Irene García

An O-acetylserine(thiol)lyase homolog with L-cysteine desulfhydrase activity regulates cysteine homeostasis in Arabidopsis.

Cysteine (Cys) occupies a central position in plant metabolism due to its biochemical functions. Arabidopsis (Arabidopsis thaliana) cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes that catalyze the biosynthesis of Cys. Because they are localized in the cytosol, plastids, and mitochondria, this results in multiple subcellular Cys pools. Much progress has been made on the most abundant OASTL enzymes; however, information on the less abundant OASTL-like proteins has been scarce. To unequivocally establish the enzymatic reaction catalyzed by the minor cytosolic OASTL isoform CS-LIKE (for Cys synthase-like; At5g28030), we expressed this enzyme in bacteria and characterized the purified recombinant protein. Our results demonstrate that CS-LIKE catalyzes the desulfuration of l-Cys to sulfide plus ammonia and pyruvate. Thus, CS-LIKE is a novel l-Cys desulfhydrase (EC 4.4.1.1), and we propose to designate it DES1. The impact and functionality of DES1 in Cys metabolism was revealed by the phenotype of the T-DNA insertion mutants des1-1 and des1-2. Mutation of the DES1 gene leads to premature leaf senescence, as demonstrated by the increased expression of senescence-associated genes and transcription factors. Also, the absence of DES1 significantly reduces the total Cys desulfuration activity in leaves, and there is a concomitant increase in the total Cys content. As a consequence, the expression levels of sulfur-responsive genes are deregulated, and the mutant plants show enhanced antioxidant defenses and tolerance to conditions that promote oxidative stress. Our results suggest that DES1 from Arabidopsis is an l-Cys desulfhydrase involved in maintaining Cys homeostasis, mainly at late developmental stages or under environmental perturbations.

Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum

A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to aminoacid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced aminoacid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.

Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic fungus Trichoderma harzianum

A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized. Partial amino-acid sequences from the purified 33-kDa chitinase were obtained. The amino-terminal peptide sequence was employed to design an oligonucleotide probe and was used as a primer to isolate a 1.2-kb cDNA. The cDNA codes for a protein of 321 amino acids, which includes a putative signal peptide of 19 amino acids. All microsequenced peptides found in this sequence, indicate that this cDNA codes for the 33-kDa chitinase. A high homology (approximately 43% identity) was found with fungal and plant chitinases, including yeast chitinases. However enzyme characteristics suggest a nutritional (saprophytic or mycoparasitic), rather than a morphogenetic, role for this chitinase. The chit33 gene appears as a single copy in the T. harzianum genome, is strongly suppressed by glucose, and de-repressed under starvation conditions as well as in the presence of autoclaved mycelia and/or fungal cell walls. The 33-kDa chitinase seems to be very stable except under starvation conditions. The independent regulation of each of the chitinases in T. harzianum indicates different specific roles.

Cysteine-Generated Sulfide in the Cytosol Negatively Regulates Autophagy and Modulates the Transcriptional Profile in Arabidopsis

This article highlights the role of hydrogen sulfide as a relevant signaling molecule in plants, of comparable importance as described in animals. This study shows the regulatory role of sulfide generated by the cytosolic l-Cys desulfhydrase 1 enzyme on autophagy in eukaryotes. In Arabidopsis thaliana, DES1 is the only identified l-Cysteine desulfhydrase located in the cytosol, and it is involved in the degradation of cysteine and the concomitant production of H2S in this cell compartment. Detailed characterization of the T-DNA insertion mutants des1-1 and des1-2 has provided insight into the role of sulfide metabolically generated in the cytosol as a signaling molecule. Mutations of L-CYS DESULFHYDRASE 1 (DES1) impede H2S generation in the Arabidopsis cytosol and strongly affect plant metabolism. Senescence-associated vacuoles are detected in mesophyll protoplasts of des1 mutants. Additionally, DES1 deficiency promotes the accumulation and lipidation of the ATG8 protein, which is associated with the process of autophagy. The transcriptional profile of the des1-1 mutant corresponds to its premature senescence and autophagy-induction phenotypes, and restoring H2S generation has been shown to eliminate the phenotypic defects of des1 mutants. Moreover, sulfide is able to reverse ATG8 accumulation and lipidation, even in wild-type plants when autophagy is induced by carbon starvation, suggesting a general effect of sulfide on autophagy regulation that is unrelated to sulfur or nitrogen limitation stress. Our results suggest that cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the transcriptional profile of Arabidopsis.

Hair removal methods: A comparative study for dermoscopy images

Abstract Removal and restoration of hair and hair-like regions within skin lesion images is needed so features within lesions can be more effectively analyzed for benign lesions, cancerous lesions, and for cancer discrimination. This paper refers to “melanoma texture” as a rationale for supporting the need for the proposed hair detection and repair techniques, which incompletely represents why hair removal is an important operation for skin lesion analysis. A comparative study of the state-of-the-art hair-repaired methods with a novel algorithm is also proposed by morphological and fast marching schemes. The hair-repaired techniques are evaluated in terms of computational, performance and tumor-disturb patterns ( TDP ) aspects. The comparisons have been done among (i) linear interpolation, inpainting by (ii) non-linear partial differential equation ( PDE ) and (iii) exemplar-based repairing techniques. The performance analysis of hair detection quality, was based on the evaluation of the hair detection error ( HDE ), quantified by statistical metrics and manually used to determine the hair lines from a dermatologist as the ground truth. The results are presented on a set of 100 dermoscopic images. For the two characteristics measured in the experiments the best method is the fast marching hair removal algorithm ( HDE : 2.98%, TDP : 4.21%). This proposed algorithm repaired the texture of the melanoma, which becomes consistent with human vision. The comparisons results obtained, indicate that hair-repairing algorithm based on the fast marching method achieve an accurate result.

