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Production and secretion of adrenomedullin from vascular smooth muscle cells: augmented production by tumor necrosis factor-alpha.

In this study, we demonstrate production and secretion of adrenomedullin (AM) from cultured vascular smooth muscle cells (VSMCs). In addition to endothelial cells (ECs), we found immunoreactive (ir-) AM in culture media of rat and bovine VSMCs in the survey for AM-producing cells. Although the secretion level of ir-AM was at most 1/6 that of rat ECs, all the examined VSMCs were shown to produce AM. By gel filtration, reverse phase high-performance liquid chromatography, and biological characterization, ir-AM in the culture medium was identified to be rat AM of 50 residues. By RNA blot analysis, a positive band of AM mRNA was detected in cultured VSMC at an intensity 3-4 fold higher than that in adrenal gland. Gene expression and production of AM were markedly augmented by tumor necrosis factor-alpha. Based on these data as well as the presence of AM specific receptors on VSMCs, AM secreted from VSMC is deduced to function as an autocrine or paracrine regulator in vascular cell communication.

Adrenomedullin↗

Oncostatin M production by human dendritic cells in response to bacterial products.

Oncostatin M (OSM) is a pleiomorphic cytokine that belongs to the IL-6 cytokine family. It is produced by activated T cells and monocytes/macrophages and plays an important role in the process of inflammatory responses. Although dendritic cells (DCs) have been shown to secrete a variety of cytokines, it is not elucidated whether DCs are able to produce OSM. To clarify this, using human DCs derived from peripheral blood cells, we measured the protein levels of OSM in the supernatants of DC cultures by ELISA and examined the expression of OSM mRNA by RT-PCR after stimulation with lipopolysaccharide (LPS) or fixed Staphylococcus aureus (SACS). Upon stimulation with bacterial products, DCs secreted a large amount of OSM protein in a dose- and time-dependent manner. Concomitantly, the expression of OSM mRNA by DCs was markedly up-regulated. Compared the ability of DCs to produce OSM with that of monocytes, which are major producers of OSM, DCs released significantly higher amounts of OSM protein in the culture supernatants than monocytes. These findings indicate for the first time that human monocyte-derived DCs can synthesize and secrete large amounts of OSM in response to bacterial products, suggesting that OSM produced by DCs at infectious sites may play a role in modulating inflammatory responses.

Base Sequence↗

Transmission of HIV-1 from productively infected mature Langerhans cells to primary CD4+ T lymphocytes results in altered T cell responses with enhanced production of IFN-gamma and IL-10.

Mature Langerhans cells (mLC), the ex vivo correlates of interdigitating dendritic cells (IDC), are susceptible to infection with HIV-1. As IDC are important activators of T helper (Th) cells in vivo, we examined the interaction of HIV-1-infected mLC with CD4+ T lymphocytes. HIV-1-infected mLC readily formed clusters with the T cells and efficiently transmitted HIV-1 to the CD4+ Th cells. Formation of syncytia between mLC and T cells was initiated by HIV-1-infected mLC. In the clusters of HIV-1-infected mLC and activated T cells a massive HIV-1 production was observed leading to the subsequent elimination of the activated and infected T helper cells. Examination of the cytokine pattern produced during interaction of infected mLC with CD4+ T cells revealed an enhanced production of IFN-gamma and IL-10 in the cocultures. These results suggest that during antigen presentation-driven T cell activation by IDC in the lymphoid tissues, HIV-1-infected IDC might efficiently transmit the virus to Th cells, leading to altered Th cell responses.

CD4-Positive T-Lymphocytes↗

Biochemical and molecular tools for the production of useful terpene products from pepper (Capsicum annuum).

Among other natural products such as colorants and flavorants, natural fungicides like the pepper phytoalexin capsidiol, and the related biochemical pathways, may be used for practical approaches. Key enzymes such as 3-hydroxy-3-methylglutaryl Coenzyme A: reductase, the farnesyl pyrophosphate synthase and and farnesyl pyrophosphate cyclases are known and some related genes have been isolated. However, specific enzymes for important and final modifications as methylation and others, are still to be studied. Construction of chimeric enzymes allowed already the synthesis of different products and the possibilities of designing new enzymes by gene manipulation to produce unknown and useful chemicals are open.

Agriculture↗

Products of cultured neuroglial cells: II. The production of fibronectin by C6 glioma cells.

