Supernatant Fraction Of Cells Research Articles

Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT.

The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP+ cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP+ cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP+ cells with ≥3nuclei, although this difference was not significant. Levels of TGF-β, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1β and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis.

The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties.

Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

Pentachlorophenol dechlorination and simultaneous Cr6+ reduction by Pseudomonas putida SKG-1 MTCC (10510): characterization of PCP dechlorination products, bacterial structure, and functional groups

It is the first report in which a novel psychrotrophic Pseudomonas putida SKG-1 strain was evaluated for simultaneous bioremediation of pentachlorophenol and Cr(6+) under various cultural and nutritional conditions. Pentachlorophenol (PCP) dechlorination products, bacterial structure, and functional groups were characterized by gas chromatography and mass spectrometry (GC-MS), scanning electron microscope and energy dispersive X-ray spectroscopy (SEM-EDS), and Fourier-transform infrared (FTIR) techniques. The strain was extremely tolerant to excessively higher individual concentration of PCP (1,400 mg l(-1)) and Cr(6+) (4,300 mg l(-1)). Increasing concentration of PCP and Cr(6+) exerted inhibitory effect on bacterial growth and toxicants' removal. The strain exhibited growth, and concomitantly remediated both the pollutants simultaneously over a broad pH (7.0-9.0) and temperature (28-32 °C) range; maximum growth, PCP dechlorination (87.5%), and Cr(6+) removal (80.0%) occurred at optimum pH 8.0 and 30 °C (from initial PCP 100 mg l(-1) and Cr(6+) 500 mg l(-1)) under shaking (150 rpm) within 72 h incubation. Optimization of agitation (125 rpm) and aeration (0.4 vvm) in bioreactor further enhanced PCP dechlorination by ~10% and Cr(6+) removal by 2%. A direct correlation existed between growth and bioremediation of both the toxicants. Among other heavy metals, mercury exerted maximum and cobalt minimum inhibitory effect on PCP dechlorination and Cr(6+) removal. Chromate reductase activity was mainly associated with the supernatant and cytosolic fraction of bacterial cells. GC-MS analysis revealed the formation of tetrachloro-p-hydroquinone, 2,4,6-trichlorophenol, and 2,6-dichlorophenol as PCP dechlorination products. FTIR spectrometry indicated likely involvement of carbonyl and amide groups in Cr(3+) adsorption, and SEM-EDS showed the presence of chromium on P. putida surface. Thus, our promising isolate can be ecofriendly employed for biotreatment of various industrial wastes contaminated with high PCP and Cr(6+) concentrations.

Protein Kinase C and NF-κB–Dependent CD4 Downregulation in Macrophages Induced by T Cell-Derived Soluble Factors: Consequences for HIV-1 Infection

Upon activation, CD4(+) T cells release cytokines, chemokines, and other soluble factors that influence the kinetics of HIV-1 replication in macrophages (M). In this article, we show that activation of human primary T cells suppresses the early stages of HIV-1 replication in human primary Mφ by downregulating the main cellular receptor for the virus CD4. The secreted factors responsible for this effect have a molecular mass greater than conventional cytokines, are independent of Th1 or Th2 polarization, and are not IFN-γ, IL-16, RANTES, or macrophage inhibitory factor, as revealed by cytokine array analysis and neutralization assays. CD4 downregulation is entirely posttranslational and involves serine phosphorylation of CD4 and its targeting to an intracellular compartment destined for acidification and degradation. CD4 downregulation is dependent on the activities of both protein kinase C and NF-κB as well as the proteasomes. Using high-resolution liquid chromatography-tandem mass spectrometry analysis in conjugation with label-free protein quantitation software, we found that proteins that promote Mφ adherence and spreading, such as attractin, fibronectin, and galectin-3-binding protein, were significantly overrepresented in the activated T cell supernatant fractions. These results reveal the existence of previously unreported anti-HIV-1 proteins, released by activated T cells that downregulate CD4 expression, and are of fundamental importance to understand the kinetics of HIV infection in vivo.

A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.

