Systematic gene deletion and functional characterization of histidine kinase phosphorelay receptors (HKRs) in the human pathogenic fungus Aspergillus fumigatus.

Histidine kinase receptors (HKRs) appear to be a common strategy for model and pathogenic fungi to sense and respond to environmental stresses. In the human pathogen Aspergillus fumigatus, which is responsible for invasive aspergillosis, 13 genes potentially encoding HKRs have been identified. Until now, only three HKRs have been functionally characterized. The aim of this study was to perform the systematic invalidation of A. fumigatus HKR genes and the careful phenotypic characterization of the relevant mutants. This study notably allowed to gain new important insights into the role of HKRs in physiology of A. fumigatus. Actually, we showed that (i) NikA/TcsC could be involved in the cell wall integrity pathway, (ii) Fhk6 and PhkA were involved in the regulation of the "fluffy" developmental program, (iii) PhkB could participate in the regulation of conidiation and (iv) PhkA was implied in the resistance of oxidative stresses.

Identification of altered gene expression in skeletal muscles from Duchenne muscular dystrophy patients.

Mutations in the dystrophin gene lead to dystrophin deficiency, which is the cause of Duchenne muscular dystrophy (DMD). This important discovery more than 10 years ago opened a new field for very productive investigations. However, the exact functions of dystrophin are still not fully understood and the complex process leading to subsequent muscle fiber necrosis has not been clearly described; hence there has not yet been any marked improvement in patient treatment. To decipher the molecular mechanisms induced by a lack of dystrophin, we started identifying genes whose expression is altered in DMD skeletal muscles. The approach was based on differential screening of a human muscle cDNA array. Nine genes were found to be up- or downregulated. Our results indicate expression alterations in mitochondrial genes, titin, a muscle transcription factor and three novel genes. First characterizations of these novel genes indicated that two of them have striated muscle tissue specificity.

Characterization of Plasmodium falciparum CDP-diacylglycerol synthase, a proteolytically cleaved enzyme.

Cytidine diphosphate-diacylglycerol (CDP-DAG), an obligatory intermediate compound in the biosynthesis of the major anionic and zwitterionic phospholipids, is synthesized by CDP-DAG synthase (CDS). The gene encoding CDS was isolated from the human malaria parasite Plasmodium falciparum, based on sequence conservation to CDS from other organisms. The P. falciparum gene is located as a single copy on chromosome 14. The open reading frame (ORF) of PfCDS gene encodes a putative protein of 667 amino acids and 78 kDa. Only the C-terminal 422 amino acids share 40% homology with eukaryotic CDSs. The very long and non-conserved N-terminal region of 245 amino acids is hydrophilic and contains asparagine-rich and repetitive sequences. Two mRNA of 3.5 and 4 kb were detected. Transcription is developmentally regulated during the asexual intraerythrocytic cycle, being the weakest in the ring-stage. PfCDS enzyme activities in infected erythrocytes correlates with the transcription pattern, consistent with an increased synthesis of phospholipids in trophozoites and schizonts. Antisera raised against two synthetic peptides from the C-terminal region of PfCDS detected a single protein of 51 kDa in Western blot analysis, specific for parasitized erythrocytes. A protein of 28 kDa was recognized by an antiserum against an N-terminal peptide, indicating that PfCDS is proteolytically processed. Expression of 51- and 28-kDa proteins was developmentally regulated similar to regulation of the transcripts and the enzyme activity. The conserved C-terminal region of PfCDS, cloned into a eukaryote expression vector and transfected in COS-7 cells, showed a two-fold increase CDP-DAG synthase activities, indicating that the isolated gene most likely encoded the P. falciparum CDS enzyme.

Expression of lactate dehydrogenase, myosin heavy chain and myogenic regulatory factor genes in rabbit embryonic muscle cell cultures.

