Enzymatic Mechanisms Involved in Evasion of Fungi to the Oxidative Stress: Focus on Scedosporium apiospermum.

The airways of patients with cystic fibrosis (CF) are frequently colonized by various filamentous fungi, mainly Aspergillus fumigatus and Scedosporium species. To establish within the respiratory tract and cause an infection, these opportunistic fungi express pathogenic factors allowing adherence to the host tissues, uptake of extracellular iron, or evasion to the host immune response. During the colonization process, inhaled conidia and the subsequent hyphae are exposed to reactive oxygen species (ROS) and reactive nitrogen species (RNS) released by phagocytic cells, which cause in the fungal cells an oxidative stress and a nitrosative stress, respectively. To cope with these constraints, fungal pathogens have developed various mechanisms that protect the fungus against ROS and RNS, including enzymatic antioxidant systems. In this review, we summarize the different works performed on ROS- and RNS-detoxifying enzymes in fungi commonly encountered in the airways of CF patients and highlight their role in pathogenesis of the airway colonization or respiratory infections. The potential of these enzymes as serodiagnostic tools is also emphasized. In addition, taking advantage of the recent availability of the whole genome sequence of S. apiospermum, we identified the various genes encoding ROS- and RNS-detoxifying enzymes, which pave the way for future investigations on the role of these enzymes in pathogenesis of these emerging species since they may constitute new therapeutics targets.

Promoter sequences involved in transforming growth factor beta1 gene induction in HaCaT keratinocytes after gamma irradiation.

Transforming growth factor beta 1 (TGFB1) is a cytokine involved in the development of both acute and late cutaneous radiation syndromes. We previously demonstrated that ionizing radiation induces TGFB1 expression in vivo in pig skin within a few hours. The purpose of the present study was to develop an in vitro human model to identify the mechanisms of this early activation. Accordingly, human HaCaT keratinocytes were irradiated with a single dose of 20 Gy. First, radiation-induced TGFB1 overexpression was checked at both the transcriptional and transductional levels in HaCaT cells. Then electrophoretic mobility shift assays (EMSA) and transient transfection with various TGFB1 promoter constructs were used to identify the sequences involved in regulating this promoter. EMSA analysis showed the induction of nuclear protein binding activity by gamma irradiation to the -365 AP1 sequence (TGTCTCA), suggesting the involvement of AP1 sequences in the regulation of TGFB1 transcription. In gene reporter assays, maximal TGFB1 promoter activation was found for the longest construct, which contains two AP1 sequences. However, assays with constructs including deletions showed that these two AP1 sequences were not sufficient to confer TGFB1 inducibility. These results showed for the first time, to our knowledge, that transcriptional regulation is involved in radiation-induced activation of TGFB1 gene expression.

Immunohistochemical analysis of cerebral cortical and vascular lesions in the primate Microcebus murinus reveal distinct amyloid beta1-42 and beta1-40 immunoreactivity profiles.

Recent reports have shown that amyloid beta deposits in the brains of Alzheimer's disease patients consist mainly of two distinct species of amyloid beta protein (Abeta) with different C-termini, Abeta1-42 (Abeta42) and Abeta1-40 (Abeta40). The nature of the Abeta species in Microcebus murinus brain was investigated immunocytochemically using polyclonal antibodies with clear specificity for the Abeta42 and Abeta40 C-termini. The cortical vascular deposits were immunopositive for both Abeta42 and Abeta40. However, most of the diffuse plaques were strongly positive for Abeta42 whereas only a subset of deposits were positive for Abeta40. Numerous cortical plaques were Abeta42-immunopositive but tested negative for Abeta40. This suggests that Abeta42 is probably associated with early stages of plaque maturation. This neuropathological feature reminiscent of that observed in brains affected by Alzheimer's disease further supports the idea that M. murinus could be used as a potential model of the early stages of this neurological disease.

Fc-receptor-mediated intracellular delivery of Cu/Zn-superoxide dismutase (SOD1) protects against redox-induced apoptosis through a nitric oxide dependent mechanism.

