Biomedical subjects

Michael J Hynes

Publications and source records attributed to Michael J Hynes.

28 records · Page 2Linked to original sources

PubMed2003

A toxicokinetic study of nickel-induced immunosuppression in rats.

The aim of the present study was to investigate dose- and time-dependent effects of NiCl2 on T-lymphocyte and macrophage-derived cytokine production in rats. Moreover we have determined the concentrations of nickel in the plasma that are required to elicit alterations in T-lymphocyte and macrophage function. NiCl2 suppressed T-lymphocyte proliferation and Th1 (IFN-gamma) and Th2 (IL-10) cytokine production in a dose- and time-dependent fashion. In addition, NiCl2 inhibited production of the pro-inflammatory cytokine TNF-alpha and increased production of the anti-inflammatory cytokine IL-10 from lipopolysaccharide (LPS) stimulated cultures. We have determined that the minimal plasma concentrations of nickel required to provoke immunosuppression are in the range 209-585 ng/mL. In the time-course study NiCl2 (3.3 mg/kg) provoked immunological changes that were maximal 1 h following administration, and some of these changes persisted for up to 24 h post administration. Overall these data clearly demonstrate that NiCl2 suppresses T-cell function and promotes an immunosuppressive macrophage phenotype in rats. This study also indicates that measuring T-cell proliferation is as sensitive a marker of NiCl2-induced immunotoxicity as measuring T-cell or macrophage cytokine production. Co-measurement of circulating nickel concentrations and immune parameters yields valuable information with regard to the potency of nickel to alter immune function in vivo. These data also suggest that quite a large quantity of nickel needs to reach the systemic circulation before any adverse effects on immune function are observed.

Animals↗

PubMed2003

The G-protein alpha-subunit GasC plays a major role in germination in the dimorphic fungus Penicillium marneffei.

The opportunistic human pathogen Penicillium marneffei exhibits a temperature-dependent dimorphic switch. At 25 degrees, multinucleate, septate hyphae that can undergo differentiation to produce asexual spores (conidia) are produced. At 37 degrees hyphae undergo arthroconidiation to produce uninucleate yeast cells that divide by fission. This work describes the cloning of the P. marneffei gasC gene encoding a G-protein alpha-subunit that shows high homology to members of the class III fungal Galpha-subunits. Characterization of a DeltagasC mutant and strains carrying a dominant-activating gasC(G45R) or a dominant-interfering gasC(G207R) allele show that GasC is a crucial regulator of germination. A DeltagasC mutant is severely delayed in germination, whereas strains carrying a dominant-activating gasC(G45R) allele show a significantly accelerated germination rate. Additionally, GasC signaling positively affects the production of the red pigment by P. marneffei at 25 degrees and negatively affects the onset of conidiation and the conidial yield, showing that GasC function overlaps with functions of the previously described Galpha-subunit GasA. In contrast to the S. cerevisiae ortholog Gpa2, our data indicate that GasC is not involved in carbon or nitrogen source sensing and plays no major role in either hyphal or yeast growth or in the switch between these two forms.

Alleles↗

PubMed2002

Investigation of the release of iron from ferritin by naturally occurring antioxidants.

Ferritin is the main intracellular iron storage protein. The release of iron from ferritin in the presence of a number of phenolic based compounds of nutritional significance was studied at physiological pH. The release of iron was measured by monitoring the formation of the iron(II)-ferrozine complex. The kinetics of this process were studied in Hepes buffer (pH 7.00), at 37 degrees C. The order of ability to remove iron from ferritin is epigallocatechin>gallic acid methyl ester approximately equal to sinapic acid>ferulic acid. The presence of the oxyradical scavenger urea resulted in a slight inhibition in the release of iron from ferritin by both gallic acid methyl ester and epigallocatechin. The ability of each reagent to release iron is interpreted on the basis of their ability to (a) reduce the bound iron and (b) complex the iron with the oxidised form of the phenol, thus mobilising it from the protein. These studies indicate that some phenolic based compounds that have been epidemiologically associated with a negative effect on iron absorption in man, can individually mobilise and release iron from ferritin under suitable conditions.