The expression of myogenic regulatory factors (MRFs), lactate dehydrogenase (LDH) and myosin heavy chains (MyHC), as markers of myogenesis, metabolism and contractility respectively, were investigated during differentiation of rabbit embryonic muscle cells in primary culture. Myf5, MyoD and myogenin mRNAs were abundantly expressed at day 1 of culture. The expression of Myf5 and MyoD mRNA transcripts decreased sharply as myoblasts fused and differentiated into myotubes, whilst myogenin mRNA was maintained throughout the duration of the culture. In contrast, MRF4 mRNA was weakly expressed on day 1 of culture, its expression increased slightly as myoblasts fused and reached a maximum level in 7-day-old cultures containing striated myofibres. The specific activity of LDH increased linearly during myoblast proliferation and fusion. In 7-day-old cultures, LDH-M mRNA (dominant in glycolytic muscles) and LDH-H mRNA (predominant in perinatal and oxidative muscles) represented 38% and 62% of total LDH mRNA respectively. At this stage, immunocytochemical staining with perinatal and adult-type MyHC antibodies showed that embryonic and perinatal MyHC isoforms were expressed in all myotubes, while few of them were stained by type I MyHC antibody. However, none of them expressed adult type II MyHC. The latter results were further supported by RT-PCR analysis of adult-type MyHC mRNA which showed that only the type I MyHC mRNA transcript was expressed. These data were in agreement with those reported in vivo on perinatal rabbit muscles. They differed from those obtained on cultured satellite cells isolated from adult rabbit fast-twitch or slow-twitch muscles which did not express embryonic MyHC, and instead expressed fast- or slow-type MyHC according to their muscle origin. Taken together, these results further suggest that myogenic mononucleated cells express different properties in vitro according to their developmental origin as well as properties related to those of the muscles from which they were isolated.

Apoptotic effects of imidazo[1,2-a]pyrazine derivatives in the human Dami cell line.

cAMP-elevating agents like phosphodiesterase inhibitors and purines have been shown to induce apoptosis. In the present work we have studied the effects of imidazo[1,2-a]pyrazine derivatives with a purine-like structure: PAB13 (6-bromo-8-(methylamino)imidazo[1,2-a] pyrazine), PAB15 (6-bromo-8-(ethylamino)imidazo[1,2-a]pyrazine), PAB23 (3-bromo-8-(methylamino)imidazo[1,2-a]pyrazine) on the growth of the Dami cell line in comparison to that of adenosine. The growth effect of PAB13, PAB15 and PAB23 was investigated in relation to their phosphodiesterase-inhibitory action and their activity on purinoceptors. Inhibition in cell growth was up to 71.0%, 76.3% and 89.7% for PAB23, PAB13 and PAB15, respectively and 100% for adenosine. Cell viability was affected in a concentration-dependent manner by PAB13, PAB15 and adenosine, with a correlation between growth inhibition and cytotoxicity. These effects of imidazo[1,2-a]pyrazine derivatives were found to be unrelated to an action on purinoceptors, but rather appear quantitatively linked to their ability in inducing apoptosis through their cAMP-increasing and phosphodiesterase-inhibitory potency.

Chemical labelling of arginyl-residues involved in anion transport mediated by human band 3 protein and some aspects of its location in the peptide chain.

The anion exchange system of human red blood cells can be completely inhibited by a large number of arginine-specific reagents. Kinetic studies have shown that these reagents act on the substrate binding site. Complete inhibition of the transport system with 14C phenylglyoxal is accompanied by modification of 2 to 3 arginine residues on the transmembrane segment of band 3. The inhibition and the binding of 14C phenylglyoxal to band 3 is reduced significantly in presence of chloride ions. The interactions between the reversible competitive inhibitor, 4-hydroxy-3-nitrophenylglyoxal (HNPG) and other irreversible anion transport inhibitors with well localized binding sites have been studied. A positive cooperativity between HNPG binding and the inhibition caused by eosin-5-maleimide (EMA), has been measured. Nearly no interactions have been found between HNPG binding site and the histidine residue(s) which react with diethylpyrocarbonate (DEPC). Sulphate self-exchange is irreversibly inhibited by two carboxyl-group reagents, 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimde (EDC) and N'-(3-dimethylaminopropyl)-N-ethylcarbodiimid methoiodide (EAC), studies on their interaction with the HNPG binding site gave results showing that the reaction site(s) of these reagents do not overlap and are not adjacent to the essential arginine(s). A model concerning such interactions is discussed.

Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: evidence for intracellular complexes.