BACKGROUND: Using specific antibodies against bovine Cu/Zn-superoxide dismutase (EC 1.15.1.1, SOD1) we demonstrated that anti-SOD antibodies (IgG1) are able to promote the intracellular translocation of the antioxidant enzyme. The transduction signalling mediated by IgG1 immune complexes are known to promote a concomitant production of superoxide and nitric oxide leading to the production of peroxynitrites and cell death by apoptosis. The Fc-mediated intracellular delivery of SOD1 thus limited the endogenous production of superoxide. It was thus of interest to confirm that in the absence of superoxide anion, the production of nitric oxide protected cells against apoptosis. Study in greater detail clearly stated that under superoxide anion-free conditions, nitric oxide promoted the cell antioxidant armature and thus protected cells against redox-induced apoptosis. MATERIALS AND METHODS: The murine macrophage cell-lines J774 A1 were preactivated or not with interferon-gamma and were then stimulated by IgG1 immune complexes (IC), free SOD1 or SOD1 IC and superoxide anion, nitric oxide, peroxynitrite, and tumor necrosis factor-alpha (TNF-alpha) production was evaluated. The redox consequences of these activation processes were also evaluated on mitochondrial respiration and apoptosis as well as on the controlled expression of the cellular antioxidant armature. RESULTS: We demonstrated that SOD1 IC induced a Fcgamma receptor (FcgammaR)-dependent intracellular delivery of the antioxidant enzyme in IFN-gamma activated murine macrophages (the J774 AI cell line). The concomitant stimulation of the FcyR and the translocation of the SOD1 in the cytoplasm of IFN-gamma-activated macrophages not only reduced the production of superoxide anion but also induced the expression of the inducible form of nitric oxide synthase (iNOS) and the related NO production. This inducing effect in the absence of superoxide anion production reduced mitochondrial damages and cell death by apoptosis and promoted the intracellular antioxidant armature. CONCLUSIONS: To define the pharmacologic mechanism of action of bovine SOD1, we attempted to identify the second messengers that are induced by SOD1 IC. In this work, we propose that Fc-mediated intracellular delivery of the SOD1 that reduced the production of superoxide anion and of peroxynitrite, promoted a NO-induced protective effect in inducing the antioxidant armature of the cells. Taken together, these data suggested that specific immune responses against antigenic SOD1 could promote the pharmacological properties of the antioxidant enzyme likely via a NO-dependent mechanism.

Genetic and physical analyses of the centromeric and pericentromeric regions of human chromosome 5: recombination across 5cen.

Human centromeres are poorly understood at both the genetic and the physical level. In this paper, we have been able to distinguish the alphoid centromeric sequences of chromosome 5 from those of chromosome 19. This result was obtained by pulsed-field gel electrophoresis after cutting genomic DNA with restriction endonucleases NcoI (chromosome 5) and BamHI (chromosome 19). We could thus define a highly polymorphic marker, representing length variations of the D5Z1 domain located at the q arm boundary of the chromosome 5 centromere. The centromeric region of chromosome 5 was then analyzed in full detail. We established an approximately 4.6-Mb physical map of the whole region with five rare-cutting enzymes by using nonchimeric YACs, two of which were shown to contain the very ends of 5cen on both sides. The p-arm side of 5cen was shown to contain an alphoid subset (D5Z12) different from those described thus far. Two genes and several putative cDNAs could be precisely located close to the centromere. Several L1 elements were shown to be present within alpha satellites at the boundary between alphoid and nonalphoid sequences on both sides of 5cen. They were used to define STSs that could serve as physical anchor points at the junction of 5cen with the p and q arms. Some STSs were placed on a radiation hybrid map. One was polymorphic and could therefore be used as a second centromeric genetic marker at the p arm boundary of 5cen. We could thus estimate recombination rates within and around the centromeric region of chromosome 5. Recombination is highly reduced within 5cen, with zero recombinants in 58 meioses being detected between the two markers located at the two extremities of the centromere. In its immediate vicinity, 5cen indeed exerts a direct negative effect on meiotic recombination within the proximal chromosomal DNA. This effect is, however, less important than expected and is polarized, as different rates are observed on both arms if one compares the 0 cM/Mb of the p proximal first 5.5 Mb and the 0.64 cM/Mb of the q proximal first 5 Mb to the sex-average 1.02 cM/Mb found throughout the entire chromosome 5. Rates then become close to the average when one goes further within the arms. Finally, most recombinants (21/22), irrespective of the arm, are of female origin, thus showing that recombination around 5cen is essentially occurring in the female lineage.