Ganglioside composition of differentiated Caco-2 cells resembles human colostrum and neonatal rat intestine

Gangliosides are glycosphingolipids found in cell membranes and human milk with important roles in cell proliferation, differentiation, growth, adhesion, migration, signalling and apoptosis. Similar changes in ganglioside composition occur during embryonic development, lactation and cancer cell differentiation. It is not known, however, whether ganglioside compositional changes that occur in differentiating colon cancer cells reflect changes that occur during intestinal development. The Caco-2 cell line is commonly used to study physiological and pathophysiological processes in the small intestine and colon. Therefore, to examine this question, undifferentiated and differentiated Caco-2 cells were grown and total lipid was extracted from cell supernatant fractions using the Folch method. The upper aqueous phase containing gangliosides was collected and purified. Total gangliosides were measured as ganglioside-bound N-acetyl neuraminic acid, while individual ganglioside content was quantified via a colorimetric assay for sialic acid and scanning densitometry. The total ganglioside content of differentiated Caco-2 cells was 2.5 times higher compared with undifferentiated cells. Differentiated Caco-2 cells had significantly more (N-acetylneuraminyl) 2-galactosylglucosyl ceramide (GD3) and polar gangliosides, and a lower N-acetylneuraminylgalactosylglucosylceramide (GM3):GD3 ratio than undifferentiated cells. The present study demonstrates that the total ganglioside content and individual ganglioside composition of differentiated Caco-2 cells are similar to those of human colostrum and neonatal rat intestine. Differentiated Caco-2 cells may therefore be an alternative model for studying physiological and pathological processes in the small intestine and colon, and may help to elucidate possible functions for specific gangliosides in development and differentiation. Further research using more sensitive techniques of ganglioside analysis is needed to confirm these findings.

The Effect of Polysulphated Glycosaminoglycan on the Production of Proteoglycan by Equine Tendon Fibroblasts in Monolayer Culture

SummaryEquine tendon fibroblasts were isolated from tissue fragments of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were treated with two dosage regimes of polysulphated glycosaminoglycan (PSGAG). The high dose group received 0,25, 50 or 200 μg/ml of PSGAG and the low dose group received 0,0.3,1 or 2 μg/ml of PSGAG. Proteoglycan production was measured via liquid scintillation counting, following radiolabel with 35SO4 (40 μCi/ml). The counts were measured in both the cell associated fraction and the supernatant fraction.The addition of PSGAG to the culture system did not adversely affect cell viability at any treatment level. The cells in all of treatment groups produced measurable amounts of proteoglycan in both the cell associated and supernatant fractions. There were not any differences between the treatment means in any of the four groups (high dose cell-associated, high dose supernatant, low dose cell-associated, low dose supernatant). A linear trend in proteoglycan production was not documented between treatment groups in either the cell-associated or the supernatant fractions for either of the two dosage regimes. The significance of the lack of effect is equivocal. PSGAG may influence tendon healing in a manner other than by increasing proteoglycan production.The effect of polysulphated glycosaminoglycan on the production of proteoglycans by equine tendon fibroblasts was investigated. A previously described model was utilized to grow the tendon fibroblasts in monolayer cultures. Significant treatment effects were not seen at either a high or low dose of PSGAG. Addition of the PSGAG did not adversely affect cell viability at any treatment level.

Changes in microtubular tau protein after morphine in a cultured human neuroblastoma cell line

1. 1. Cultured human SH-SY5Y adrenergic neuroblastoma cells were used to examine the action of morphine sulfate on microtubular tau protein. 2. 2. After 48 hr treatment morphine sulfate (200 μM) reduced tau protein in the cytoplasmic (supernatant) fraction of undifferentiated cells, and in the cytoplasmic as well as membrane (pellet) fractions of differentiated cells. 3. 3. A 71% increase ( P<0.05) in total protein in the membrane (pellet) fraction of undifferentiated cells and a 188% increase ( P<0.01) in that of differentiated cells accompanied the decrease in tau protein. 4. 4. A 51% reduction ( P<0.01) in the number of undifferentiated (but not differentiated) cells was seen after this drug (200 μM).

Portal venous transfusion up-regulates Kupffer cell cyclooxygenase activity: a mechanism of immunosuppression in organ transplantation.