The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37 degrees C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Fig receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4 degrees C with 125I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with 125I-HSAB-FGF-2 at 4 degrees C and then washed, were shifted to 37 degrees C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of 125I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex.

Towards the localization of the essential arginine residues in the band 3 protein of human red blood cell membranes.

The effects of 4-hydroxy-3-nitrophenylglyoxal (HNPG), on the binding of eosin-5-maleimide (EMA), and diethyl pyrocarbonate (DEPC) to the anion transport system in the human red blood cell membrane, have been investigated. HNPG is a reversibly binding, arginine-specific, anion transport competitive inhibitor, known to act on the anion binding site. The EMA reaction site is an external facing lysine residue (Lys-430) in the 17 kDa transmembrane segment. The DEPC reaction site is an intracellular histidine (His-819) in the 35 kDa fragment. The results show that inhibition of the transport system with EMA increases in presence of HNPG to about 2.3 times. This finding suggests a positive cooperativity between the HNPG and EMA binding site and give evidence that the essential arginine is either nearby or allosterically linked to Lys-430. The inhibition of the cells with DEPC was nearly unchanged or slightly decreased in the presence of 10 mM HNPG. These results suggest that the intracellular His-residue which reacts with DEPC is not a part of the transport pathway. Our experiments with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) have shown that its affinity to the transport system does not change after pre-treatment with phenylglyoxal (PG). We also found that the binding of [14C]phenylglyoxal (PG) to band 3 reduces significantly in presence of chloride. This is another evidence for the direct involvement of arginine residues in substrate binding.

Down-regulation of mitochondrial mRNAs in the mdx mouse model for Duchenne muscular dystrophy.

In our search for genes up- or down-regulated genes in the mdx mouse model for Duchenne muscular dystrophy, we isolated a down-regulated mitochondrial DNA clone. In addition to this clone, all protein-coding mitochondrial genes tested had tissue-specific and age independent down-regulated expression. This implied mechanisms at the RNA level since no change in the mitochondrial DNA contents were detected. Cytochrome c oxidase activity showed the same range of down-regulated expression. These data provide a molecular basis for energetic metabolism modifications in mdx mice.

E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway.

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

Interaction of basic fibroblast growth factor (FGF-2) with nonresponsive HeLa cells.

Human HeLa adenocarcinoma cells did not respond to basic fibroblast growth factor (bFGF or FGF-2) but did bind the same amount of bFGF as responsive Chinese hamster lung fibroblasts (CCL 39). Heparinase II treatment of HeLa and CCL 39 cells resulted in a decrease of bFGF binding by 96 and 57%, respectively, indicating that heparan sulfate molecules were involved in bFGF binding. On HeLa cells, bFGF bound to a single family of low-affinity sites. Cross-linking experiments of 125I-bFGF to HeLa cells yielded several labeled complexes. Cell-associated 125I-bFGF was internalized in both cell types either by high-affinity receptors and heparitinase-sensitive sites in CCL 39 cells or by heparitinase-sensitive binding sites only in HeLa cells. The binding of bFGF to nonresponsive HeLa cells and its internalization via a family of heparitinase-sensitive binding sites might illustrate other functions of bFGF unrelated to cell proliferation.

Localization of angiotensin-converting enzyme (ACE) mRNA in rabbit gastric mucosa by in situ hybridization.

In a previous study we showed, by immunohistochemical analysis on rabbit fundic mucosa, that in addition to its usual presence on the luminal plasma membrane of endothelial cells, angiotensin converting-enzyme (ACE) was localized inside granules of surface and neck mucous cells and within granules of chief cells. The aim of the present study was to localize ACE mRNA in cells of the rabbit fundic mucosa by in situ hybridization with a 35S-labeled probe. This probe was a cDNA fragment (406 BP) encoding a portion of the rabbit ACE mRNA obtained by reverse transcription followed by polymerase chain reaction on total RNA extracted from fundic mucosa. ACE mRNAs were detected in mucous and chief cells and in endothelial cells of the mucosal vasculature. These results are in complete agreement with our prior studies which showed by immunohistochemical analysis that ACE is present in these cells. Our findings therefore suggest that ACE previously detected in epithelial cells of the rabbit gastric mucosa is actually synthesized within these cells.

