalpha1-3Gal epitope as a host modification factor eliciting natural humoral immunity to enveloped viruses.

Human sera contain high levels of natural antibody (Ab) to Galalpha1-3Gal, a terminal glycosidic structure expressed on the surface of cells of mammals other than Old World primates. Incorporation of this determinant onto retroviral membranes by passage of viruses in cells encoding alpha-1-3-galactosyltransferase (GT) renders retroviruses sensitive to lysis by natural Ab and complement in normal human serum (NHS). Plasma membrane-budding viruses representing four additional virus groups were examined for their sensitivities to serum inactivation after passage through human cell lines that lack a functional GT or human cells expressing recombinant porcine GT. The inactivation of lymphocytic choriomeningitis virus (LCMV) by NHS directly correlated with host modification of the virus via expression of Galalpha1-3Gal and was blocked by incorporation of soluble Galalpha1-3Gal disaccharide into the inactivation assay. GT-deficient mice immunized to make high levels of Ab to Galalpha1-3Gal (anti-Gal Ab) were tested for resistance to LCMV passaged in GT-expressing cells. Resistance was not observed, but in vitro analyses of the mouse immune sera revealed that the antiviral activity of the sera was insufficient to eliminate LCMV infectivity on its natural targets of infection, macrophages, which express receptors for Ab and complement. Newcastle disease virus and vesicular stomatitis virus (VSV) were inactivated by NHS regardless of cell passage history, whereas Sindbis virus (SV) passaged in human cells resisted inactivation. Both VSV and SV passaged in Galalpha1-3Gal-expressing human cells incorporated this sugar moiety onto their major envelope glycoproteins. SV passaged in mouse cells expressing Galalpha1-3Gal was moderately sensitive to inactivation by NHS. These results indicate that enveloped viruses expressing Galalpha1-3Gal differ in their sensitivities to NHS and that a potent complement source, such as that in NHS, is required for efficient inactivation of sensitive viruses in vitro and in vivo.

Animals↗
PubMed1997

Alteration of glycosylation renders HIV sensitive to inactivation by normal human serum.

Retroviruses from various mammalian species, excluding humans, are effectively inactivated in normal human serum (NHS). Recent studies have shown that NHS inactivation of retroviruses occurs through natural Ab recognition of a terminal glycosidic moiety on the viral envelope that is acquired during replication in the host cell. This carbohydrate structure (the alpha-galactosyl epitope) is expressed on the cells of most mammals, with the exception of humans and other Old World primates. In this study, NHS sensitivity of HIV was assessed following viral propagation in human cells that were manipulated to express the alpha-galactosyl epitope. HUT-78 cells were transduced with an exogenous alpha1-3-galactosyl transferase gene, which codes for the terminal glycosyl transferase responsible for generation of the alpha-galactosyl epitope. The transduced HUT-78 cells expressed high levels of the alpha-galactosyl epitope on their membrane surface, rendering them sensitive to killing in NHS. Similarly, HIV passaged through these cells acquired the alpha-galactosyl epitope in association with the envelope glycoprotein gp120 and was also effectively inactivated in NHS. Viral inactivation was abolished by the addition of a synthetic disaccharide that contains the alpha-galactosyl epitope, indicating that virolysis is mediated by anti-alpha-galactosyl natural Ab. These results demonstrate that, like other retroviruses bearing the alpha-galactosyl epitope, HIV modified to express this epitope is inactivated in NHS. Furthermore, these data suggest that expression of the alpha-galactosyl epitope on the surface of viruses may have implications in the interspecies transmission of such viruses to humans.

Animals↗
PubMed1996

Reaction of human hemoglobin toward the alkylating agent S-(2-chloroethyl)glutathione.