Animals↗

PubMed2002

The genes gmdA, encoding an amidase, and bzuA, encoding a cytochrome P450, are required for benzamide utilization in Aspergillus nidulans.

Two unlinked loci, gmdA and bzuA, have previously been identified as being required for the utilization of benzamide as the sole nitrogen source by Aspergillus nidulans. We have cloned each of these genes via direct complementation. The gmdA gene encodes a predicted product belonging to the amidase signature sequence family that displays similarity to AmdS from A. nidulans. However, identity is significantly higher to the amdS gene from Aspergillus niger. The bzuA gene encodes a protein belonging to the cytochrome P450 superfamily and is orthologous to the benzoate para-hydroxylase-encoding gene bphA of A. niger. The bzuA1 mutation prevents the use of benzoate as a carbon source and intracellular accumulation of benzoate results in growth inhibition on benzamide. Northern blot analysis has shown that gmdA expression is subject solely to AreA-dependent nitrogen metabolite repression while bzuA is strongly benzoate inducible and subject to CreA-mediated carbon catabolite repression and a probable inactivation of benzoate induction by glucose. Fluorescence microscopy of a fusion of the N-terminal end of BzuA to green fluorescent protein revealed that this protein localizes to the endoplasmic reticulum.

Amidohydrolases↗

PubMed2002

Mutation and functional analysis of the Aspergillus nidulans ammonium permease MeaA and evidence for interaction with itself and MepA.

The movement of ammonium across biological membranes is mediated in both prokaryotic and eukaryotic systems by ammonium transport proteins which constitute a family of related sequences (called the AMT/MEP family). Interestingly, recent evidence suggests that human and mouse Rhesus proteins which display significant relatedness to AMT/MEP sequences may function as ammonium transporters. To add to the functional understanding of ammonium transport proteins, the sequence changes in 37 loss-of-function mutations within the Aspergillus nidulans ammonium permease gene, meaA, were characterized. Together with the identification of conserved AMT/MEP residues and regions, the mutational analysis predicted regions important for uptake activity. Specifically, a major facilitator superfamily like motif (161-GAVAERGR-168 in MeaA) may be important for the translocation of ammonium across the membrane as may the conserved Pro186 residue. A specific Gly447 to Asp mutation was introduced into MeaA and this mutant protein was found to trans-inhibit the activity of endogenous MeaA and the other A. nidulans ammonium transporter, MepA. These results suggest that MeaA may interact with itself and with MepA, although any hetero-interaction is not required for ammonium transport function. In addition, cross-feeding studies showed that MeaA and to a lesser extent MepA are also required for the retention of intracellular ammonium.

Alleles↗

PubMed2002

A basic helix-loop-helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei.

Members of the APSES protein group are basic helix-loop-helix (bHLH) proteins that regulate processes such as mating, asexual sporulation and dimorphic growth in fungi. Penicillium marneffei is a human pathogen and is the only member of its genus to display a dimorphic growth transition. At 25 degrees C, P. marneffei grows with a filamentous morphology and produces asexual spores from multicellular con-idiophores. At 37 degrees C, the filamentous morphology is replaced by yeast cells that reproduce by fission. We have cloned and characterized an APSES protein-encoding gene from P. marneffei that has a high degree of similarity to Aspergillus nidulans stuA. Deletion of stuA in P. marneffei showed that it is required for metula and phialide formation during conidiation but is not required for dimorphic growth. This suggests that APSES proteins may control processes that require budding (formation of the metulae and phialides, pseudohyphal growth in Saccharomyces cerevisiae and dimorphic growth in Candida albicans) but not those that require fission (dimorphic growth in P. marneffei). The A. nidulans DeltastuA mutant has defects in both conidiation and mating. The P. marneffei stuA gene was capable of complementing the conidiation defect but could only inefficiently complement the sexual defects of the A. nidulans mutant. This suggests that the P. marneffei gene, which comes from an asexual species, has diverged significantly from the A. nidulans gene with respect to sexual but not asexual development.