The possibility of fibronectin production by C6 glioma cells was examined with assays which require protein synthesis. Proteins produced by C6 cells using radiolabeled amino acid precursors were tested for affinity to collagen by binding to immobilized gelatin. The predominant collagen binding protein made by C6 coelectrophoresed with fibronectin synthesized by control fibroblasts and with the larger of the two proteins in unlabeled fibronectin when applied to polyacrylamide gels with sodium dodecyl sulfate (SDS). In addition, C6 produced a larger collagen binding protein of approximately 270,000 molecular weight. Solubilities in urea solutions of the collagen-binding proteins made by C6 cells and fibroblasts were similar. Immunofluorescence showed fibronectin associated with the C6 cell monolayer, but less abundant than the fibronectin associated with fibroblasts. Results provide evidence for the production of fibronectin by the C6 glioma cell line.

Animals↗

Stimulation by lipoxygenase products of superoxide anion production in FMLP-treated neutrophils.

Our recent observation that leukotriene B4 (10(-9)M) is a potent enhancer of FMLP-initiated neutrophil superoxide anion formation prompted an evaluation of the ability of other lipoxygenase products and related compounds to modulate this response. The results indicate that FMLP-evoked O-2 may be enhanced by 10(-8)-10(-7)M levels of a number of lipids, in addition to LTB4, including 5-HPETE, 5-HETE, 5,15-DiHPETE and by higher levels of other 15-series lipoxygenase products and arachidonic acid. It is of interest that the relative potency of these agents in potentiating the superoxide response to FMLP approximately parallels their reported ability to induce chemotactic activity in leukocytes.

Adult↗

Retinoic acid inhibits CD40 plus IL-4 mediated IgE production through alterations of sCD23, sCD54 and IL-6 production.

BACKGROUND: All-trans retinoic acid (ATRA) inhibits IgE synthesis from anti-CD40 plus IL-4 stimulated human B lymphocytes. OBJECTIVE: To study the underlying mechanisms, we examined here molecules which are known to have an impact on IgE production, namely CD23, CD54 and IL-6. METHODS: Human anti-CD40 plus IL-4 stimulated B cells were cultured in the absence and presence of ATRA (10(-6)-10(-10) M). ELISAs were performed to determine soluble (s) CD23 and sCD54, IL-6 and IgE-levels. CD23 and CD54 surface expression were determined by flow cytometric analysis. Semiquantitative-RT-PCR was employed to analyse IL-6, CD23 and CD54 mRNA expression. RESULTS: ATRA induced a dose-dependent increase of percent CD23 (3.4 fold) or CD54 (1.6 fold) positive B cells. At the mRNA level, this was reflected by a modest increase of CD54 mRNA (46.5 +/- 15.8%) only. By contrast, levels of sCD54 were decreased dose-dependently in the presence of ATRA (56.6 +/- 7.6%). Cytokine analysis showed that IL-6 secretion was significantly inhibited by ATRA (53.6 +/- 0.6%) and also IL-6 mRNA synthesis was reduced (66.3 +/- 11.6%). The observed inhibition of IgE production mediated by ATRA was significantly reversed to 90.5 +/- 12% by the addition of 100 pg/mL recombinant IL-6. CONCLUSIONS: ATRA interferes through several pathways with the anti-CD40 plus IL-4 mediated B cell activation, namely IL-6, CD23 and CD54.

Antigens, Differentiation, B-Lymphocyte↗

Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production.

Screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth. Ralstonia eutropha H16 and Pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (PHA). R. eutropha and P. oleovorans accumulated PHA amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in mineral salt medium with the oil from the rhamnose production as the sole carbon source. The accumulated PHA isolated from R. eutropha consisted of only 3-hydroxybutyric acid, whereas the PHA isolated from P. oleovorans consisted of 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, and 3-hydroxydodecanoic acid. The composition was confirmed by gas chromatography of the isolated polyesters. Batch and fed-batch cultivations in stirred-tank reactors were done.

Bioreactors↗

Production of sophorolipids from whey. II. Product composition, surface active properties, cytotoxicity and stability against hydrolases by enzymatic treatment.

Sophorolipids, obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1',4"-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFTmin 39 mN m-1, CMC 130 mg l-1), water solubilities (2-3 g l-1) and low cytotoxicities (LC50 300-700 mg l-1). In contrast, purified sophorolipids were more surface active (SFTmin 36 mN m-1, CMC 10 mg l-1), less water soluble (max. 70 mg l-1) and showed stronger cytotoxic effects (LC50 15 mg l-1). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect.

Animals↗

Anthraquinones production, hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies.

The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H2O2) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l(-1), intracellular AQ content at 42 mg g(-1) DW, and H2O2 level at 9 micromol g(-1) FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g(-1) FW and total carotenoids at 13.3 mg g(-1) FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H2O2 levels. Vitamin C content at 30-120 microg g(-1) FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400-500 microg g(-1) FW in 7-day-old cultures from all medium strategies and reduced to 50-150 microg g(-1) FW on day 14 and 21; as compared to 60 microg g(-1) FW in callus and 200 microg g(-1) FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.