Cloning of the presenilin 2 cDNA and its distribution in brain of the primate, Microcebus murinus: coexpression with betaAPP and Tau proteins.

A 1340-bp cDNA fragment encoding the lemurian presenilin 2 protein (PS2) was isolated from a Microcebus murinus brain cDNA library by PCR using oligonucleotide primers based on the nucleotide sequence of the human gene. Analysis of five isolated clones showed that the sequence encoded a 448-amino-acid open reading frame, 95.5% identical to the human and 93.5% identical to the mouse presenilin 2 sequences. However, neither the localization of the 2 positions in PS2 nor that of the 43 positions in PS1 associated with early onset Alzheimer's disease were changed. Expression of the presenilin 2 was detected by RT-PCR and compared with that of presenilin 1 and betaAPP in the brain and in peripheral tissues (liver, kidney, and spleen). Immunohistochemistry with a specific polyclonal antiserum raised against a synthetic peptide from the N-terminal part of the human PS2 showed that the protein is distributed throughout the microcebe brain, in vascular and nerve structures. In cortical and in subcortical areas, PS2 labeling was weak and granular in appearance and was scattered throughout the cytoplasm of many neurones, extending into neurites. The gene expression of PS2 increased with age but was not affected by the presence of numerous amyloid plaques. Double labeling immunocytochemistry detected very few neurones with combined immunoreactivity PS2 and APP, or PS2 and Tau.

Molecular cloning, sequencing, and brain expression of the presenilin 1 gene in Microcebus murinus.

The cDNA encoding the Microcebus murinus presenilin 1 protein (PSI) was cloned by RT-PCR from a brain cDNA library using various combinations of oligonucleotide primers designed on the basis of the human nucleotide sequence. Analysis of five clones isolated from two positive combinations revealed that the deduced open reading frame encodes two protein isoforms of 467 and 463 amino acid residues. The shorter isoform lacked the four residues VRSQ in the N-terminal region and like the 467 amino acid isoform presented 22 substitutions with its human homologue. The 12 bp nucleotide deletion evidenced in the cDNA encoding the shorter isoform is consistent with the use of an alternative 5' splice donor site identified at the end of the human exon 3. The immunohistochemistry performed with a specific polyclonal antiserum raised against a synthetic peptide located in the human large hydrophilic loop of PS1 revealed that the protein is widely distributed independently of age or of pathology in the microcebe brain. PS1 is found predominantly in neurons of the different cortical layers and hippocampus but also in subcortical structures. The PS1 labelling appeared as thin granulations scattered throughout the cytoplasm of numerous neurons and sometimes in neurites.

Role of nitric oxide in the anti-tumoral effect of retinoic acid and 1,25-dihydroxyvitamin D3 on human promonocytic leukemic cells.

All trans retinoic acid and vitamin D3 derivatives are well known for their antileukemic activity, while the precise mechanism of this effect remains to be clarified. Using human leukemic U937 and THP-1 promonocytic cell lines, we analyzed the effect of all-trans retinoic acid (RA) and/or 1,25-dihydroxyvitamin D3 (VD) on the generation of nitric oxide (NO), a potent antitumoral mediator. U937 cell differentiation with VD or with both RA and VD (RA/VD) correlated with gene transcription and functional expression of inducible nitric oxide synthase (iNOS). Nitrites and L-citrulline were also detected in U937 cell supernatants as soon as 24 hours following cell incubation with VD or RA/VD, but not in cells treated with RA alone. Inhibition of iNOS activity by NG-monomethyl-L-arginine (LNMMA) significantly decreased in vitro U937 cell differentiation with VD and RA/VD as shown by the expression of cell differentiation markers (CD14 and CD68) and by the capacity of these cells to undergo a luminol-dependent chemiluminescence in response to opsonized zymosan. Similar results were obtained using the THP-1 cell line strengthening the role of NO in the VD- and RA/VD-induced growth arrest and terminal differentiation of promonocytic leukemia cells.

