Functional characterization of the Aspergillus nidulans glucosylceramide pathway reveals that LCB Δ8-desaturation and C9-methylation are relevant to filamentous growth, lipid raft localization and Psd1 defensin activity.

C8-desaturated and C9-methylated glucosylceramide (GlcCer) is a fungal-specific sphingolipid that plays an important role in the growth and virulence of many species. In this work, we investigated the contribution of Aspergillus nidulans sphingolipid Δ8-desaturase (SdeA), sphingolipid C9-methyltransferases (SmtA/SmtB) and glucosylceramide synthase (GcsA) to fungal phenotypes, sensitivity to Psd1 defensin and Galleria mellonella virulence. We showed that ΔsdeA accumulated C8-saturated and unmethylated GlcCer, while gcsA deletion impaired GlcCer synthesis. Although increased levels of unmethylated GlcCer were observed in smtA and smtB mutants, ΔsmtA and wild-type cells showed a similar 9,Me-GlcCer content, reduced by 50% in the smtB disruptant. The compromised 9,Me-GlcCer production in the ΔsmtB strain was not accompanied by reduced filamentation or defects in cell polarity. When combined with the smtA deletion, smtB repression significantly increased unmethylated GlcCer levels and compromised filamentous growth. Furthermore, sdeA and gcsA mutants displayed growth defects and raft mislocalization, which were accompanied by reduced neutral lipids levels and attenuated G. mellonella virulence in the ΔgcsA strain. Finally, ΔsdeA and ΔgcsA showed increased resistance to Psd1, suggesting that GlcCer synthesis and fungal sphingoid base structure specificities are relevant not only to differentiation but also to proper recognition by this antifungal defensin.

Evolutionary relationship between defensins in the Poaceae family strengthened by the characterization of new sugarcane defensins.

Plant defensins are small (45-54 amino acids), highly basic, cysteine-rich peptides structurally related to defensins of other organisms, including insects and mammals. Small putative proteins (MW < 10 kDa) containing eight cysteines were screened based on the sugarcane expressed sequence tag (EST) database. We selected ORFs that exhibited 25-100% similarity in primary sequence with other defensins in the NCBI database and that contained eight cysteines. This similarity is sufficient for folding prediction, but not enough for biological activity inference. Six putative defensins (Sd1-6) were selected, and activity assays showed that recombinant Sd1, Sd3 and Sd5 are active against fungi, but not against bacteria. Structural characterization, based on circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy showed that the structures of these Sds were compatible with alpha/beta proteins, a feature expected for plant defensins. Phylogenetic analysis revealed that sugarcane defensins could clearly be grouped within defensins from Poaceae family and Andropogoneae tribe. Our work demonstrates that defensins show strong conservation in the Poaceae family and may indicate that the same conservation occurs in other families. We suggest that evolutionary relationships within plant families can be used as a procedure to predict and annotate new defensins in genomes and group them in evolutionary classes to help in the investigation of their biological function.

Pressure response in the yeast Saccharomyces cerevisiae: from cellular to molecular approaches.

Yeasts are unicellular organisms that are exposed to a highly variable environment, concerning the availability of nutrients, temperature, pH, radiation, access to oxygen and, specially, water activity. Evolution has selected yeasts to tolerate, to a certain extent, these environmental stresses. High hydrostatic pressure (HHP) exerts a broad effect upon yeast cells, interfering with the cell membranes, cellular architecture and in processes ofpolymerisation and denaturation of proteins. Gene expression patterns in response to HHP revealed a stress response profile. The majority of the upregulated genes were involved in stress defence and carbohydrate metabolism while most of the repressed ones were in cell cycle progression and protein synthesis categories. In addition, in the present work it was seen that mild pressure induced cell cycle arrest and protection against severe stresses, such as high temperature, high pressure and ultra cold shock. Nevertheless, this protection was only significant if the cells were incubated at atmospheric pressure after the HHP treatment. Expression of genes that were upregulated by HHP and are related to resistance to this stresses were also analyzed, and, for the majority of them, higher induction was attained after 15 min post-pressurization. Taken together, the results imply an interconnection among stresses.

cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris.

In the present work, we describe the cDNA cloning, expression in Pichia pastoris, purification, and characterization of the recombinant Pisum sativum defensin 1 (rPsd1), a novel Cys-rich protein presenting four disulfide bridges and high antifungal activity. Several parameters that affect the level of protein expression were assayed. The best condition yielded 13.8 mg/L (1.50 microg/10(8) cells) of active rPsd1. The recombinant rPsd1 was purified to homogeneity by cation exchange, followed by reversed-phase HPLC, and subjected to automated amino acid sequencing, which revealed four additional amino acids (EAEA) at the N-terminal region. Circular dichroism, intrinsic fluorescence, and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein has a very similar folding and a correct disulfide-bonding pattern when compared to native Psd1. Nevertheless, the rPsd1 presented a more species-specific antifungal activity. The importance of the N- and C-termini for Psd1 activity is pointed out.

