Orientation of electron transport activities in the membrane of intact glyoxysomes isolated from castor bean endosperm

Intact glyoxysomes were isolated from castor bean endosperm on isometric Percoll gradients. The matrix enzyme, malate dehydrogenase, was 80% latent in the intact glyoxysomes. NADH:ferricyanide and NADH:cytochrome c reductase activities were measured in intact and deliberately broken organelles. The latencies of these redox activities were found to be about half the malate dehydrogenase latency. Incubation of intact organelles with trypsin eliminated NADH:cytochrome c reductase activity, but did not affect NADH:ferricyanide reductase activity. NADH oxidase and transhydrogenase activities were negligible in isolated glyoxysomes. Mersalyl and Cibacron blue 3GA were potent inhibitors of NADH:cytochrome c reductase. Quinacrine, Ca²⁺ and Mg²⁺ stimulated NADH:cytochrome c reductase activity in intact glyoxysomes. The data suggest that some electron donor sites are on the matrix side and some electron acceptor sites are on the cytosolic side of the membrane.     

12S,19- and 12S,20-dihydroxyeicosanoids: novel 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid metabolites formed by hydroxylation and reduction in murine lymphocytes

Murine spleen cells and purified B lymphocytes oxidized arachidonic acid via the lipoxygenase pathway. The major metabolite of both the whole spleen and enriched B lymphocytes was 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid. A novel metabolite was observed that did not have an absorbance from 210 to 400 nm, indicating the absence of a conjugated double bond system. The new metabolite was converted to the methyl ester, reduced by platinum oxide, derivatized to the trimethylsilyl ether, and analyzed by gas chromatography-mass spectrometry. A major and a minor component were observed in the analysis of the new compound. The major component had major diagnostic ions indicating the presence of hydroxyl groups at C-12 and C-19. The minor component had major diagnostic ions indicating the presence of hydroxyl groups at C-12 and C-20. The new metabolites are characterized as a mixture of 12S,19- and 12S,20-dihydroxyeicosanoids presumably formed by hydroxylation and reduction of one or more double bonds of 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid. These metabolites were formed predominantly with whole spleen lymphocytes but could be detected at longer incubation times or by using 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid as the starting substrate with highly enriched B lymphocytes.     

Lack of a relationship between immune function and chemically induced hepatocarcinogenesis in B6C3F1 mice

Summary The relationship between immune function and chemically induced hepatocarcinogenesis was studied employing an in vivo murine model. Neonatal B6C3F₁ mice were given a single carcinogenic dose of diethylnitrosamine (DEN) and the time-response kinetics for the early (foci of alteration) and late (adenomas/carcinomas) phases of hepatocellular carcinogenesis were compared to changes in hematopoiesis and immune functions associated with immune surveillance and natural resistance. Increases in hematopoiesis occurred just prior to or concurrent with the appearance of hepatocellular carcinomas, while increased macrophage and natural killer cell cytotoxicity and suppression of cell-mediated immunity occurred following tumor appearance and progressed with increasing tumor burden. Neither immunological nor hematopoietic changes were associated with early phases of hepatocarcinogenesis, as monitored by the appearance of altered hepatocellular foci. Although changes in hematopoiesis may represent an early indicator for hepatocarcinogenesis in the mouse tumor model, the data suggest that altered immune surveillance and natural resistance are not factors in the development of chemically induced hepatocellular tumors, and the changes in immune function are probably secondary to tumor development.     

Activated conformations of the ras-gene-encoded p21 protein. 1. An energy-refined structure for the normal p21 protein complexed with GDP.

A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gln 61, bound to GDP, has been constructed in four stages using the available alpha-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 61 likewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the alpha-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no alpha-carbon coordinates for residues 60-65 have been determined, these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factor Tu (EF-Tu) to which p21 bears an overall 40% sequence homology, were determined from their corresponding positions in a high-resolution structure of EF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the alpha-carbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the non-homologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of membrane-bound electron transport enzymes from castor bean glyoxysomes and endoplasmic reticulum