Inhibition of anion transport in the human red blood cell membrane with para- and meta-methoxyphenylglyoxal.

The positional isomers para-methoxyphenylglyoxal and meta-methoxyphenylglyoxal were newly synthesized and found to be potent inhibitors of sulfate exchange in the red blood cell membrane. The rate of inactivation of the transport system with both reagents obeys pseudo-first-order kinetics and increases with increasing pH and reagent concentration. The degree of inhibition of the transport system with the meta-isomer exceeds the inhibition caused by the para-isomer. At 2 mM 3-methoxyphenylglyoxal (3-MOPG) and 37 degrees C the half-lifetime of the anion transporter is 5.4 min at pH 8.0. Under the same experimental conditions the half-lifetime of the transporter at 2 mM 4-methoxyphenylglyoxal (4-MOPG) is found to be 24.7 min. The binding site of these reagents is found to be the same as binding site of the reversibly acting phenylglyoxal derivative 4-hydroxy-3-nitrophenylglyoxal (HNPG). Chloride ions are able to protect the transporter against inhibition with both reagents. Anion transport inhibitors like 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) and flufenemate, which are known to act on band 3 protein, are able to interact with the binding of the newly synthesised reagents. Phloretin and phloridzin show no interaction.

Interactions of FGFs with target cells.

Growth factors play a key role in cellular communication, a necessary step for the development of pluricellular organisms. The fibroblast growth factors (FGF) are among these polypeptides and have seven known members: FGF 1 to FGF 7 which are also known as acidic FGF, basic FGF, translation products of oncogenes hst, int 2, FGF 5, FGF 6 and FGF 7 or keratinocyte growth factor (KGF) respectively [1]. The best known and the most abundant in normal adult tissues are acidic and basic FGFs, or FGF 1 and 2 respectively, which have been subjected to extensive studies both in vitro and in vivo. These two factors have almost ubiquitous distribution and a wide spectrum of biological activity including action on cellular proliferation and differentiation, as well as neurotrophic and angiogenic properties [1]. These different activities are induced by triggering specific receptors present at the surface of the target cell. Following this interaction, the FGF-receptor complexes are internalized and activate intracellular pathways. An important effort of investigations has been produced to characterize these receptors and intracellular pathways. It is the purpose of this review to present this work which will focus on FGFs 1 and 2. The existence of two classes of interactions has been reported as early as 1987 [52, 53, 54] suggesting the presence of high and low affinity receptors for FGFs.

Internalization of basic fibroblast growth factor by Chinese hamster lung fibroblast cells: involvement of several pathways.

Subconfluent Chinese hamster lung fibroblast cells (CCL39) which express high- and low-affinity binding sites for basic fibroblast growth factor (bFGF) were used to study bFGF internalization. Kinetics at 37 degrees C indicated that this process was complex and involved various pathways with regard to the ligand concentration used. Internalization with 6 to 45 pM of 125I-r-bFGF led to a steady state that lasted up to 3 h without any appearance of 125I-labeled degradation products in the cell-culture medium, suggesting that the endocytosis reached equilibrium. Furthermore, binding data at steady state, at 37 degrees C, revealed a two-phase Scatchard curve suggesting the involvement of two families of interaction sites in the process of internalization. Apparent dissociation constants were estimated to be 20 pM and 58 nM, respectively, and the number of bFGF molecules involved per cell, 4300 and 1.3 x 10(6), respectively. These data were in good agreement with those obtained from binding experiments at equilibrium at 4 degrees C. Besides, higher concentrations of 125I-r-bFGF (greater than 47 pM) induced an internalization process which did not reach steady state and was not saturable. These results suggest that CCL39 cells could internalize bFGF by various pathways involving high- and low-affinity binding sites.

Chemical properties of the anion transport inhibitory binding site of arginine-specific reagents in human red blood cell membranes.