In order to investigate if hemoglobin might serve as a biomarker of exposure for 1,2-dichloroethane (DCE) encountered in the workplace, human hemoglobin was alkylated at physiologic pH by the episulfonium ion of S-(2-chloroethyl)glutathione (CEG). In vitro alkylation resulted in three alkylation products on the alpha chain and at least two alkylation products on the beta chain as determined directly by matrix-assisted laser desorption-ionization mass spectrometry. To ascertain if the site of alkylation was the reactive sulfhydryl present at cysteine-93 on the beta chain of hemoglobin (beta-93 Cys), a spectrophotometric assay using 4,4'-dithiodipyridine was used to measure the free sulfhydryl groups before and after treatment of hemoglobin with various amounts of CEG. Results indicate that the episulfonium ion did not react substantially at beta-93 Cys, as there was no measurable decrease in the sulfhydryl to hemoglobin ratio, even with a large excess of CEG. In contrast, iodoacetamide did react with the sulfhydryl groups and gave a dose-dependent decrease in the sulfhydryl to hemoglobin ratio as measured by this assay. CEG-treated hemoglobin was digested with Staphylococcus aureus endoproteinase Glu-C and the digest was analyzed by fast atom bombardment mass spectrometry. Only one peak in the FAB mass spectrum could correspond to a peptide modified by the episulfonium ion of CEG. These results indicate that although the episulfonium ion of CEG does alkylate human hemoglobin, beta-93 Cys is not the major alkylation target.

Alkylating Agents↗
PubMed1996

Colchicine and vinblastine prevent the piperonyl butoxide-induced increase in rat biliary output of alpha-tocopherol.

alpha-Tocopherol, a lipid-soluble chain-breaking antioxidant, is important in the protection of biologic membranes and is transported in the body in association with lipids. It has been estimated that the liver contains up to 29% of the alpha-tocopherol stores of the body. Piperonyl butoxide (PIP) is a widely used synthetic methylenedioxyphenyl insecticide synergist. Bile was collected for 1 hr prior to, and 5 hr following, i.p. injection of 1 g/kg PIP. Within 3 hr of PIP administration, biliary alpha-tocopherol output increased to 132% of pretreatment measurements. Hepatic alpha-tocopherol levels of PIP-treated rats decreased to 26% below those of controls at the end of the bile collection period. Biliary bile acid and phospholipid output also increased significantly following PIP treatment. In a second set of experiments, rats were pretreated with either colchicine or one of two doses of vinblastine; both colchicine and the higher dose of vinblastine prevented the PIP-induced decrease in hepatic alpha-tocopherol and the increase in biliary output of alpha-tocopherol, bile acids, and phospholipids. These results suggest that (i) PIP-induced biliary secretion of alpha-tocopherol is associated with altered biliary phospholipid output, (ii) a significant portion of hepatic alpha-tocopherol may function as a highly mobilizable source of antioxidant for possible rapid enterohepatic distribution, and (iii) microtubules may be involved in this process.

Animals↗
PubMed1995

Glutathione redox cycle-driven recovery of reduced glutathione after oxidation by tertiary-butyl hydroperoxide in preimplantation mouse embryos.

The purpose of this study was to determine whether mouse embryos at the two-cell to blastocyst stage have the capacity to reduce glutathione disulfide (GSSG) and to elucidate the mechanism that embryos utilize to recover from tertiary-butyl hydroperoxide (tBH)-induced oxidative stress. Experiments were conducted on embryos in vitro and tBH was used to induce oxidation of embryonic reduced glutathione (GSH). After derivatization of extracted embryo samples with dansyl chloride, GSH and GSSG were measured at picomole levels by fluorometric HPLC. Two-cell- and blastocyst-stage embryos were able to recover their GSH levels within 45 min after depletion of GSH by incubation in tBH for 15 min. Addition of 1,3-bis(2-chloroethyl)-1-nitrosourea to the culture medium blocked recovery of GSH and resulted in continued elevation of GSSG. Addition of buthionine sulfoximine (BSO) to the culture medium did not affect GSH levels in two-cell-stage embryos, but did reduce GSH levels in blastocysts by 1.5 h. Culture of two-cell embryos in the presence of BSO for 45 h decreased embryonic GSH content and percentage of embryos developing to the blastocyst stage. These results indicate that preimplantation mouse embryos have the capacity to reduce GSSG and suggest that under normal conditions, depletion and synthesis of GSH occur to a greater extent in the blastocyst than in the two-cell-stage embryo. A major protective role for glutathione reductase during specific stages of embryo development is indicated.