Effect of hydrostatic pressure on the morphology and ultrastructure of wild-type and trehalose synthase mutant cells of Saccharomyces cerevisiae.

AIMS: Saccharomyces cerevisiae was used for studying the physiological effects of hydrostatic pressure. METHODS AND RESULTS: The effects of hydrostatic pressure on the ultrastructure of wild-type and trehalose-6-phosphate synthase (tps1) mutant cells were investigated by transmission electron microscopy. Pressure induced several morphological changes in wild-type and tps1 cells, the latter showing greater structural alterations. When the cells were submitted to a preheat treatment they both acquired resistance to the pressure treatment. CONCLUSION: As the tps1 mutant was 1000-fold more barosensitive than its parental strain, it showed greater structural alterations compared with the wild-type. Microscopic images of the yeast cells suggested that hydrostatic pressure induced changes in the cytoskeleton and therefore, on the cell wall and in the dynamics of the organelles. SIGNIFICANCE AND IMPACT OF THE STUDY: This work presents the effects of hydrostatic pressure on the morphology of yeast cells and confirms the importance of several different factors in the protection of cells against stress.

Differential expression of D(1A) and D(1B) dopamine receptor mRNAs in the developing avian retina.

In the chick retina, the D1 dopaminergic system differentiates very early, as shown by receptor-mediated increases in intracellular cyclic AMP concentration and the presence of [(3)H]SCH23390-specific binding sites. Here, we characterized, by RT-PCR, the expression of defined D1 receptor subtypes D(1A), D(1B), and D(1D) during the development of the chick retina. Total RNA was extracted from retinas of 6-day-old embryos (E6) to 1-day-old hatched chickens and reverse-transcribed. The resulting cDNA was amplified using D(1A)-, D(1B)-, or D(1D)-specific primers, and the PCR-amplified products were analyzed by electrophoresis. The fragment corresponding to D(1A) receptor was detected in developing retina as early as E7, whereas the fragment corresponding to D(1B) was observed starting around E10. No PCR product corresponding to D(1D) was observed in the retina, although it was detected in chick brain. As synaptogenesis in chick retina begins after E11 and [(3)H]SCH 23390 D1 binding sites increase after this stage, the present results show that expression of D(1B) receptor increases during synaptogenesis, whereas D(1A) is the receptor subtype associated with the D1-like actions of dopamine early in retina development.

Characterization of two novel defense peptides from pea (Pisum sativum) seeds.

A fraction that possesses antifungal activity against Aspergillus niger has been isolated from seeds of the pea (Pisum sativum) by ammonium sulfate fractionation followed by gel filtration on Sephadex G-75. On further purification by reverse-phase high performance liquid chromatography, two small cysteine-rich polypeptides were obtained (Psd1 and Psd2). They are localized primarily in vascular bundles and epidermis tissues of pea pods and exhibit high antifungal activity toward several fungi, displaying IC(50) values ranging from 0.04 to 22 microg/ml. This inhibitory activity decreases when A. niger growth medium is supplemented with cations such as Ca(2+), Mg(2+), Na(+), and K(+). Although the primary sequence of both Psd1 and Psd2 shows homology with other plant defensins, they cannot easily be assigned to any established group.

Presence of antibodies against the third intracellular loop of the m2 muscarinic receptor in the sera of chronic chagasic patients.

Patients in the chronic phase of Chagas' disease suffer from a slowly evolving inflammatory cardiomyopathy that can lead to severe cardiac dilatation, congestive heart failure, and death. This process appears to be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after infection with the parasite Trypanosoma cruzi. Recent evidence suggests that there are circulating antibodies in chronic chagasic patients that alter the physiological behavior of the heart on binding to G-protein-coupled cardiovascular receptors, including beta1-adrenergic and m2 muscarinic receptors. A 42 kDa fusion protein was constructed that contains the central part of the third intracellular loop (i3; Arg(267)-Arg(381)) of the human m2 muscarinic receptor, linked to glutathione S-transferase. This fusion protein was overexpressed in Escherichia coli and subsequently purified by affinity chromatography. Based on Western blots, the i3 loop is specifically recognized by the sera of chronic chagasic patients who have reached advanced stages of cardiac failure (according to the Los Andes classification). Analysis of the prevalence and distribution of these antibodies shows a strong association between seropositive patients and moderate (group II) to severe (group III) heart dysfunction.

N-terminal chimeric constructs improve the expression of sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

Wild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H(+)-ATPase were expressed in yeast under control of a heat-shock regulated promoter. The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes. Addition of the first 88 residues of H(+)-ATPase to the Ca(2+)-ATPase N-terminal end promoted a marked shift in the localization of chimeric H(+)/Ca(2+)-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER. Furthermore, there was a three-fold increase in the overall level of expression of chimeric H(+)/Ca(2+)-ATPase. Similar results were obtained for a chimeric Ca(2+)-ATPase containing a hexahistidine sequence added to its N-terminal end. Both H(+)/Ca(2+)-ATPase and 6xHis-Ca(2+)-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover. Conversely, a replacement chimera in which the N-terminal end of SR Ca(2+)-ATPase was replaced by the corresponding segment of H(+)-ATPase was not stably expressed in yeast membranes. These results indicate that the N-terminal segment of Ca(2+)-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase.