Portal venous transfusions (PVTs) of blood have been shown to induce significant immunosuppression in animal models of organ transplantation. A proposed mechanism of PVT-induced immunosuppression is via alteration of Kupffer cell arachidonic acid metabolism with increased secretion of the suppressive metabolite prostaglandin E2 (PGE2). This study assessed the hypothesis that PVT increases Kupffer cell PGE2 production via up-regulation of Kupffer cell phospholipase A2 (PLA2) as well as constitutive (COX1) and inducible (COX2) cyclooxygenase. Kupffer cells from Lewis rats were harvested 1 hr after PVT with either 1 ml of Wistar-Furth blood, systemic transfusion (SVT), or saline via portal vein (PVSal). After lipopolysaccharide stimulation, 24-hr Kupffer cell supernatant fractions were assayed for PGE2. PGE2 was increased after SVT (1465+/-234 pg/ml) compared with PVSal (597+/-99; P<0.01). PVT increased Kupffer cell PGE2 (5370+/-533; P<0.001 vs. SVT and vs. PVSal) even more substantially. Kupffer cells from PVT-treated rats were then cultured in the presence of inhibitors of PLA2, COX1, or COX2. When Kupffer cells were treated with mepacrine to inhibit PLA2 (5575+/-453 pg/ml), PGE2 production was not different from that by PVSal-treated controls (6467+/-614 pg/ml), but when Kupffer cells were incubated in the presence of the COX1 inhibitor flurbiprofen (3512+/-407 pg/ml) or the COX2 inhibitor nimesulide (2800+/-830 pg/ml), production was decreased 46.7% and 56.7%, respectively, over control activity without added inhibitor. PVT also increased Kupffer cells COX1 and COX2 mRNA as measured by Northern blot. Heart transplants were then performed from Wistar-Furth donors into Lewis recipients at the time of PVT, SVT, PVSal, or PVT + indomethacin (COX1/2 inhibitor). PVT prolonged allograft survival (12.0+/-0.9 days) compared with PVSal (6.3+/-0.3; P<0.01) or SVT (6.3+/-0.3; P<0.04). Indomethacin shortened graft survival when given with PVT (6.5+/-0.3 days). In summary, PVT increased Kupffer cell PGE2 production, up-regulated transcription of Kupffer cells COX1 and COX2 mRNA, and prolonged cardiac allograft survival. COX1/2 inhibition abrogated the effect of PVT. The results indicated that the immunosuppressive effect of PVT may be mediated by up-regulation of Kupffer cell COX1 and COX2. Manipulation of Kupffer cell arachidonic acid metabolism may be useful in augmentation of PVT-induced immunosuppression.

Critical relationship between autoantibody recognition and thyrotropin receptor maturation as reflected in the acquisition of complex carbohydrate.

Generation of large amounts of recombinant TSH receptor (TSHR) protein capable of recognition by TSHR autoantibodies is a goal of clinical importance. We expressed in Chinese hamster ovary cells the human TSHR ectodomain (ECD) with a carboxyl-terminus six-histidine tag. After transgene amplification, expressing clones were selected by nickel chelate chromatography in combination with [35S] methionine precursor labeling. An approximately 74-kDa protein was detected in the culture medium, and larger quantities of an approximately 68-kDa protein were found in the cell soluble fraction. Immunoblot analysis with a rabbit antiserum revealed that most of the TSHR-ECD was not secreted, but was retained within the soluble fraction of the cell. Nickel chelate chromatography was not effective in purifying significant quantities of this material. In contrast, with Concanavalin A, but not with wheat germ agglutinin, an approximately 50-fold purification of TSHR-ECD was achieved from the cell soluble fraction. Surprisingly, this affinity-enriched TSHR, containing high mannose carbohydrate, was not recognized by human TSHR autoantibodies in sera from six individuals. By ion exchange chromatography, the autoantibody-neutralizing TSHR in the cell supernatant fraction was found to be nonidentical with TSHR-ECD protein recognized by antisera from immunized animals. The present data indicate the critical relationship between autoantibody recognition and TSHR maturation as reflected in the acquisition of complex carbohydrate. Nonsecretion of the TSHR-ECD appears to be related to the specific protein rather than to the glycosylation apparatus of the host cell. Antibodies from immunized animals may be ineffective in monitoring purification of autoantigen-competent TSHR. Finally, the data explain why soluble recombinant TSHR generated in many expression systems is not recognized satisfactorily by human autoantibodies.