Anthraquinones↗

Secretory products of breast cancer cells upregulate hyaluronan production in a human osteoblast cell line.

Hyaluronan (HA) has been implicated in breast cancer progression and metastasis to lymph nodes. Although breast cancer has a strong propensity for metastasis to the bone, the role of HA in the development of bone metastasis in breast cancer is not well delineated. In order to determine the role of HA in breast cancer induced osteolysis, we examined the effect of secretory products in the conditioned medium of breast cancer cell lines on the expression of hyaluronan synthases (HAS), accumulation of HA in pericellular matrix, secretion of HA in the culture media, and on expression of surface HA receptors, in a human immature osteoblast cell line (Hfob). Our results show that conditioned medium derived from breast cancer cells upregulate the expression of hyaluronan synthases, HAS1 and HAS2, followed by significant increase in pericellular and secreted HA in Hfob cells. Our results further demonstrate that both CD44 and receptor for hyaluronan-mediated motility (RHAMM) are involved in binding cell surface associated HA on Hfob cells. Analysis of the growth factors in the conditioned medium implicates TGF-beta1 in the modulation of HAS1 and HAS2, as well as in the increase in pericellular and secreted HA. This report is the first to show that soluble factors produced by breast cancer cells mediate increase in HA production in osteoblasts.

Breast Neoplasms↗

Effect of palifermin in a murine model of graft-versus-host disease (GVHD) associated with Th2 cytokine production, autoantibody production, and glomerulonephritis.

Palifermin (recombinant human keratinocyte growth factor) prevents the development of acute, lethal graft-versus-host disease (GVHD). It does so, at least in part, by protecting cells from injury. Another property of Palifermin is immune regulation. How the latter influences the evolution of GVHD remains uncertain. We explored the effect of Palifermin on GVHD in the DBA/2 --> ((DBA/2)x(C57BL/6))F(1)-hybrid strain combination, a model associated with autoantibody production and glomerulonephritis. Untreated recipients survived until at least day 150 post-induction. Palifermin-treated recipients succumbed between days 50 and 90 with levels of proteinuria of up to 20 g/L, ascites, and rapidly progressive, crescentic glomerulonephritis that was most severe in mice with the greatest levels of proteinuria. Kidney sections from both Palifermin-treated and untreated recipients showed the presence of granular deposits of IgG, IgM, IgA, and C3 in the mesangium and the glomerular basement membrane. Electron microscopy confirmed the extensive glomerular immune complex deposition. Antinuclear and anti-dsDNA antibodies were present in sera from both treated and untreated recipients; however, those in the latter were only detectable if the serum was kept at 37 degrees C, indicating that they were cryoglobulins. IL-4 was detectable only in cultures from Palifermin-treated recipients and the levels of IL-5 and IL-13 were significantly higher in the Palifermin-treated group than in untreated GVHD mice. IFN-gamma was only detectable in untreated GVHD mice. These data suggest that although Palifermin can protect mice with acute GVHD, it exacerbates GVHD in a model associated with autoantibody production and a preponderance of Th2 cytokines.

Animals↗

Type I interferons attenuate T cell activating functions of human mast cells by decreasing TNF-alpha production and OX40 ligand expression while increasing IL-10 production.

Recent studies have demonstrated that mast cells not only mediate inflammatory reactions in type I allergy but also play an important role in adaptive immunity. In the present study, we investigated the effects of interferon-alpha, which shares the same receptor as IFN-beta, on human cord blood-derived mast cells. Mast cells produced TNF-alpha, and IL-10, and expressed OX40 ligand upon activation by crosslinking of FcepsilonRI. When treated with interferon-alpha, TNF-alpha production was decreased while IL-10 and TGF-beta productions were increased. Furthermore, flow cytometric analysis revealed that interferon-alpha downregulated expression OX40 ligand on mast cells which is crucial for mast cell-T cell interaction. We confirmed that the viability of mast cells was not affected by interferon-alpha treatment. Accordingly, interferon-alpha-treated mast cells induced lower levels of CD4+ T cell proliferation compared with those without interferon-alpha treatment. These results suggest that type I interferons suppress T cell immune responses through their regulatory effects on mast cells.

Cell Survival↗

Standards for product safety design: a framework for their production.

Accident reduction can be achieved through improved standards of product design and associated safety performance standards. Although many problems exist in terms of data production, reporting, collection and dissemination, it is suggested that a useful design data bank could be contructed and applied. The discussion of data requirements, classification and application is oriented particularly towards the problem of child safety.

Journal Article↗

Increased production of lipoxygenase products by cholesterol-rich mouse macrophages.

