Triplicate genes for mitochondrial ADP/ATP carriers in the aerobic yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> are regulated differentially in the absence of oxygen

Yarrowia lipolytica is a strictly aerobic fungus, which differs from the extensively studied model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe with respect to its physiology, genetics and dimorphic growth habit. We isolated and sequenced cDNA and genomic clones (YlAAC1) from Y. lipolytica that encode a mitochondrial ADP/ATP carrier. The YlAAC1 gene can complement the S. cerevisiae aac2 deletion mutant. Southern hybridization, analysis of Yarrowia clones obtained in the course of the Génolevures project, and further sequencing revealed the existence of two paralogs of the YlAAC1 gene, which were named YlAAC2 and YlAAC3, respectively. Phylogenetic analysis showed that YlAAC1 and YlAAC2 were more closely related to each other than to YlAAC3, and are likely to represent the products of a recent gene duplication. All three Y. lipolytica YlAAC genes group together on the phylogenetic tree, suggesting that YlAAC3 is derived from a more ancient duplication within the Y. lipolytica lineage. A similar branching pattern for the three ScAAC paralogs in the facultative anaerobe S. cerevisiae demonstrates that two rounds of duplication of AAC genes occurred independently at least twice in the evolution of hemiascomycetous yeasts. Surprisingly, in both the aerobic Y. lipolytica and the facultative anaerobe S. cerevisiae, the three paralogs are differentially regulated in the absence of oxygen. Apparently, Y. lipolytica can sense hypoxia and down-regulate target genes in response.     

The inhibitor protein of the F1F0-ATP synthase is associated to the external surface of endothelial cells

The ATPase inhibitor protein (IP) of mitochondria was detected in the plasma membrane of living endothelial cells by flow cytometry, competition assays, and confocal microscopy of cells exposed to IP antibodies. The plasma membranes of endothelial cells also possess beta-subunits of the mitochondrial ATPase. Plasma membranes have the capacity to bind exogenous IP. TNF-alpha decreases the level of beta-subunits and increases the amount of IP, indicating that the ratio of IP to beta-subunit exhibits significant variations. Therefore, it is probable that the function of IP in the plasma membrane of endothelial cells is not limited to regulation of catalysis.     

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.     

Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation.     

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases: assays with human cathepsin B

We have developed positional scanning synthetic combinatorial libraries to define the substrate specificity of carboxydipeptidases. The library Abz-GXXZXK(Dnp)-OH, where Abz is ortho-aminobenzoic acid, K(Dnp) is N^ε-2,4-dinitrophenyl-lysine with free carboxyl group, the Z position was successively occupied with 1 of 19 amino acids (cysteine was omitted), and X represents randomly incorporated residues, was assayed initially with human cathepsin B, and arginine was defined as one of the best residues at the P₁ position. To examine the selectivity of , S₂, and S₃ subsites, the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH were then synthesized. The peptide Abz-GIVRAK(Dnp)-OH, which contains the most favorable residues in the positions identified by screening of the libraries with cathepsin B, was hydrolyzed by this enzyme with k_cat/K_m = 7288 mM⁻¹ s⁻¹. This peptide is the most efficient substrate described for cathepsin B to this point, and it is highly selective for the enzyme among the lysosomal cysteine proteases.     

Mammalian mitochondrial and microsomal cytochromes b(5) exhibit divergent structural and biophysical characteristics

The only outer mitochondrial membrane cytochrome b(5) examined to date, from rat (rOM b(5)), exhibits greater stability than known mammalian microsomal (Mc) isoforms, as well as a much higher kinetic barrier for hemin dissociation and a more negative reduction potential. A BlastP search of available databases using the protein sequence of rOM b(5) as template revealed entries for analogous proteins from human (hOM b(5)) and mouse (mOM b(5)). We prepared a synthetic gene coding for the heme-binding domain of hOM b(5), and expressed the protein to high levels. The hOM protein exhibits stability, hemin-binding, and redox properties similar to those of rOM b(5), suggesting that they are characteristic of the OM b(5) subfamily. The divergence in properties between the OM and Mc b(5) isoforms in mammals can be attributed, at least in part, to the presence of two extended hydrophobic patches in the former. The biophysical properties characteristic of the OM proteins may be important in facilitating the two functions proposed for them so far, reduction of ascorbate radical and stimulation of androgen synthesis.     

Changing ABRA protein peptide to fit into the HLA-DRbeta1*0301 molecule renders it protection-inducing

The Plasmodium falciparum acidic-basic repeat antigen represents a potential malarial vaccine candidate. One of this protein's high activity binding peptides, named 2150 ((161)KMNMLKENVDYIQKNQNLFK(180)), is conserved, non-immunogenic, and non-protection-inducing. Analogue peptides whose critical binding residues (in bold) were replaced by amino-acids having similar mass but different charge were synthesized and tested to try to modify such immunological properties. These analogues' HLA-DRbeta1* molecule binding ability were also studied in an attempt to explain their biological mechanisms and correlate binding capacity and immunological function with their three-dimensional structure determined by (1)H NMR. A 3(10) distorted helical structure was identified in protective and immunogenic peptide 24922 whilst alpha-helical structure was found for non-immunogenic, non-protective peptides having differences in alpha-helical position. The changes performed on immunogenic, protection-inducing peptide 24922 allowed it to bind specifically to the HLA-DRbeta1*0301 molecule, suggesting that these changes may lead to better interaction with the MHC Class II-peptide-TCR complex rendering it immunogenic and protective, thus suggesting a new way of developing multi-component, sub-unit-based anti-malarial vaccines.     

Efficient cold transfection of pea ferredoxin-NADP(H) oxidoreductase into rat hepatocytes

We describe the use of a non-viral, polyethylenimine-based vector to transfect rat hepatocytes preserved under hypothermic storage. DNA sequences encoding Escherichia coli beta-galactosidase and pea ferredoxin-NADP(H) oxidoreductase (FNR), cloned into plasmids pCH110 and pKM4 respectively, were used. FNR was detected in the liver of animals transplanted with transfected cells; no reactivity was observed in endogenous parenchyma. The expression of the transgene was transient as it was detectable up to 96 h subsequently declining to undetectable levels. In contrast to non-transfected cells, the engraftment of FNR-positive cells was not associated with inflammatory reaction. The percentage of FNR-positive implanted hepatocytes was at least five times higher than the original transfection efficiency measured in vitro, while the percentage of beta-galactosidase-positive cells was similar for both methods. These data indicate that the transfection system is effective in the transfer of plasmid DNA into hepatocytes under cold preservation and suggest the advantage of pKM4-transfected hepatocytes on engraftment in the recipient parenchyma.     

Potentially toxic and harmful microalgae from coastal waters of the Campania region (Tyrrhenian Sea, Mediterranean Sea)

A total number of 40 potentially toxic microalgae and 5 taxa causing discolorations have been identified along the coasts of the Campania region (South Tyrrhenian Sea, Mediterranean Sea). This number is based on results of over 20 years of research at a coastal station in the Gulf of Naples, 4 years of monitoring activity along the Campania coasts and of a series of scattered and sporadic observations. Several species of the recently erected genera Karenia and Takayama are reported for the first time in the area. Information on the period of highest abundances or most probable period of occurrence indicates the late spring and summer as the periods of maximum risk of harmful events. Despite the variety of potentially toxic species, no human health problems nor fish kills have ever been recorded in Campania. The reasons for this apparent paradox are probably to be found in the ecological factors that regulate the abundance, toxicity and spatial distribution of the potentially harmful species and reflect as well the relatively low number of aquaculture plants in the area.