Animals↗
PubMed1995

Influence of metabolic inhibitors on mitochondrial permeability transition and glutathione status.

Treatment of isolated mitochondria with Ca2+ and inorganic phosphate (Pi) induces an inner membrane permeability that appears to be mediated through a cyclosporin A (CsA)-inhibitable Ca(2+)-dependent pore. Isolated mitochondria during inner membrane permeability undergo rapid efflux of matrix solutes such as glutathione as GSH and Ca2+, loss of coupled functions, and large amplitude swelling. Permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability, has been observed. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing Ca2+ and Pi abolished large amplitude swelling of mitochondria. The GSH status during a Ca(2+)- and Pi-dependent mechanism of mitochondrial GSH release in isolated mitochondria was influenced significantly by metabolic inhibitors of the respiratory chain but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial GSH and Ca2+. The release of GSH was inhibited by the addition of CsA, a potent inhibitor of permeability transition. Under these conditions we did not find GSSG; however, rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP occurred. The addition of CsA, prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP. Since NADH and NADPH were extensively oxidized, protection against oxidative stress is reflected in maintenance of GSH and not observable lipid peroxidation. Evidence from transmission electron microscopy analysis, combined with the GSH release data, indicate that permeability transition can be observed in the absence of large amplitude swelling.

Adenine Nucleotides↗
PubMed1995

Synthesis of glutathione in the preimplantation mouse embryo.

Depletion and repletion of glutathione in two-cell to blastocyst stage mouse embryos was examined. Reduced (GSH) and oxidized glutathione (GSSG) were measured by fluorimetric HPLC after derivatization of extracted embryo samples with dansyl chloride. Addition of buthionine sulfoximine (BSO) to culture medium for 16 h decreased GSH levels in both two-cell and blastocyst stage embryos; however, GSH decreased more drastically in blastocysts. Addition of diethyl maleate (DEM) to culture medium depleted GSH in both two-cell and blastocyst stage embryos. After removal of DEM, GSH levels returned to normal in blastocysts, and addition of BSO or removal of cystine from medium blocked GSH repletion. Two-cell stage embryos were unable to recover GSH levels after depletion and exhibited decreased in vitro development. Addition of methionine to culture medium was unable to substitute for cystine as a source of cysteine in glutathione synthesis, indicating that the embryos do not use the cystathionine pathway. Embryos collected early on Day 3 of development were unable to recover GSH levels within 5 h, whereas embryos collected late on Day 3 recovered GSH within 5 h. Addition of cycloheximide to culture medium at noon on Day 3 of development decreased the ability of blastocysts to recover their GSH levels late on Day 3. These data indicate that GSH turnover and synthesis increases between the two-cell and blastocyst stages. The increase in the ability of embryos to synthesize GSH on Day 3 is dependent on protein synthesis. Cleavage stage embryos have limited capacity to synthesize GSH and appear susceptible to adverse effects of toxicants or conditions that deplete glutathione.

Animals↗
PubMed1995

Impact of nutrients on cellular lipid peroxidation and antioxidant defense system.

A symposium entitled Impact of Nutrients on Cellular Lipid Peroxidation and Antioxidant Defense System was held at the 33rd Annual Meeting of the Society of Toxicology (SOT) at the Loews Anatole Hotel in Dallas, Texas. The symposium was sponsored by the Food Safety Specialty Section and focused on the role of particular nutrients in cellular lipid peroxidation and antioxidant defense system. Emphasis was placed on defining underlying mechanisms for damage and protection, as well as potential ramification in human health issues. The following are extractions of some of the highlights from each presentation.

Antioxidants↗
PubMed1995

Adenovirus-based p53 gene therapy in ovarian cancer.