Compartmentalization of Cholesterol Biosynthesis: CONVERSION OF MEVALONATE TO FARNESYL DIPHOSPHATE OCCURS IN THE PEROXISOMES

We have recently demonstrated that mevalonate kinase and farnesyl diphosphate (FPP) synthase are localized predominantly in peroxisomes. This observation raises the question regarding the subcellular localization of the enzymes that catalyze the individual steps in the pathway between mevalonate kinase and FPP synthase (phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and isopentenyl diphosphate isomerase). These enzyme are found in the 100,000 x g supernatant fraction of cells or tissues and have been considered to be cytoplasmic proteins. In the current studies, we show that the activities of mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase are equal in extracts prepared from intact cells and selectively permeabilized cells, which lack cytosolic enzymes. We also demonstrate structure-linked latency of phosphomevalonate kinase and mevalonate diphosphate decarboxylase that is consistent with a peroxisomal localization of these enzymes. Finally, we show that cholesterol biosynthesis from mevalonate can occur in selectively permeabilized cells lacking cytosolic components. These results suggest that the peroxisome is the major site of the synthesis of FPP from mevalonate, since all of the cholestrogenic enzymes involved in this conversion are localized in the peroxisome.

Farnesyl-diphosphate synthase is localized in peroxisomes.

In this study, we have investigated the subcellular localization of farnesyl-diphosphate synthase (FPP synthase). FPP synthase produces FPP, which is utilized in the synthesis of squalene, cholesterol, farnesylated and geranylgeranylated proteins, dolichols, coenzyme Q, and the isoprenoid moiety of heme a. This enzyme is found in the 100,000 x g supernatant fraction of cells or tissues and has been considered to be a cytoplasmic protein. In this study, analysis of FPP synthase activity and protein in fractionated rat liver together with immunofluorescent and immunoelectron microscopy studies demonstrated unequivocally that FPP synthase is largely localized in peroxisomes. These data, in combination with the previous observation that mevalonate kinase is predominantly localized in peroxisomes, suggest that peroxisomes are the major site of synthesis of FPP from mevalonate. We also demonstrate that in liver tissue obtained from patients with peroxisomal deficiency diseases (Zellweger syndrome and neonatal adrenoleukodystrophy), the activities of five enzymes involved in isoprenoid synthesis, namely mevalonate kinase, phosphomevalonate kinase, mevalonate-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, and FPP synthase, are significantly reduced, consistent with a peroxisomal localization of these enzymes.

Distinct 19 S and 20 S subcomplexes of the 26 S proteasome and their distribution in the nucleus and the cytoplasm.

The 26 S proteolytic complex ("26 S proteasome") is a macromolecular assembly thought to be involved in ATP- and ubiquitin-dependent protein degradation in the cytoplasm of higher eukaryotic cells. This complex is composed of one 20 S cylinder particle (multicatalytic proteinase, 20 S proteasome) and two cap-shaped 19 S particles comprising a set of polypeptides in the M(r) range of 35,000-110,000. Here we show that cell supernatant fractions contain both these two subunit complexes as distinct particles as well as assembled to 26 S proteasomes. We have separated and purified all three forms from Xenopus laevis oocytes and have determined their peptidase and protease activities. Using various antibodies specific for either a constitutive p52 polypeptide of the 19 S cap complex or for proteins of the 20 S cylinder particle, we have immunolocalized these complexes in both the cytoplasm and the nucleus of diverse species and cell types. The occurrence of all three forms, the 26 S proteasome, the 20 S cylinder particle, and the 19 S cap complex in the nucleoplasm has also been demonstrated in analyses of isolated giant nuclei from Xenopus oocytes. In addition, we show that the 19 S and 20 S subcomplexes can be released from 26 S proteasomes by ATP depletion and that readdition of ATP to 19 S and 20 S particles in cell extracts leads to the reformation of 26 S proteasomes. We discuss that all three particles (19 S, 20 S, and 26 S) exist in a dynamic equilibrium in both cell compartments and serve cytoplasmic as well as nucleus-specific functions.

Intermediates in translocation of diphtheria toxin across the plasma membrane.