The metabolism of arachidonic acid by cholesterol-enriched resident mouse peritoneal macrophages was investigated. The amounts of monohydroxyeicosatetraenoic acid (mono-HETE) produced by the cholesterol-rich macrophages were 2.5-fold greater when compared to control macrophages. The major lipoxygenase product, identified by high-performance liquid chromatography in both macrophages was 12-HETE. Since macrophages are important participants in the formation of atheromatous lesions, the increased metabolism of arachidonic acid to HETE products by cholesterol-rich macrophages could contribute to the initiation and progression of the atherosclerotic process.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

Mosaicism in female hybrid hares heterozygous for glucose-6-phosphate dehydrogenase. VI. Production of monotypism in the aortas of 4 of 10 mosaic hares fed cholesterol oxidation products.

The normal aortic tissue of black women heterozygous for glucose-6-phosphate dehydrogenase (G-6-PD) usually consists of two cell phenotypes (mosaicism). By electrophoresis two G-6-PD types are demonstrated (ditypism). Advanced atherosclerotic lesions from such women not infrequently yield samples displaying only one G-6-PD type (monotypism). Possible causes for the monotypism include (1) monoclonal origin of the lesion and (2) selective growth and/or survival advantage of one phenotype over the other. We have been attempting to produce monotypism in the aortas of a hybrid hare model that displays G-6-PD mosaicism in the normal state. In the current study 14 G-6-PD mosaic hares were fed a high-cholesterol diet for 6 to 17 months. All developed extensive moderately severe (as compared to humans) atherosclerotic lesions. No monotypic samples were found. Ten hares were fed (in addition to the standard high-cholesterol diet) small doses of one of two cholesterol oxidation products (25-hydroxycholesterol or triol) for 6 to 21 months. Four of the ten developed monotypic foci in either atherosclerotic lesions or normal aortic tissue or both. The reason for giving the oxidation products was because they are cytotoxic for cells in vitro and one (25-hydroxycholesterol) had been shown to be more toxic for one phenotype than the other with cultured fibroblasts from the mosaic hares (not infrequently resulting in the culture becoming monotypic). We suggest that the monotypism observed in vivo was probably produced by a mechanism similar to that operating in vitro.

Animals↗

Quantification of superantigen induced IFN-gamma production by computerised image analysis--inhibition of cytokine production and blast transformation by pooled human IgG.

A quantitative image analysis technique was developed to assess the cytokine content of immunocytochemically stained cytokine producing cells. Peripheral blood mononuclear cells were stimulated to induce cytokine production with the superantigen streptococcal pyrogenic exotoxin-A. We have developed a method based on indirect immunocytochemistry which identifies IFN-gamma producing cells by a characteristic morphology generated by the accumulation of IFN-gamma in the Golgi organelle. An image analysis technique permitted discrimination between these producer cells and IFN-gamma binding target cells, which showed a different appearance, with staining restricted to the cell surface membrane. A semi-automated routine programme allowed the signal from a video camera to be processed by computerised image analysis methodology. This enabled us to measure the number of cytokine producing cells, the cytokine staining intensity in individual cells and the cell size expressed in actual cell area. The incidence of IFN-gamma producing cells determined by image analysis measurement was compared to results obtained using manual microscopy. Cell size was assessed by the image analysis system as well as by flow cytometry. Administration of pooled human IgG for intravenous use (IVIg) to the superantigen stimulated cells significantly down-regulated IFN-gamma production, both in terms of the numbers of producer cells and in terms of cytokine staining intensity in individual cells. In addition blast transformation of cells was substantially reduced. These effects, mediated by IVIg, were also evident following delayed IVIg administration 24 h after the initial cell stimulation.

Adult↗

Production of 19-oic-11-deoxycorticosterone from 19-oxo-11-deoxycorticosterone by cytochrome P-450(11)beta and nonenzymatic production of 19-nor-11-deoxycorticosterone from 19-oic-11-deoxycorticosterone.

19-Oxo-11-deoxycorticosterone was incubated with a cytochrome P-450(11)beta-reconstituted system, and the metabolites were analyzed by high performance liquid chromatography(HPLC). The main product found after chromatography was collected and treated with diazomethane. HPLC and 1H-NMR analysis of the methylated derivative indicated that it was 19-oic-11-deoxycorticosterone methyl ester. When 19-oic-11-deoxycorticosterone was stored at -20 degrees C for 1 month, it was spontaneously converted to other steroids. Structural analysis of the main degradation product indicated that it was 19-nor-11-deoxycorticosterone. These results suggest that the conversion of 19-oxo-11-deoxycorticosterone to 19-oic-11-deoxycorticosterone occurs through the P-450(11)beta-catalyzed reaction, and that the 19-oic-11-deoxycorticosterone thus formed is nonenzymatically converted into 19-nor-11-deoxycorticosterone.

Adrenal Cortex↗