No founder effect in three novel Alzheimer's disease families with APP 717 Val-->Ile mutation. Clerget-darpoux. French Alzheimer's Disease Study Group.

We sequenced exons 16 and 17 of the APP (amyloid precursor protein) gene in 18 unrelated French Alzheimer's disease (AD) patients. These patients had an onset before the age of 60 and belonged to families with autosomal dominant transmission of the disease. We detected the APP 717 Val-->Ile mutation in three out of 18 (16.6%) families. In these three families, all affected subjects had the APOE 3/3 genotype, but their ages of onset ranged from 38 to 60 years, indicating that factors other than the APOE genotype influence age of onset. Analysis of two polymorphic loci adjacent to the APP gene showed that at least two independent mutational events had occurred within these pedigrees, in spite of their origin in the same region of France.

beta-Amyloid protein precursor in Microcebus murinus: genotyping and brain localization.

Senile plaques characterized by beta-amyloid protein (A beta) deposits around dystrophic neurites and glial cells are more abundant in the cerebral cortical parenchyma of Alzheimer's disease (AD) patients than in the aged population. Four different mutations in the amyloid precursor protein (APP) gene have been directly involved in a few cases of familial AD with early onset (before 60 years). Previous studies have shown that Microcebus murinus, a nonhuman primate, also develops analogous deposits of A beta in the cortical parenchyma and blood vessel walls in the brain. Sequence analysis of exons 16 and 17 of the APP gene, encoding for A beta, revealed that even if nucleotide divergences occurred, the resulting peptide is completely homologous with the human A beta. The systematic comparison of the A beta nucleotide sequence in microcebus with or without amyloid deposits revealed that neither the presence of mutations involved in some cases of early onset familial AD nor the presence of a mutational founder effect can explain the amyloidosis observed in some old microcebus of our breeding. Localization of the APP was performed by immunocytochemistry in the brains of adult microcebus (1 to 11 years of age) using two antibodies raised against the C-terminus and N-terminus portions of APP. Microscopic examinations revealed that in the microcebus the APP distribution was similar to that observed in the human: (1) A beta and its precursor were simultaneously observed in amyloid plaques (AP) of the cortical parenchyma; (2) APP was localized in cell bodies and proximal dendrites of neurons, in astrocytes and oligodendrocytes, and in blood vessel and capillary walls; (3) labeling of APP in these structures was correlated with the presence of AP; and (4) labeling of APP increased with the age of the animal.

Identification of a unique apolipoprotein E allele in Microcebus murinus; ApoE brain distribution and co-localization with beta-amyloid and tau proteins.

This report is devoted to the characterization of the apolipoprotein E (ApoE) in Microcebus murinus. Only one allele homologous to the human ApoE4 allele was evidenced. The distribution of the corresponding ApoE protein in the brain was found in association with the pathological proteins characteristic of Alzheimer's disease (AD). Immunocytochemistry revealed brain deposits of ApoE in: (1) the cortical amyloid plaques; (2) the neurones of the various cortical lobes, the hippocampus and the brainstem; (3) the glial cells, astrocytes of the cortical parenchym and oligodendrocytes of the corpus callosum; and (4) the vessel walls. Most ApoE, beta-amyloid protein, abnormally phosphorylated Tau proteins and gliofilament acid proteins were seen in the same cortical areas. These findings for ApoE report the view that Microcebus murinus, in captivity, presents a pathological profile very similar to that observed in AD.