Active diphtheria toxin consists of two parts, fragments A and B. Fragment A has enzymatic activity and inhibits protein synthesis. Fragment B binds to cellular receptors, and upon exposure to low pH it inserts into the membrane and facilitates translocation of the A fragment into the cytosol, concomitantly with formation of cation-selective channels. Reduction of the interfragment disulfide bridge is required for release of fragment A and intoxication. In cells treated with N-ethylmaleimide (NEM), which inhibits reduction of the disulfide bridge, fragment A was translocated to the cytosol but not released from fragment B. In the presence of NEM a peptide larger than fragment A was protected against extracellularly added Pronase. This peptide (M(r) approximately 24,000) was released to the supernatant fraction of saponin-treated cells. This indicates that fragment A, which is 21 kDa, is covalently attached via a disulfide bond to an N-terminal (M(r) approximately 3,000) piece of fragment B. The 24-kDa fragment disappeared upon reduction, and the 21-kDa fragment A appeared instead. NEM did not prevent channel activity by fragment B in the context of full-length toxin, demonstrating that channel formation occurs in spite of inhibited reduction of the disulfide bond. Thus, channel formation is not dependent on release of fragment A from the toxin-receptor complex.

Cyclic AMP regulation of messenger RNA level of the stimulatory GTP-binding protein Gs alpha. Isoproterenol, forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate increase the level of Gs alpha mRNA in cultured astroglial cells.

The existence of a cAMP-dependent regulation of the expression of the alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gs) in a well characterized astroglial cells culture was established. The culture of astroglial cells for 3-6 h with isoproterenol (10 microM) or forskolin (10 microM) (a cAMP-inducing agent) increased (200-400%) the response of adenylylcyclase to agents which bypass the receptor; GTP, GTP[S] or forskolin. For prolonged exposure times (15 h or more) to isoproterenol or forskolin, the adenylycyclase activity decreased to the value observed in control cells. The same biphasic response of adenylylcyclase to isoproterenol (10 microM) plus GTP (10 microM) occurred in membrane fractions from cells cultured with forskolin, whereas a diminished response to isoproterenol was observed in isoproterenol-treated cells, indicating that the beta-adrenergic receptor was desensitized. To understand the molecular mechanism of these phenomena, we measured the levels of the alpha subunits of the guanine-nucleotide binding protein (Gs and Gi) by Western-blot analysis. The culture of astroglial cells with isoproterenol or forskolin (3-24 h) resulted in a transient increase of both the Gs alpha and the Gi alpha protein levels, while the level of G beta subunits was unaffected. We also identified Gs alpha protein (about 40% of the total cellular protein) in the supernatant fraction of astroglial cells but its level was not modified by the stimulation of cells by forskolin. The level of Gs alpha mRNA measured by Northern-blot analysis was transiently increased (200%) after stimulation of astroglial cells with isoproterenol or forskolin for an incubation period of 6-9 h, then returned to that of control cells for longer period of time. In addition, the Gs alpha mRNA level was threefold increased when cells were cultured for 2-6 h with 8-bromoadenosine 3':5'-cyclic monophosphate (10 microM), a permeant analogue of cAMP. These results indicate that cAMP induces a time-dependent increase of Gs alpha mRNA. The half-life of Gs alpha protein and Gs alpha mRNA were determined. Pulse-chase studies revealed that the decay of Gs alpha protein was clearly biphasic with an early phase (5-6 h) and a slower second phase (20-25 h) but the treatment of cells with forskolin did not accelerate or slow down the turnover of Gs alpha protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Mast cells contain spleen-type prostaglandin D synthetase.

