Biomedical subjects

A Leite

Publications and source records attributed to A Leite.

At least 19 recordsLinked to original sources

PubMed2006

Psychological aspects of pre-symptomatic testing for Machado-Joseph disease and familial amyloid polyneuropathy type I.

Machado-Joseph disease [MJD, also spinocerebellar ataxia type 3 (SCA3)] and familial amyloid polyneuropathy type I (FAP-I or ATTR V30M) are neurodegenerative disorders, inherited in an autosomal dominant fashion, which have a high prevalence in Portugal, probably due to a founder effect. MJD and FAP-I are late-onset diseases, with symptoms emerging usually during adulthood. CGPP, which is the national reference centre for these disorders, has a genetic lab that offers diagnostic, pre-symptomatic and prenatal testing and an outpatient clinic to counsel and follow relatives at risk for hereditary ataxias, FAP-I and Huntington disease (HD). The present work is a review of our 10-year experience with psychological counselling of individuals at risk for MJD and FAP-I. Persons at risk for FAP-I may show a better response to pre-symptomatic testing than those who are at risk for MJD and HD because of the availability of liver transplantation, which may improve their health and life expectancy. Psychological well-being and specific distress of MJD and FAP-I test applicants, before undergoing genetic testing (baseline level) and 3 to 6 months after disclosure of test results, have shown a low level of change, both in identified carriers and non-carriers. A major goal of psychological characterization of at-risk individuals for MJD and FAP-I is to determine the factors that influence the uptake of genetic testing.

Amyloid Neuropathies, Familial↗

PubMed2002

Expression of a thioredoxin peroxidase in insulin-producing cells.

The presence of thioredoxin peroxidase (TPx), also known as thiol specific antioxidant (TSA), was investigated in neonatal and adult rat islets, and in the beta-cell line HIT-T15. Western blotting of extracts from neonatal and adult pancreatic islets and from the tumoral cell line HIT-T15 revealed the presence of a 25 kDa protein that comigrated with purified yeast TPx. Endocrine pancreatic TPx accounted for approximately 0.01% of the total protein content. Treatment with H2O2 for 3 h increased the expression of TPx in HIT-T15 cells. The distribution of TPx throughout the islet cells was confirmed by immunocytochemistry. Since pancreatic beta-cells possess a weak antioxidant enzyme defense system, especially with regard to hydrogen peroxidase-decomposing enzymes, the presence of a TPx analog in islets suggests that this enzyme may play a role in protecting pancreatic cells against reactive oxygen species.

Aging↗

PubMed2001

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity.

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

Bacterial Proteins↗

PubMed2001

Insulin modulates leptin-induced STAT3 activation in rat hypothalamus.

Insulin and leptin have overlapping effects in the control of energy homeostasis, but the molecular basis of this synergism is unknown. Insulin signals through a receptor tyrosine kinase that phosphorylates and activates the docking proteins IRSs (insulin receptor substrates), whereas the leptin receptor and its associated protein tyrosine kinase JAK2 (Janus kinase 2) mediate phosphorylation and activation of the transcription factor STAT3 (signal transducer and activator of transcription). Here, we present evidence for the integration of leptin and insulin signals in the hypothalamus. Insulin induced JAK2 tyrosine phosphorylation, leptin receptor phosphorylation which, in the presence of leptin, augmented the interaction between STAT3 and this receptor. Insulin also increased the leptin-induced phosphorylation of STAT3 and its activation. These results indicate that insulin modulates the leptin signal transduction pathway, and may provide a molecular basis for the coordinated effects of insulin and leptin in feeding behavior and weight control.

Animals↗

PubMed2001

Selectively labeling the heterologous protein in Escherichia coli for NMR studies: a strategy to speed up NMR spectroscopy.

Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The (1)H/(15)N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective (15)N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good (1)H/(15)N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The (15)N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the (1)H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.

DNA-Binding Proteins↗

PubMed2000

Molecular characterization of a new arcelin-5 gene.

