The Saccharomyces cerevisiae COQ10 gene encodes a START domain protein required for function of coenzyme Q in respiration

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q(2). Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q(6), the coq10 mutant has near normal amounts of Q(6) in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the aromatic-rich protein family Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q(6)/mol of protein. We propose that Coq10p is a Q(6)-binding protein and that in the coq10 mutant Q(6) it is not able to act as an electron carrier, possibly because of improper localization.

Production of pro- and anti-inflammatory cytokines by monocytes from patients with paracoccidioidomycosis

Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases. We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis. Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 mug/ml of lipopolysaccharide (LPS) for 18 h at 37 degreesC, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay. The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls. These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities. However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation. Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls. These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and-4, in bovine antral follicles

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.

Chloroquine is therapeutic in murine experimental model of paracoccidioidomycosis

Chloroquine, due to its basic properties, has been shown to prevent the release of iron from holotransferrin, thereby interfering with normal iron metabolism in a variety of cell types. We have studied the effects of chloroquine on the evolution of experimental paracoccidioidomycosis by evaluating the viable fungal recovery from lung, liver and spleen from infected mice and H2O2, NO production, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10 levels and transferrin receptor (TfR) expression from uninfected and infected peritoneal macrophages. Chloroquine caused a significant decrease in the viable fungal recovery from all organs tested, during all periods of evaluation. Peritoneal macrophages from chloroquine-treated infected mice showed higher H2O2 production and TfR expression, and decreased levels of NO, endogenous and stimulated-TNF-alpha, IL-6 and IL-10 during the three evaluated periods. However, despite its suppressor effects on the macrophage function, the chloroquine therapeutic effect upon murine paracoccidioidomycosis was probably due to its effect on iron metabolism, blocking iron uptake by cells, and consequently restricting iron to fungus growth and survival.

Conversion of immunosuppressive monotherapy from cyclosporin A to tacrolimus reverses bone loss in rats

Tacrolimus is used for transplant patients with refractory graft rejection and those with intolerance to cyclosporin (CsA), without the disfiguring adverse effects frequently attributed to CsA therapy. Since we have shown that CsA-associated bone loss can also affect alveolar bone, the purpose of this study was to evaluate the effects of conversion of monotherapy from CsA to tacrolimus on alveolar bone loss in rats. Groups of rats were treated with either CsA (10 mg/kg/day, s.c.), tacrolimus (I mg/kg/day, s.c.), or drug vehicle for 60 and 120 days, and an additional group received CsA for 60 days followed by conversion to tacrolimus for a further 60-day period. Bone-specific alkaline phosphatase (BALP), tartrate-resistent acid phosphatase (TRAP-5b), calcium (Ca2+), interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) concentrations were evaluated in the serum. Analyses of bone volume, bone surface, number of osteblasts, and osteoclasts were performed. Treatment with CsA for either 60 or 120 days was associated with bone resorption, represented by lower bone volume and increased number of osteoclasts; serum BALP, TRAP-5b, IL-1 beta, IL-6, and TNF-alpha were also higher in these animals. After conversion from CsA to tacrolimus, all the altered serum markers returned to control values in addition to a significant increase of bone volume and a lower number of osteoclasts. This study shows that conversion from CsA to tacrolimus therapy leads to a reversal of the CsA-induced bone loss, which can probably be mediated by downregulation of IL-1 beta, IL-6, and TNF-alpha production.

Low intensity laser therapy (LILT) in vivo acts on the neutrophils recruitment and chemokines/cytokines levels in a model of acute pulmonary inflammation induced by aerosol of lipopolysaccharide from Escherichia coli in rat

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Downregulation of nuclear factor-kappa B (NF-kappa B) pathway by silibinin in human monocytes challenged with Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metacyclogenesis modulates the ability of Leishmania promastigotes to induce IL-12 production in human mononuclear cells

Immunity against the intracellular protozoan Leishmania species is highly dependent on Th1 development. We have previously shown that IL-12 is a powerful and probably obligatory factor for Th1 cell generation and proliferation. We also observed that the experimental infection of C3H and BALB/c mice with Leishmania major is associated with IL-12 production in vivo. Now we demonstrate that metacyclic L. major promastigotes are poor inducers of IL-12 in vitro in fresh human PBMC and monocytes. In addition, we show that the ability of this pathogen to induce IL-12 and other cytokines is modulated by the metacyclogenic process, which had previously not been recognized. In contrast to the infective parasites (metacyclic promastigotes), the procyclic promastigotes collected at the logarithmic phase of the culture displayed a striking ability to induce IL-12, IFN-γ, TNF-α, and IL-10. Despite this differential effect of procyclic and metacyclic parasites in terms of IL-12 induction, both stages were inhibitory for IL-12 production induced by Staphylococcus aureus. The ability of procyclic promastigotes and, to a much lesser extent, that of metacyclic promastigotes to induce IL-12 were enhanced by pretreatment of monocytes in a cytokine milieu containing IFN-γ, IL-4, IL-13, or granulocyte-macrophage CSF or. by neutralization of endogenous IL-10. Our results suggest the development of an evasion mechanism as the Leishmania promastigotes mature to infectious forms and the possi-bility of using Ags derived from procyclic promastigotes for immunization procedures. Copyright © 1997 by The American Association of Immunologists.