Prostaglandin D synthetase activity in the cytosol (100,000 x g, 1-h supernatant) fraction of peritoneal mast cells of adult rats (105.0 nmol/min/mg protein) was the highest among such activities in various rat tissues and cells. As judged by the absolute requirement for glutathione for the reaction (Km = 300 microM), the Km value for prostaglandin H2 (200 microM), and insensitivity of the activity to 1 mM 1-chloro-2,4-dinitrobenzene, the enzyme in mast cells was similar to rat spleen prostaglandin D synthetase and differed from rat brain prostaglandin D synthetase or glutathione S-transferase, all of which catalyze the isomerase reaction from prostaglandin H2 to prostaglandin D2. In immunotitration analyses, the activity in mast cells showed a titration curve exactly identical with that of the purified spleen-type enzyme and almost completely absorbed by an excess amount of antibody against this enzyme, but it remained unchanged after incubation with antibodies against the brain-type enzyme and glutathione S-transferase isozymes thus far purified. In Western blot after two-dimensional electrophoresis of crude extracts of mast cells, a single immunoreactive spot was observed with antibody against the spleen-type enzyme at the same position as that of the purified enzyme (Mr = 26,000, pI = 5.2). Furthermore, the immunoreactive protein obtained from mast cells showed the same peptide fingerprints as those of the purified spleen-type enzyme, after partial digestion with Staphylococcus aureus V8 protease or trypsin. In immunoperoxidase staining, the immunoreactivity of the spleen-type enzyme was found in the cytosol of tissue mast cells in various organs such as thymus, intestine, stomach, and skin of adult rats. These findings indicate that prostaglandin D2 is produced by the spleen-type synthetase in mast cells of various tissues.

Differential biotransformation of glyceryl trinitrate by red blood cell – supernatant fraction and pulmonary vein homogenate

We have demonstrated previously that glyceryl trinitrate (GTN) undergoes biotransformation to two glyceryl dinitrate (GDN) metabolites in the human red blood cell-supernatant fraction (RBC-SF) by hemoglobin-mediated and sulfhydryl-dependent enzymatic mechanisms. In the present study, we have shown that biotransformation of GTN in rabbit RBC-SF yields a glyceryl-1,2-dinitrate (1,2-GDN)/glyceryl-1,3-dinitrate (1,3-GDN) ratio of 5.3. Following inhibition of hemoglobin-mediated biotransformation of GTN by carbon monoxide (CO), the 1,2-GDN/1,3-GDN ratio was 2.1. Following inhibition of sulfhydryl-dependent biotransformation by N-ethylmaleimide (NEM), the 1,2-GDN/1,3-GDN ratio was 30.0. We have demonstrated previously that for GTN-induced vasodilation of isolated bovine pulmonary vein (BPV), the 1,2-GDN/1,3-GDN ratio was 7.1, which indicated that a hemoprotein-dependent process was involved in GTN biotransformation. To determine if this was the case, the biotransformation of GTN (0.51 microM) was studied in BPV homogenates; 31.1 pmol GDN/mg BPV protein was formed in 20 min. The 1,2-GDN/1,3-GDN ratio was 1.1, which indicated that hemoprotein-mediated biotransformation did not occur. This conclusion was supported by the fact that CO did not inhibit GTN biotransformation. GTN biotransformation by BPV homogenate was inhibited 62% by NEM, 89% by boiling of the homogenate, and almost completely by boiling plus NEM. These results indicated that biotransformation of GTN by the BPV homogenate involved in a combination of enzymatic and nonenzymatic processes that were mostly sulfhydryl dependent. It is concluded that the mechanism for GTN biotransformation in isolated intact BPV, which yielded preferential formation of 1,2-GDN, was rendered nonfunctional upon tissue homogenization.

SK&F 86002: A structurally novel anti-inflammatory agent that inhibits lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid

The effects of SK&F 86002 [5-(4-pyridyl)-6 (4-fluorophenyl)-2,3-dihydroimidazo (2,1-b) thiazole] on the generation of eicosanoids in vitro and on inflammatory responses in vivo are described and compared to other non-steroidal anti-inflammatory drugs. SK&F 86002 inhibited prostaglandin H 2 (PGH 2) synthase activity ( ic 50 120 μM) as well as prostanoid production by rat basophilic leukemia (RBL-1) cells ( ic 50 70 μM) and its sonicate ( ic 50 100 μM) and human monocytes ( ic 50 1μM). In addition, SK&F 86002 inhibited the generation of dihydroxyeicosatetraenoic acid (diHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE) by a high speed supernatant fraction of RBL-1 cells ( ic 50 10 μM). Cellular production of 5-lipoxygenase products was inhibited by SK&F 86002 as measured by leukotriene B 4 (LTB 4) generation from human neutrophils ( ic 50 20 μM), leukotriene C 4 (LTC 4) generation by human monocytes ( ic 50 20 μM), and 5-HETE production by RBL-1 cells ( ic 50 40 μM). The in vivo profile of anti-inflammatory activity of SK&F 86002 supports the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. The responses of arachidonic-acid-induced edema in the mouse ear and rat paw, as well as the cell infiltration induced by carrageenan in the mouse peritoneum and by arachidonic acid in the rat air pouch, were inhibited by SK&F 86002 and phenidone but not by the selective cyclooxygenase inhibitors naproxen and indomethacin.