A large pedigree with early-onset Alzheimer's disease: clinical, neuropathologic, and genetic characterization.

We present clinical, neuropsychological, and neuropathologic data on a large pedigree including 34 subjects with early-onset progressive dementia. The mean (+/- SD) age at onset was 46 +/- 3.5 years and the mean age at death 52.6 +/- 5.7 years. Twelve patients were clinically diagnosed as having probable Alzheimer's disease (AD) according to the NINCDS-ADRDA criteria. Neuropsychological evaluation, performed at a moderate stage of the disease, was available in six subjects and showed a classic pattern of cognitive deficit. Myoclonus and extrapyramidal signs were common, and seizures were present in all affected subjects. There were neuropathologic changes typical of AD in two brains. A significant lod score of 5.48 was observed at a recombination fraction of theta = 0.0 with the genetic marker D14S43, thereby establishing that the responsible gene was located on chromosome 14q24.3. These results suggest that epilepsy could represent a particular feature in AD families linked to chromosome 14q.

The meiosis-specific Xmr gene product is homologous to the lymphocyte Xlr protein and is a component of the XY body.

The Xlr (X-chromosome linked, lymphocyte regulated) multigene family was previously found to determine, in the lymphoid cell lineage, the stage-specific expression of a nuclear protein with a primary sequence suggestive of a transcriptional activator function. We report here the characterization of a second functional member of the Xlr gene family that is abundantly transcribed in testis in a tissue-specific and developmentally regulated manner. The protein product of this newly identified gene, called Xmr (Xlr-related, meiosis regulated), is located in the nuclei of spermatocytes, early in the prophase of the first meiotic division, and later becomes concentrated in the XY nuclear subregion where it is in particular associated with the axes of sex chromosomes. The Xmr protein provides a new tool for the investigation of sex chromosome behaviour during meiosis in mammals.

Functional interaction between beta 2-adrenoceptor agonists and interleukin-4 in the regulation of CD23 expression and release and IgE production in human.

Normal human peripheral blood mononuclear cells (PBMC) produced IgE when stimulated with IL-4. In the present report it was shown that beta 2-adrenoceptor agonists, salbutamol and fenoterol, potentiated the IL-4-induced IgE production without significantly affecting the expression of the low affinity receptor for IgE at the cell surface of monocytes and B lymphocytes. However, beta 2-adrenoceptor agonists were shown to enhance at day 7 the IL-4-induced release of the soluble form of CD23 (sCD23) by PBMC. This effect was specific since a beta-adrenoceptor antagonist, D,L-propranolol, inhibited the IL-4-induced IgE production by these cells. Alternatively, the beta 2-adrenoceptor agonists inhibited the production by these cells of interferon-gamma (IFN-gamma) but did not affect the production of IL-4 when stimulated with phytohemagglutinin A + a phorbol ester. These data suggest that beta 2-adrenoceptor agonists influence the IL-4-induced IgE production in humans by enhancing the release of sCD23 and inhibiting the production of endogenous IFN-gamma. In addition to the effect on the IL-4-induced IgE production it was shown that beta 2-adrenoceptor agonists potentiated the effect of IL-4 on a human promonocytic cell line, U 937, by enhancing CD23 expression and release and by inducing the differentiation of these cells into monocyte-like cells. Taken together, these data indicate that beta 2-adrenoceptor agonists potentiated the effect of IL-4 and that this functional interaction is different considering the cell-lineage and the stage of differentiation of these cells.

Leukotriene B4 potentiates the expression and release of Fc epsilon RII/CD23, and proliferation and differentiation of human B lymphocytes induced by IL-4.