Molecular mechanisms of the induction of IL-12 and its inhibition by IL- 10

Exogenously added IL-10 rapidly inhibited Staphylococcus aureus- or LPS- induced cytokine mRNA expression in human PBMCs and monocytes, with a maximal effect observed when IL-10 was added from 20 h before until 1 h after the addition of the inducers. Nuclear run-on assays revealed that the inhibition of IL-12 p40, IL-12 p35, and TNF-α was at the gene transcriptional level and that the addition of IL-10 to S. aureus- or LPS-treated PBMCs did not affect mRNA stability. The inhibitory activity of IL-10 was abrogated by cycloheximide (CHX), suggesting the involvement of a newly synthesized protein(s). The addition of CHX at 2 h before S. aureus or LPS also inhibited the accumulation of IL-12 p40 mRNA, but did not inhibit IL-12 p35 and TNF-α mRNA. This finding suggests that p40 transcription is regulated through a de novo synthesized protein factor(s), whereas the addition of CHX at 2 h after S. aureus activation caused superinduction of the IL-12 p40, IL-12 p35, and TNF-α genes. These results indicate that in human monocytes, the mechanism(s) of IL-10 suppression of both IL-12 p40 and IL-12 p35 genes is primarily seen at the transcriptional level, and that the induction of the IL-12 p40 and p35 genes have different requirements for de novo protein synthesis.

p38 MAPK regulates IL-1β induced IL-6 expression through mRNA stability in osteoblasts

Osteoblast-derived IL-6 functions in coupled bone turnover by supporting osteoclastogenesis favoring bone resorption instead of bone deposition. Gene regulation of IL-6 is complex occurring both at transcription and post-transcription levels. The focus of this paper is at the level of mRNA stability, which is important in IL-6 gene regulation. Using the MC3T3-E1 as an osteoblastic model, IL-6 secretion was dose dependently decreased by SB203580, a p38 MAPK inhibitor. Steady state IL-6 mRNA was decreased with SB203580 (2 μM) ca. 85% when stimulated by IL-1β (1-5 ng/ ml). These effects require de novo protein synthesis as they were inhibited by cycloheximide. p38 MAPK had minor effects on proximal IL-6 promoter activity in reporter gene assays. A more significant effect on IL-6 mRNA stability was observed in the presence of SB203580. Western blot analysis confirmed that SB203580 inhibited p38 MAP kinase, in response to IL-1β in a dose dependent manner in MC3T3-E1 cells. Stably transfected MC3T3-E1 reporter cell lines (MC6) containing green fluorescent protein (GFP) with the 3′untranslated region of IL-6 were constructed. Results indicated that IL-1β, TNFα, LPS but not parathyroid hormone (PTH) could increase GFP expression of these reporter cell lines. Endogenous IL-6 and reporter gene eGFP-IL-6 3′UTR mRNA was regulated by p38 in MC6 cells. In addition, transient transfection of IL-6 3′UTR reporter cells with immediate upstream MAP kinase kinase-3 and -6 increased GFP expression compared to mock transfected controls. These results indicate that p38 MAPK regulates IL-1β-stimulated IL-6 at a post transcriptional mechanism and one of the primary targets of IL-6 gene regulation is the 3′UTR of IL-6.

Cytokine and antibody production during murine leptospirosis

The aim of the present study was to investigate the kinetics of humoral and cellular responses during leptospirosis. We observed that the presence of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) was associated with antibody production and bacterial recovery, and the compromising of both TNF-α and IL-6 in the immunopathogenesis of leptospirosis during an experimental infection of BALB/c mice inoculated with Leptospira interrogans serovar Canicola was verified. Results showed higher levels of TNF-α and IL-6 in the initial phase of infection, in which the greatest bacterial clearance was observed. However, when the bacterial recovery was compared with the kinetics of the production of antibodies, the results revealed a kinetics proportionally inverted to antibody production. This fact may be related to some inhibitory factor which could be responsible for the selective suppression of the cellular immune response. We concluded that during leptospirosis there was a greater mobilization of the cellular immune response activity, mainly in the initial phase of the infectious process, for posterior involvement of the humoral response, and that both TNF-α and IL-6 could be associated with the immunopathogenesis of the disease.

Pro- and anti-inflammatory cytokines produced by human monocytes challenged in vitro with Paracoccidioides brasiliensis

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1β, IL-6, IL-10 and TNF-α. Pb18 induced higher levels of IL-1β, IL-6, and IL-10 than Pb265. IL-8 and TGF-β1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.