Increased susceptibility to mouse hepatitis virus 3 of peritoneal macrophages exposed to dieldrin

Interaction of a single dose (36 mg/kg body wt) of the organochlorine pesticide dieldrin with mouse peritoneal macrophages was examined in C57Bl 6 , ( C57Bl 6 × A J )F 1 , and A J strains of different genetic resistance to mouse hepatitis virus 3 (MHV3) infection. In vivo studies showed increased susceptibility to MHV3 acute disease of C57Bl 6 and ( C57Bl 6 × A J )F 1 animals challenged with the pesticide. Significant decrease of mean time of death in dieldrin-exposed, MHV3-infected susceptible C57Bl 6 mice was observed similarly upon po or ip administration of a single, sublethal dose of dieldrin. In addition, decrease of humoral response to the virus was quantified by determination of anti-MHV3 IgG antibodies in spleen cell supernatant fractions and in blood sera of dieldrin-exposed C57Bl 6 mice. A single dose of dieldrin did not alter the in vivo resistance of A J animals to acute MHV3 disease. The resistant A J mice, however, showed increased mortality upon two subsequent exposures to dieldrin followed by infection with high lethal doses of MHV3. Phagocytic activity, cell adherence capacity, and attachment and uptake of 3H-radiolabeled MHV3 by C57Bl 6 peritoneal macrophages were determined by in vitro studies. These affector activities of peritoneal macrophages were slightly decreased or unchanged in cells originating from animals exposed to the pesticide. However, the intrinsic activity of MHV3 restriction appeared to be affected in macrophages derived from dieldrin-treated animals: (i) peritoneal C57Bl 6 macrophages collected from the early phase of acute MHV3 disease contained increased MHV3 antigen and (ii) increased cytolysis was observed after in vitro MHV3 infection of macrophages originating from dieldrin-exposed C57Bl 6 mice.

Biosynthesis of UDP-N-acetyl-D-glucosaminuronic acid and UDP-N-acetyl-D-mannosaminuronic acid in Micrococcus luteus.

The occurrence and formation of UDP-N-acetyl-D-glucosaminuronic acid (UDP-GlcNAcA) and UDP-N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA) were studied in Micrococcus luteus ATCC 4698. UDP-N-acetylhexosaminuronic acid separated from D-cycloserine-inhibited cells was shown to be a mixture of UDP-GlcNAcA and UDP-ManNAcA in the ratio of 87:13, whereas that obtained from untreated cells was a 96:4 mixture of these two nucleotides. Crude enzyme preparations obtained from the supernatant fraction of cells catalyzed the NAD+-dependent conversion of UDP-GlcNAc into UDP-GlcNAcA and UDP-ManNAcA. Studies on the partial separation and properties of enzymes revealed that UDP-GlcNAcA is synthesized directly from UDP-GlcNAc by the action of UDP-GlcNAc dehydrogenase and that UDP-ManNAcA is synthesized from UDP-GlcNAc through the successive actions of UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase. However, enzymatic conversion of UDP-GlcNAcA to UDP-ManNAcA was not detected. Ammonium sulfate protects both dehydrogenases from inactivation during storage and incubation. Partially purified UDP-GlcNAc dehydrogenase required dithiothreitol and the particulate fraction for its full activity. The apparent Km values of UDP-GlcNAc dehydrogenase for UDP-GlcNAc and NAD+ were 0.28 and 1.43 mM, respectively. The optimum pH of this enzyme was higher than 9 in Tris-HCl buffer. p-Chloromercuribenzoate at 27 microM as well as 10 mM ethanol almost completely inhibited the UDP-GlcNAc dehydrogenase reaction.