Arcelins are insecticidal proteins found in some wild accessions of the common bean, Phaseolus vulgaris. They are grouped in six allelic variants and arcelin-5 is the variant with the highest inhibitory effect on the development of Zabrotes subfasciatus larvae. Characterization of the protein and its genes resulted in the identification of three polypeptides and the isolation of two genes that encode the Arc5a and Arc5b polypeptides. Here we describe a new gene, Arc5-III. The protein it encodes has 81% amino acid identity with the derived amino acid sequences of Arc5-I and Arc5-II. The Arc5-III gene is highly expressed in developing seeds and at a much lower level in roots. Data obtained by a combination of two-dimensional gel electrophoresis, protein sequencing and MALDI-TOF mass spectrometry analysis support the conclusion that Arc5-III encodes a polypeptide present in Arc5c band. Using ion-exchange chromatography, three fractions containing arcelin-5 polypeptides were eluted by increasing the salt concentration. The three fractions contain various amounts of the three arc-5 polypeptides and inhibit the growth of Zabrotes subfasciatus larvae differentially, suggesting differences in insecticidal activity among the arcelin-5 isoforms.

Amino Acid Sequence↗

PubMed2000

Regulation of lysine catabolism in higher plants.

Lysine is an essential amino acid for mammals but its concentration in cereals, one of our main food sources, is low. Research over the past 40 years has unraveled many biochemical and molecular details of the aspartic acid pathway, which is the main route of lysine biosynthesis in plants. However, genetic manipulation of this pathway has not been successful at producing high-lysine seeds. This is because lysine, instead of being accumulated, is degraded via the saccharopine pathway. Recent work has increased our knowledge of this pathway, including both the enzymes involved and their regulation.

Lysine↗

PubMed1999

Plant uncoupling mitochondrial protein activity in mitochondria isolated from tomatoes at different stages of ripening.

In the present study we have observed a higher state of coupling in respiring mitochondria isolated from green as compared to red tomatoes (Lycopersicon esculentum, Mill.). Green tomato mitochondria produced a membrane potential (deltapsi) high enough to phosphorylate ADP, whereas in red tomato mitochondria, BSA and ATP were required to restore deltapsi to the level of that obtained with green tomato mitochondria. This supports the notion that such uncoupling in red tomato mitochondria is mediated by a plant uncoupling mitochondrial protein (PUMP; cf. Vercesi et al., 1995). Nevertheless, mitochondria from both green and red tomatoes exhibited an ATP-sensitive linoleic acid (LA)-induced deltapsi decrease providing evidence that PUMP is also present in green tomatoes. Indeed, proteoliposomes containing reconstituted green or red tomato PUMP showed LA uniport and LA-induced H+ transport. It is suggested that the higher concentration of free fatty acids (PUMP substrates) in red tomatoes could explain the lower coupling state in mitochondria isolated from these fruits.

Animals↗

PubMed1999

Identification of a DNA-binding factor that recognizes an alpha-coixin promoter and interacts with a Coix Opaque-2 like protein.

Transient expression and electrophoretic mobility shift assay were used to investigate the cis elements and the DNA-binding proteins involved in the regulation of expression of a 22 kDa zein-like alpha-coixin gene. A set of unidirectional deletions was generated in a 962 bp fragment of the alpha-coixin promoter that had been previously fused to the reporter gene GUS. The constructs were assayed by transient expression in immature maize endosperm. There was no significant decrease in GUS activity as deletions progressed from -1084 to -238. However, deletion from -238 to -158, which partially deleted the O2c box, resulted in a dramatic decrease in GUS activity emphasizing the importance of the O2 box in the quantitative expression of the gene. The -238 promoter fragment interacted with Coix endosperm nuclear proteins to form 5 DNA-protein complexes, C1-C5, as detected by EMSA. The same retarded complexes were observed when the -158 promoter fragment was used in the binding reactions. Reactions with nuclear extracts isolated from Coix endosperms harvested from 6 to 35 days after pollination revealed that the 5 DNA-protein complexes that interact with the alpha-coixin promoter are differentially assembled during seed development. Deletion analysis carried out on the -238/ATG promoter fragment showed that a 35 bp region from -86 to -51 is essential for the formation of the complexes observed. When nuclear extracts were incubated with an antiserum raised against the maize Opaque-2 protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented indicating that an Opaque-2 like protein participates in the formation of those complexes. Complex C2 was not affected by the addition of the O2 antibody, suggesting the existence of a novel nuclear factor, CBF1, that binds to the promoter and makes protein-protein associations with other proteins present in Coix endosperm nuclei.

DNA-Binding Proteins↗

PubMed1999

The DapA gene encoding the lysine biosynthetic enzyme dihydrodipicolinate synthase from Coix lacryma-jobi: cloning, characterization, and expression analysis.