Effect of hydrostatic pressure of a mutant of Saccharomyces cerevisiae deleted in the trehalose-6-phosphate synthase gene.

Mutants Saccharomyces cerevisiae deleted on the trehalose-6-phosphate synthase gene (tps1) and their parental wild-type cells were submitted to hydrostatic pressure in the range of 0-200 MPa. Experimental evidence showed that viability for both strains decreased with increasing pressure and that tps1 mutants, unable to accumulate trehalose, were more sensitive to hydrostatic pressure than the wild-type cells. Additionally, both tps1 and wild-type cells in the stationary phase, when there is an accumulation of endogenous trehalose, were more resistant to pressure than proliferating cells. Under these conditions, mutant cells were also more sensitive to pressure treatment than the wild type. The present work also showed that mild pressure pretreatment did not induce hydrostatic pressure resistance (barotolerance) in yeast cells.

Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation.

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.

Beneficial effects of anti-growth hormone antiserum in avian muscular dystrophy.

Human subjects and mice have been found to have a milder progression of muscular dystrophy when the disease is associated with genotypically determined dwarfism. In this paper we describe an experimental test for reducing growth hormone in dystrophic chickens that uses rabbit anti-chicken growth hormone anti-serum (anti-cGH). Antiserum was injected daily into dystrophic (line 413) male chickens from day 1 to day 8 after hatching. Dystrophic chickens injected with anti-cGH maintained a significantly higher score in the standardized test for righting ability (P less than 0.001-0.051) from 3 to 9 1/2 wk after hatching when compared with dystrophic controls. The observed prolongation of the functional ability of injected dystrophic animals suggests that growth hormone plays a role in potentiating the symptoms of dystrophy in chickens.

Pressure-induced dissociation of solubilized sarcoplasmic reticulum ATPase.

The effect of hydrostatic pressure on the self-association of sarcoplasmic reticulum ATPase solubilized by nonionic detergent was studied in the pressure range of 1 atm up to 2 kilobars. Polarization of intrinsic tryptophan fluorescence or of fluorescence of a pyrene probe covalently attached to the ATPase was measured. An increase in hydrostatic pressure promoted dissociation of the protein into monomers. For a midpoint dissociation pressure of 1.3 kilobars, the standard volume change in the dissociation reaction was delta Vop = -167 ml/mol. Full reversibility of the pressure effects was shown to occur, as seen by recovery of polarization. An increase in Ca2+ concentration from 50 microM to 5 mM and of pH from 6.9 to 8.6 were found to increase the midpoint dissociation pressure, indicating that these factors stabilize the dimeric state. The hydrolytic activity of the ATPase was measured under pressure. The activity was inhibited by pressure increase. It was found that an irreversible inactivation of the solubilized enzyme occurred during turnover and that increasing pressure added to this instability. Reversibility of the activity was critically dependent on the presence of 10 mM Ca2+ in the assay medium.

Labeling of a thiol residue in sarcoplasmic reticulum ATPase by pyrene maleimide. Solvent accessibility studied by fluorescence quenching.

Sarcoplasmic reticulum ATPase was specifically labeled by the fluorescent probe N-(1-pyrene)maleimide which modified 1 mol of a highly reactive thiol residue per mol of ATPase under appropriate conditions, when the probe concentration was varied in the range 0.1-1.5 microM. Addition of inorganic phosphate to the labeling medium increased both the rate of labeling and the number of modified thiol residues. Addition of ATP gave a marked kinetic protection from labeling, suggesting that the label was attached to a protein domain which is sensitive to changes at the catalytic site. Quenching of pyrene fluorescence emission of labeled ATPase by acrylamide and cesium chloride gave linear Stern-Volmer plots. The Stern-Volmer quenching constants of pyrene-ATPase fluorescence were 10 times lower than the constant obtained for acrylamide quenching of the fluorescent adduct of pyrene-maleimide-cystein used as a control, indicating that the pyrene moiety of the probe was considerably shielded from the medium solvent when covalently attached to the ATPase. The efficiency of quenching of pyrene-ATPase fluorescence increased by a significant amount upon addition of 100 microM Ca2+, when compared to the quenching in the presence of a Ca2+ chelator. It suggests that occupancy of the high affinity Ca2+ sites of the ATPase increases the accessibility of medium solvent into hydrophobic domains of the enzyme. The fluorescence lifetime of the solubilized pyrene-ATPase emission was 144-149 ns. The fluorescence polarization of pyrene-ATPase solubilized by nonionic detergent C12E8 was rho = 0.10 and it increased with an increase in the viscosity of the medium yielding a linear Perrin plot. The rotational correlation time for the soluble ATPase was 532 ns, corresponding to the overall rotation of a detergent-pyrene-ATPase particle with radius of 87A.