Membranes purified from castor bean endosperm glyoxysomes by washing with sodium carbonate exhibited integral NADH:ferricyanide and NADH:cytochrome c reductase activities. The enzyme activities could not be attributed to contamination by other endomembranes. Purified endoplasmic reticulum membranes also contained the redox activities; and marker enzyme analysis indicated minimum cross contamination between glyoxysomal and endoplasmic reticulum fractions. The glyoxysomal redox activities were optimally solubilized at detergent to protein ratios (weight to weight) of 10 (Triton X-100), 50 (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and 100 (octylglucoside). Detergent in excess of the solubilization optimum was stimulatory to NADH:ferricyanide reductase and inhibitory to NADH:cytochrome c reductase. Endoplasmic reticulum redox activity solubilization profiles were similar to those obtained for glyoxysomal enzymes using Triton X-100. Purification of the glyoxysomal and endoplasmic reticulum NADH:ferricyanide reductases was accomplished using dye-ligand affinity chromatography on Cibacron blue 3GA agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of NADH:ferricyanide reductase preparations purified by rate-zonal density gradient centrifugation, affinity chromatography, and nondenaturing electrophoresis of detergent-solubilized glyoxysomal and endoplasmic reticulum membranes consistently displayed 32- and 33-kDa silver-stained polypeptide bands, respectively.     

Chemical induction of interleukin-8, a proinflammatory chemokine,in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 mol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001–100 ng/ml), cycloheximice (CHX) (0.01–50 g), lipopolysaccharide (0.1–100 g/ml), tumor necrosis factor- (TNF) (3.13–50 ng/ml), or interleukin-1 (IL-1) (1–182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNF induced IL-8 production that coincided with significant cell cycle inhibition. IL-1 had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31–10 g/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNF reduced the mitotic index by 45%, slowed cell cycle progression by 3.5 h, and induced a flat, albeit large, IL-8 response at concentrations 12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.Abbreviations B(a)P benzo(a)pyrene - BrdU 5-bromo-2-deoxyuridine - CHX cycloheximide - ICAM intercellular adhesion molecules - IL-1 interleukin-1 - IL-8 interleukin-8 - KGM keratinocyte growth medium - LPS lipopolysaccharide - PKC protein kinase C - PMA phorbol-13-myristate-12-acetate - PMN polymorphonuclear neutrophil - ROS reactive oxygen species - SCE sister chromatid exchange - TNF tumor necrosis factor      

Chemical separation of helper cell function and delayed hypersensitivity responses

Studies were performed to investigate the effects of the immunosuppressive chemical TCDD. Fetal and neonatal rats were exposed to TCDD through maternal dosing (5 μg/Kg) at Day 18 of gestation and on Days 0, 7, and 14 of postnatal life. Another group of neonatal rats were exposed to TCDD through maternal dosing on Days 0, 7, and 14 of postnatal life only. Parameters of cell-mediated and humoral immune function were investgiated. TCDD suppressed delayed hypersensitivity responses and responses to the mitogens Con A and PHA without affecting humoral immune function. Suppression of T-cell function was selective in that helper function was not suppressed. Transfer of primed T-lymphocytes from TCDD treated and non-treated animals into neonatally thymectomized animals confirmed this. Results indicate that delayed hypersensitivity function and helper function reside in distinct T-cell subsets.     

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.     

Genotyping of single nucleotide polymorphisms in cytokine genes using real-time PCR allelic discrimination technology

Single nucleotide polymorphisms (SNPs), particularly those within regulatory regions of genes that code for cytokines often impact expression levels and can be disease modifiers. Investigating associations between cytokine genotype and disease outcome provides valuable insight into disease etiology and potential therapeutic intervention. Traditionally, genotyping for cytokine SNPs has been conducted using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), a low throughput technique not amenable for use in large-scale cytokine SNP association studies. Recently, Taqman real-time PCR chemistry has been adapted for use in allelic discrimination assays. The present study validated the accuracy and utility of real-time PCR technology for a number of commonly studied cytokine polymorphisms known to influence chronic inflammatory diseases. We show that this technique is amenable to high-throughput genotyping and overcomes many of the problematic features associated with PCR-RFLP including post-PCR manipulation, non-standardized assay conditions, manual allelic identification and false allelic identification due to incomplete enzyme digestion. The real-time PCR assays are highly accurate with an error rate in the present study of <1% and concordance rate with PCR-RFLP genotyping of 99.4%. The public databases of cytokine polymorphisms and validated genotyping assays highlighted in the present study will greatly benefit this important field of research.