Amino Acid Sequence↗

PubMed2002

A gene from Aspergillus nidulans with similarity to URE2 of Saccharomyces cerevisiae encodes a glutathione S-transferase which contributes to heavy metal and xenobiotic resistance.

Aspergillus nidulans is a saprophytic ascomycete that utilizes a wide variety of nitrogen sources. We identified a sequence from A. nidulans similar to the glutathione S-transferase-like nitrogen regulatory domain of Saccharomyces cerevisiae Ure2. Cloning and sequencing of the gene, designated gstA, revealed it to be more similar to URE2 than the S. cerevisiae glutathione S-transferases. However, creation and analysis of a gstA deletion mutant revealed that the gene does not participate in nitrogen metabolite repression. Instead, it encodes a functional theta class glutathione S-transferase that is involved in resistance to a variety of xenobiotics and metals and confers susceptibility to the systemic fungicide carboxin. Northern analysis showed that gstA transcription is strongly activated upon exposure to 1-chloro-2,4-dinitrobenzene and weakly activated by oxidative stress or growth on galactose as a carbon source. These results suggest that nitrogen metabolite repression in A. nidulans does not involve a homolog of the S. cerevisiae URE2 gene and that the global nitrogen regulatory system differs significantly in these two fungi.

Aspergillus nidulans↗

PubMed2002

Isolation and characterization of two ammonium permease genes, meaA and mepA, from Aspergillus nidulans.

Ammonium and the analogue methylammonium are taken into the cell by active transport systems which constitute a family of transmembrane proteins that have been identified in fungi, bacteria, plants, and animals. Two genes from Aspergillus nidulans, mepA and meaA, which encode ammonium transporters with different affinities have been characterized. The MepA transporter exhibits the highest affinity for methylammonium (Km, 44.3 microM); in comparison, the Km for MeaA is 3.04 mM. By use of targeted gene replacement strategies, meaA and mepA deletion mutants were created. Deletion of both meaA and mepA resulted in the inability of the strain to grow on ammonium concentrations of less than 10 mM. The single meaA deletion mutant exhibited reduced growth at the same concentrations, whereas the mepA deletion mutant displayed wild-type growth. Interestingly, multiple copies of mepA were found to complement the methylammonium resistance phenotype conferred by the deletion of meaA. The expression profiles for mepA and meaA differed; the mepA transcript was detected only in nitrogen-starved cultures, whereas meaA was expressed under both ammonium-sufficient and nitrogen starvation conditions. Together, these results indicate that MeaA constitutes the major ammonium transport activity and is required for the optimal growth of A. nidulans on ammonium as the sole nitrogen source and that MepA probably functions in scavenging low concentrations of ammonium under nitrogen starvation conditions.

Animals↗

PubMed2002

G-protein signaling mediates asexual development at 25 degrees C but has no effect on yeast-like growth at 37 degrees C in the dimorphic fungus Penicillium mameffei.

The ascomycete Penicillium marneffei is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch. At 25 degrees C, P. marneffei grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores. At 37 degrees C, it forms uninucleate yeast cells which divide by fission. We have cloned a gene encoding a G alpha subunit of a heterotrimeric G protein from P. marneffei named gasA with high similarity to fadA in Aspergillus nidulans. Through the characterization of a delta gasA strain and mutants carrying a dominant activating or a dominant interfering gasA allele, we show that GasA is a key regulator of asexual development but seems to play no role in the regulation of growth. A dominant activating gasA mutant whose mutation results in a G42-to-R change (gasA(G42R)) does not express brlA, the conidiation-specific regulatory gene, and is locked in vegetative growth, while a dominant interfering gasA(G203R) mutant shows inappropriate brlA expression and conidiation. Interestingly, the gasA mutants have no apparent defect in dimorphic switching or yeast-like growth at 37 degrees C. Growth tests on dibutyryl cyclic AMP (dbcAMP) and theophylline suggest that a cAMP-protein kinase A cascade may be involved in the GasA signaling pathway.

Aspergillus nidulans↗

PubMed2002

Regulation of the acuF gene, encoding phosphoenolpyruvate carboxykinase in the filamentous fungus Aspergillus nidulans.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.

Amino Acid Sequence↗