Mutations of the p53 tumor suppressor gene are the most common molecular genetic abnormality to be described in ovarian cancer. To determine the feasibility of mutant p53 as a molecular target for gene therapy in ovarian cancer, we constructed an adenovirus vector containing the wild-type p53 gene. The ability of this adenovirus construct (Ad-CMV-p53) to express p53 protein was examined by Western blot analysis in the H358 lung cancer cell line, which has a homozygous deletion of the p53 gene. The ability of the adenovirus vector system to infect ovarian cancer cells was tested using an adenovirus containing the beta-galactosidase reporter gene under the control of the CMV promoter (Ad-CMV-beta gal). The ovarian cancer cell line 2774, which contains an Arg273His p53 mutation, was infected with Ad-CMV-beta gal, and the infected cells were assayed for beta-galactosidase activity after 24 hr. To test the ability of wild-type p53 to inhibit cell growth, the 2774 cell line was infected with Ad-CMV-p53 or Ad-CMV-beta gal, and the effect of these agents on the growth of 2774 cells was determined using an in vitro growth inhibition assay. Western blot analysis of lysates from H358 cells infected with Ad-CMV-p53 showed expression of wild-type p53 protein. When 2774 cells were infected with Ad-CMV-beta gal at a multiplicity of infection (m.o.i.) of 10 PFU/cell, > 90% of cells showed beta-galactosidase activity, demonstrating that these cells are capable of efficient infection by the adenovirus vector. Growth of 2774 cells infected with Ad-CMV-p53 was inhibited by > 90% compared to noninfected cells. The ability of the adenovirus vector to mediate high-level expression of infected genes and the inhibitory effect of Ad-CMV-p53 on the 2774 cell line suggests that the Ad-CMV-p53 could be further developed into a therapeutic agent for ovarian cancer.

Adenoviridae↗
PubMed1995

Cystathionine.

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Alkynes↗
PubMed1994

Release of mitochondrial glutathione and calcium by a cyclosporin A-sensitive mechanism occurs without large amplitude swelling.

Treatment of isolated mitochondria with calcium and inorganic phosphate induces inner membrane permeability that is thought to be mediated through a non-selective, calcium-dependent pore. The inner membrane permeability results in the rapid efflux of small matrix solutes such as glutathione and calcium, loss of coupled functions, and large amplitude swelling. We have identified conditions of permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing calcium and inorganic phosphate abolished large-amplitude swelling of mitochondria but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial glutathione and calcium. The release of both glutathione and calcium was inhibited by the addition of cyclosporin A, a potent inhibitor of permeability transition. Transmission electron microscopy analysis, combined with the glutathione and calcium release data, indicate that permeability transition can be observed in the absence of large-amplitude swelling. Permeability transition occurring both with and without large-amplitude swelling was accompanied by a collapse of the membrane potential. We conclude that cyclosporin A-sensitive permeability transition can occur without obvious morphological changes such as large-amplitude swelling. Monitoring the cyclosporin A-sensitive release of concentrated endogenous matrix solutes, such as GSH, may be a sensitive and useful indicator of permeability transition.

Animals↗
PubMed1994

Role of differential mRNA stability in the regulated expression of IgM and IgD.

The expression of IgM and IgD heavy chain mRNA in resting vs activated B cells offers a unique tool for the assessment of the effect of translation on mRNA stability because mu and delta mRNA have identical VDJ sequences but differ substantially in the rest of the molecule. We have shown that despite the 5' UTR identity that allows equal access to the translation machinery, mu mRNA has a significantly higher turnover rate than delta mRNA. However, the short t1/2 of mu mRNA increases significantly after B cell activation. Furthermore, the induction of microS mRNA after B cell activation provides yet another related molecule for comparison. Thus, despite the fact that microS and microM mRNA differ at their 3' ends, they have identical turnover rates in activated B cells. In addition, because the turnover rates of delta mRNA and beta 2 and GAPDH mRNA remain unchanged, these experiments suggest that B cell activation results in the induction of regulatory factor(s) that target specific sequences within mRNA-mu to confer greater stability. They also argue against a more passive regulation of mRNA stability that is a consequence of alterations in the secretory machinery.

Animals↗
PubMed1994

Oxidation of pyridine nucleotides and depletion of ATP and ADP during calcium- and inorganic phosphate-induced mitochondrial permeability transition.

We have examined the pyridine and adenine nucleotide status during calcium- and inorganic phosphate-induced permeability transition. Calcium- and inorganic phosphate-induced permeability transition is accompanied by the rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP. The addition of cyclosporin A, a potent inhibitor of the permeability transition prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP.

Adenine Nucleotides↗