Mitochondrial β-Cyanoalanine Synthase Is Essential for Root Hair Formation in Arabidopsis thaliana

The β-cyanoalanine synthase enzyme is responsible for detoxifying the cyanide generated during cellular metabolism, primarily in the synthesis of ethylene. This analysis of a null mutant of this enzyme indicates that cyanide can act in some developmental processes as a signaling molecule. Cyanide is stoichiometrically produced as a coproduct of the ethylene biosynthesis pathway and is detoxified by β-cyanoalanine synthase enzymes. The molecular and phenotypical analysis of T-DNA insertion mutants of the mitochondrial β-cyanoalanine synthase CYS-C1 suggests that discrete accumulation of cyanide is not toxic for the plant and does not alter mitochondrial respiration rates but does act as a strong inhibitor of root hair development. The cys-c1 null allele is defective in root hair formation and accumulates cyanide in root tissues. The root hair defect is phenocopied in wild-type plants by the exogenous addition of cyanide to the growth medium and is reversed by the addition of hydroxocobalamin or by genetic complementation with the CYS-C1 gene. Hydroxocobalamin not only recovers the root phenotype of the mutant but also the formation of reactive oxygen species at the initial step of root hair tip growth. Transcriptional profiling of the cys-c1 mutant reveals that cyanide accumulation acts as a repressive signal for several genes encoding enzymes involved in cell wall rebuilding and the formation of the root hair tip as well as genes involved in ethylene signaling and metabolism. Our results demonstrate that mitochondrial β-cyanoalanine synthase activity is essential to maintain a low level of cyanide for proper root hair development.

Cysteine and Cysteine-Related Signaling Pathways in Arabidopsis thaliana

Cysteine occupies a central position in plant metabolism because it is a reduced sulfur donor molecule involved in the synthesis of essential biomolecules and defense compounds. Moreover, cysteine per se and its derivative molecules play roles in the redox signaling of processes occurring in various cellular compartments. Cysteine is synthesized during the sulfate assimilation pathway via the incorporation of sulfide to O-acetylserine, catalyzed by O-acetylserine(thiol)lyase (OASTL). Plant cells contain OASTLs in the mitochondria, chloroplasts, and cytosol, resulting in a complex array of isoforms and subcellular cysteine pools. In recent years, significant progress has been made in Arabidopsis, in determining the specific roles of the OASTLs and the metabolites produced by them. Thus, the discovery of novel enzymatic activities of the less-abundant, like DES1 with L-cysteine desulfhydrase activity and SCS with S-sulfocysteine synthase activity, has provided new perspectives on their roles, besides their metabolic functions. Thereby, the research has been demonstrated that cytosolic sulfide and chloroplastic S-sulfocysteine act as signaling molecules regulating autophagy and protecting the photosystems, respectively. In the cytosol, cysteine plays an essential role in plant immunity; in the mitochondria, this molecule plays a central role in the detoxification of cyanide, which is essential for root hair development and plant responses to pathogens.

Lesion border detection in dermoscopy images using dynamic programming

Background/purpose: Automated border detection is an important and challenging task in the computerized analysis of dermoscopy images. However, dermoscopic images often contain artifacts such as illumination, dermoscopic gel, and outline (hair, skin lines, ruler markings, and blood vessels). As a result, there is a need for robust methods to remove artifacts and detect lesion borders in dermoscopy images.

Histone H1 Is Required for Proper Regulation of Pyruvate Decarboxylase Gene Expression in Neurospora crassa

ABSTRACT We show that Neurosporacrassa has a single histone H1 gene, hH1, which encodes a typical linker histone with highly basic N- and C-terminal tails and a central globular domain. A green fluorescent protein-tagged histone H1 chimeric protein was localized exclusively to nuclei. Mutation of hH1 by repeat-induced point mutation (RIP) did not result in detectable defects in morphology, DNA methylation, mutagen sensitivity, DNA repair, fertility, RIP, chromosome pairing, or chromosome segregation. Nevertheless, hH1 mutants had mycelial elongation rates that were lower than normal on all tested carbon sources. This slow linear growth phenotype, however, was less evident on medium containing ethanol. The pyruvate decarboxylase gene, cfp, was abnormally derepressed in hH1 mutants on ethanol-containing medium. This derepression was also found when an ectopically integrated fusion of the cfp gene promoter to the reporter gene hph was analyzed. Thus, Neurospora histone H1 is required for the proper regulation of cfp, a gene with a key role in the respiratory-fermentative pathway.

A feature-preserving hair removal algorithm for dermoscopy images.

Accurate segmentation and repair of hair‐occluded information from dermoscopy images are challenging tasks for computer‐aided detection (CAD) of melanoma. Currently, many hair‐restoration algorithms have been developed, but most of these fail to identify hairs accurately and their removal technique is slow and disturbs the lesions pattern.