A series of arginine-specific reagents with different size and polarity have been synthesized and their inhibitory potency on sulfate exchange in resealed ghosts has been investigated. The synthesized phenylglyoxal derivatives p-nitro-, p-methyl-, p-hydroxy-, p-carboxy-, p-sulfo-, and p-azido-phenylglyoxal are found to be potent inhibitors of anion transport. The reaction between the cells and azidophenylglyoxal was performed in the dark. Exposure of the modified cells to the light was not followed by an increase in the inhibition. No cross-linking products were visible after gel electrophoresis. The rate of inactivation of sulfate flux with these reagents obeyed pseudo-first-order kinetics and increases with increasing reagents concentration and pH. Prolonged incubation of the cells with these reagents results in almost complete inhibition of the transport system. The positively charged phenylglyoxal derivative 4-(trimethylammonioacetylamido)phenylglyoxal was not able to inhibit the transport system. The hydrophobic character and the electronic properties of these reagents do not correlate with their inhibitory potency. Their electrostatic and steric effects seem to play the major role in their action.

Dose-response analysis of infants prenatally exposed to methyl mercury: an application of a single compartment model to single-strand hair analysis.

A new method of estimating fetal exposure is used in a dose-response analysis of data from the 1971 outbreak of methyl mercury poisoning in rural Iraq. An X-ray fluorescence instrument for the measurement of single strands of human hair was employed to obtain longitudinal profiles recapitulating fetal exposure. Logit and hockey-stick models as well as nonparametric smoothing are used to describe data on delayed development and central nervous system abnormality.

Internalization and limited processing of basic fibroblast growth factor on Chinese hamster lung fibroblasts.

Using either acidic (pH 2.5) or trypsic treatments, we demonstrated that 125I-labeled basic Fibroblast Growth Factor (125I-bFGF) was submitted to an internalization process on responsive Chinese hamster lung fibroblasts (CCL39) at 37 degrees C. Various experiments based on the measurement of cell-associated radioactivity, as well as on research of degradated products of 125I-bFGF in cellular supernatants, showed that most of the internalized radioactivity remained intracellularly located after up to 5 hr of incubation. Analyses of this radioactivity by NaDodSO4-PAGE revealed the presence of labeled peptides issued from the limited processing of the native 125I-bFGF form (17 kD) and whose molecular weights were estimated to be 9 and 6 kD. Kinetic experiments indicated that proteolysis of the 125I-bFGF began early on incubation (less than 30 min) and led to a prolonged preservation of the 9- and 6-kD peptides which were still detectable after 13 hr of incubation. Preincubation of the cells with different lysosomotropic agents completely inhibited the proteolysis, indicating that this event occurred probably in an intracellular acidic compartment. Two enzyme inhibitors, leupeptin and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), were also shown to interfere with the formation of both 9- and 6-kD peptides, thus suggesting a way to control the appearance of these fragments, and hence to determine their potential intracellular role.

Studies on inactivation of anion transport in human red blood cell membrane by reversibly and irreversibly acting arginine-specific reagents.

A chromophoric derivative of phenylglyoxal, 4-hydroxy-3-nitrophenylglyoxal (HNPG), known to be highly selective for modification of arginine residues in aqueous solution is found to be a potent inhibitor of anion transport across the red cell membrane. In contrast to the action of all other arginine-specific reagents used under the experimental conditions in this laboratory, the action of HNPG on sulfate transport is completely reversible. Hence, a kinetic analysis of its inhibitory effect on SO4(2-) self-exchange could be performed. The effect of increasing chloride concentration on the inhibitory potency of HNPG is consistent with the concept that Cl- and HNPG compete for the same site on the anion transporter. The IC50 value for the inhibition of SO4(2-) exchange with HNPG is about 0.13 mM at pH 8.0 and 0.36 mM at pH 7.4, and the Hill coefficient for the interaction between the transporter and the inhibitor is near one at both pH's. HNPG is able to protect the transport system against inhibition with the (under our experimental conditions) irreversibly acting arginine specific reagent, phenylglyoxal. Partial inactivation of the transport system with phenylglyoxal lowers the maximal rates of SO4(2-) and chloride exchange but does not modify the apparent KS for the substrate anions. Reversibly acting anion transport inhibitors known to interact with the DIDS binding site like salicylate, tetrathionate, APMB, DNDS, and flufenamate are able to protect the transport system against phenylglyoxalation. Other inhibitors like phloretin and phlorizin have no effect.