This study documents the influence of leukotriene (LT) B4 on human B lymphocyte responses. Incubation of freshly isolated B lymphocytes with LTB4, but not LTC4, induced a slight but significant, time- and dose-dependent increase in the surface expression of Fc epsilon RII/CD23 and class II MHC Ag and in the release of soluble CD23. These changes were maximal at 10 nM LTB4 after an incubation period of 48 h. When B lymphocytes were preactivated in vitro with Staphylococcus aureus Cowan strain I (SAC), neither LTB4 nor LTC4 was able to promote proliferation and/or IgG and IgM secretion. In contrast, when resting B lymphocytes were stimulated with a suboptimal concentration (3 U/ml) of IL-4, LTB4, but not LTC4, potentiated both the Fc epsilon RII/CD23 and the class II MHC antigen expression, and the release of soluble CD23 in a dose-dependent manner, without affecting the kinetics of these responses. Furthermore, LTB4, but not LTC4, amplified both the proliferative response and the IgG and IgM secretion induced by addition of a suboptimal dose of IL-4 (3 U/ml) to SAC-preactivated B lymphocytes. Again, LTB4 did not modify the kinetics of the proliferative response promoted by IL-4. Although LTB4 potentiated IL-4-induced IgG and IgM secretion from SAC-activated B lymphocytes, no production of IgE was observed. These data indicate that LTB4 could play a regulatory role in the modulation of IL-4-induced signaling in human B lymphocytes.

Activation and infection of B cells by Epstein-Barr virus. Role of calcium mobilization and of protein kinase C translocation.

Both the activation and the transformation of human B cells by EBV were inhibited by either the Ca2+ channel blocking agent verapamil or the combination of theophylline and dibutyryl cAMP: the day 4 and day 20 peaks of [3H]TdR incorporation were abolished; the EBNA marker was not expressed by day 10; lymphoblastoid cell lines did not arise. Short term incubation of B cells with EBV or verapamil showed that the effect of verapamil was reversible and took place early in the interaction between EBV and B cells. The effect of EBV on the early metabolic events of B cell response was thus examined in the presence and in the absence of the drugs. Compared to anti-mu stimulation, supernatant of the transforming B95-8 strain as well as that of the non-transforming P3HR1 strain induced a drug sensitive increase of the free cytosolic Ca2+ concentration. This increase was associated with a protein kinase C translocation from the cytosol to a membrane bound compartment. Moreover, B95-8 supernatant induced phosphatidyl inositol metabolism by human B cells but at least four times less than that induced by anti-mu antibody. These metabolic events induced by EBV were significantly inhibited by anti-CD21 antibodies whereas anti-mu induced metabolic events were not. The infection of EBV negative Ramos cell line was prevented by verapamil or by theophylline + dibutyryl cAMP. Verapamil did not modify the density of EBV receptors but negatively interfered with the penetration of the virus into B cells. Thus B cell activation through the EBV receptor and virus penetration share a common metabolic pathway which is also used for transduction of the signal delivered through the membrane Ig.

The cytosolic free calcium in anti-mu-stimulated human B cells is derived partly from extracellular medium and partly from intracellular stores.

The inositol phospholipid metabolism and the increase in cytosolic free Ca2+ concentration ([Ca2+]i) into the cell are recognized as two important events in the anti-mu-induced B cell activation. The anti-mu stimulation caused the [3H]inositol incorporation and also a rapid increase in [Ca2+]i from 85 nM to 285 nM. This signal returned to baseline a few minutes after stimulation. By using the fluorescent indicator quin-2 we demonstrated that this [Ca2+]i uptake was derived part from extracellular medium and part from intracellular stores. Both EGTA (a calcium chelator) and TMB.8 (a drug which interferes with Ca2+ sequestration by smooth endoplasmic reticulum) partially suppressed the intracellular Ca2+ uptake and were fully inhibitory when added together. The role of Ca2+ from intracellular stores may also be evidenced in calcium-free experiments, or in permeabilized experiments using exogenous inositol 1,4,5-trisphosphate (IP3, the putative mobilizer of intracellular Ca2+). Preventing the increase in [Ca2+]i also prevents the apparition of early activation makers. These results are consistent with the hypothesis that the Ca2+ increase in B cells stimulated by anti-mu is caused by the generation of IP3 during the phosphatidyl-inositol metabolism and also by the entry of extracellular Ca2+ through the plasma membrane.