Dihydrodipicolinate synthase (DHPS) is the main enzyme of a specific branch of the aspartate pathway leading to lysine biosynthesis in higher plants. We have cloned and characterized the DHPS-encoding Dap)A gene from the maize-related grass Coix lacryiana-jobi. The DapA open reading frame is interrupted by two introns and encodes the 326 amino acid-long Coix DHPS protein, which is 95% identical to the maize DHPS protein. Coix DNA gel blot analysis with maize DHPS cDNA as a probe showed a single strongly hybridizing band along with faint bands. RNA gel blot analysis showed that DHPS transcripts are present in coleoptiles, embryos, endosperms, and roots but are almost undetectable in blades of young leaves of both Coix and maize. The 5'-flanking region of the DapA gene contains a TGACTC GCN4-like element located 372 bp upstream the putative translation start codon. Steady-state levels of DHPS mRNA were slightly reduced in the endosperms and embryos of the maize lysine-rich opaque2 mutants when compared with those in normal kernels. Selective binding assay with the maize Opaque2 protein (O2) showed that the GCN4-like element is not an O2 binding site, suggesting that the DHPS gene is not under the control of O2.

Amino Acid Sequence↗

PubMed1999

The role of opaque2 in the control of lysine-degrading activities in developing maize endosperm.

We have isolated a cDNA clone, designated ZLKRSDH, encoding the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) from maize. The predicted polypeptide has an N-terminal LKR domain and a C-terminal SDH domain that are similar to the yeast LYS1 and LYS9 monofunctional proteins, respectively. The maize LKR/SDH protein is located in the cytoplasm of subaleurone endosperm cell layers. Transcripts and polypeptides as well as enzyme activities showed an upregulation and downregulation during endosperm development. The developmental expression of ZLKRSDH was examined in normal and opaque2 seeds. In the mutant endosperm, mRNA levels were reduced by >90%, with concomitant reductions in polypeptide levels and LKR/SDH activity. These results suggest that lysine levels in the endosperm are likely to be controlled at the transcriptional level by the Opaque2 transcription factor.

Amino Acid Sequence↗

PubMed1998

AtPUMP: an Arabidopsis gene encoding a plant uncoupling mitochondrial protein.

A cDNA clone (AtPUMP) encoding a plant uncoupling mitochondrial protein was isolated from Arabidopsis thaliana. The cDNA contains an open reading frame of 921 nucleotides encoding 306 amino acids (predicted molecular weight 32,708). The predicted polypeptide is 81% identical and 89% similar to the potato UCP-like protein, and includes an energy transfer protein motif common to mitochondrial transporters. The AtPUMP gene exists as a single copy in the Arabidopsis genome. The corresponding transcript was expressed in all tissues and was strongly induced by cold treatment. We suggest that the putative AtPUMP protein may play a role in heat-requiring physiological events in Arabidopsis.

Amino Acid Sequence↗

PubMed1998

The molecular and functional characterization of an Opaque2 homologue gene from Coix and a new classification of plant bZIP proteins.

The seed storage proteins of Coix, sorghum and maize are codified by homologous genes which are coordinately expressed in the endosperm in a temporal-specific fashion. Opaque2 (O2), a bZIP protein originally isolated from maize, has been described as a transcription activator of alpha- and beta-prolamin genes. The isolation and characterization of cDNA and genomic clones encoding the Opaque2 homologue from Coix are reported here. The coding region of the Coix O2 gene is interrupted by five introns and codifies a polypeptide of 408 amino acids. Comparison of the deduced amino acid sequence with two different sequences of maize O2 protein showed that the Coix O2 protein is similar to the maize O2 isolated from W22 maize inbred line. The Coix O2 protein has the same binding specificity and expression pattern of the maize O2. The O2 proteins together with OHP1, OsBZIPPA, SPA, CPRF2 and RITA1 were assigned to one of the five bZIP plant families in an updated classification of plant bZIP according to bZIP domain similarity.

Amino Acid Sequence↗

PubMed1998

Cooperative DNA binding and sequence discrimination by the Opaque2 bZIP factor.

The maize Opaque2 (O2) protein is a basic leucine zipper transcription factor that controls the expression of distinct classes of endosperm genes through the recognition of different cis-acting elements in their promoters. The O2 target region in the promoter of the alpha-coixin gene was analyzed in detail and shown to comprise two closely adjacent binding sites, named O2u and O2d, which are related in sequence to the GCN4 binding site. Quantitative DNase footprint analysis indicated that O2 binding to alpha-coixin target sites is best described by a cooperative model. Transient expression assays showed that the two adjacent sites act synergistically. This synergy is mediated in part by cooperative DNA binding. In tobacco protoplasts, O2 binding at the O2u site is more important for enhancer activity than is binding at the O2d site, suggesting that the architecture of the O2-DNA complex is important for interaction with the transcriptional machinery.

Base Sequence↗

PubMed1997

ACGT and vicilin core sequences in a promoter domain required for seed-specific expression of a 2S storage protein gene are recognized by the opaque-2 regulatory protein.

The expression of Brazil nut storage albumin genes is highly regulated during seed development. Several sequences in the promoter of one of these genes show homologies with the target sites of the maize O2 bZIP regulatory protein. We therefore asked whether the O2 protein would recognize these promoter sequences. We show that the O2 protein binds to three different sequences (F1, F2 and F3). F1 and F3 are hybrid C/G and A/G boxes, respectively, that are homologous to the O2-binding site of a maize alpha-zein gene. F2 is a new O2-binding sequence related to the O2 target sites of the Coix alpha-coxin, the maize b-32 genes and the AP-1 pseudopalindrome. Molecular modelling showed that an Asn and a Ser in the 02 DNA binding domain make different base-specific contacts with each operator. 5' Promoter deletions of the be2S1 gene showed that the domain containing the O2 target sites F1 and F2 is required for detectable reporter gene expression in transgenic tobacco seeds. Moreover, the homologous coix O2 protein was shown to in situ transactivate the promoter region encompassing the three O2-binding sites F1, F2 and F3. Thus, these sites may be in vivo regulatory sequences mediating activation by bZIP regulatory proteins.

2S Albumins, Plant↗

PubMed1996

Transcriptional control of the cellulase genes in Trichoderma reesei.

The expression of the cellulase transcripts of Trichoderma reesei is controlled by the nature of the energy carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Evidence is presented suggesting that a low constitutive extracellular cellulolytic system catalyzes the formation of a soluble inducer from cellulose, and this inducer triggers the expression of the cellulase transcripts. This basal and cellulose-induced expression of the cellobiohydrolase I mRNAs (cbh1), the major member of the cellulase system, is transcriptionally controlled by two independent cis-acting DNA regions. In addition, expression of the cbh1 transcript is influenced by the physiological state of the mitochondria and this sensitivity is controlled through the 5'-flanking DNA sequence of this gene.

Cellulase↗

PubMed1995

Structure, organization and promoter expression of the actin-encoding gene in Trichoderma reesei.

The single gene encoding actin (Act) in the cellulolytic filamentous fungus Trichoderma reesei (Tr) has been isolated and characterized. The gene contains five introns located in identical positions when compared to the putative ancestral actin genes (act) present in Thermomyces lanuginosus and Aspergillus nidulans. The 5' untranslated region (UTR) of the gene contains a TATA-like sequence (TAATA), a C + T-rich region and a potential CCAAT motif. This region was used as a homologous promoter to direct expression of hygromycin-B-resistance-encoding gene as a dominant-selectable Tr marker.

Actins↗

PubMed1995

The involvement of Opaque 2 on beta-prolamin gene regulation in maize and Coix suggests a more general role for this transcriptional activator.

The maize opaque 2 (o2) mutation is known to have numerous pleiotropic effects. Some polypeptides have their expression depressed while others are enhanced. The best characterized effects of the o2 mutation are those exerted on endosperm genes encoding the storage protein class of the 22 kDa alpha-zeins and the ribosome inactivating protein b-32. The Opaque 2 (O2) locus encodes a basic domain-leucine zipper DNA-binding factor, O2, which transcriptionally regulates these genes. In the maize-related grass Coix lacryma-jobi, an O2-homologous protein regulates the 25 kDa alpha-coixin family. We show in this paper that O2 transcriptionally regulates the structurally and developmentally different class of the beta-prolamins. A new O2-binding box was identified in beta-prolamin genes from maize and Coix that, together with the boxes previously identified in other endosperm expressed genes, forms a curious collection of O2 cis elements. This may have regulatory implications on the role of O2 in the mechanism that controls coordinated gene expression in the developing endosperm. Considering that the O2 locus controls at least three distinct classes of genes in maize endosperm, we propose that the O2 protein may play a more general role in maize endosperm development than previously conceived.

Base Sequence↗