Paracoccidoides brasiliensis 30 kDa Adhesin: Identification as a 14-3-3 Protein, Cloning and Subcellular Localization in Infection Models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis. © 2013 Silva et al.

Paracoccidoides brasiliensis 30 kDa adhesin: identification as a 14-3-3 protein, cloning and subcellular localization in infection models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.

Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein

The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

Differential display and suppression subtractive hybridization analysis of the pulp of ripening banana

The fruit of banana undergoes several important physico-chemical changes during ripening. Analysis of gene expression would permit identification of important genes and regulatory elements involved in this process. Therefore, transcript profiling of preclimacteric and climacteric fruit was performed using differential display and Suppression subtractive hybridization. Our analyses resulted in the isolation of 12 differentially expressed cDNAs, which were confirmed by dot-blots and northern blots. Among the sequences identified were sequences homologous to plant aquaporins, adenine nucleotide translocator, immunophilin, legumin-like proteins, deoxyguanosine kinase and omega-3 fatty acid desaturase. Some of these cDNAs correspond to newly isolated genes involved in changes related to the respiratory climacteric, or stress-defense responses. Functional characterization of ripening-associated genes could provide information useful in controlling biochemical pathways that would have an impact on banana quality and shelf life. (C) 2009 Elsevier B.V. All rights reserved.

Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators

The recent identification of several additional members of the family of sugar transport facilitators (gene symbol SLC2A, protein symbol GLUT) has created a heterogeneous and, in part, confusing nomenclature. Therefore, this letter provides a summary of the family members and suggests a systematic nomenclature for SLC2A and GLUT symbols.

Fishing the key biological components of the bacterium Geobacter metallireducens for optimal biotechnological applications

Os resultados apresentados no capítulo 2 foram incluídos no artigo Dantas JM, Campelo LM, Duke NEC, Salgueiro CA, Pokkuluri PR (2015) "The structure of PccH from Geobacter sulfurreducens – a novel low reduction potential monoheme cytochrome essential for accepting electrons from an electrode", FEBS Journal, 282, 2215-2231.

Caracterização molecular de dois isolados brasileiros de Lettuce mosaic virus apresentando propriedades biológicas distintas

Efetuou-se a clonagem e seqüenciamento do gene que codifica a proteína capsidial de dois isolados do vírus do mosaico da alface (Lettuce mosaic virus, LMV) provenientes do estado de São Paulo, previamente caracterizados como pertencentes aos patótipos II (AF198, incapaz de infetar cultivares com os genes de resistência mo1¹ ou mo1²) e IV (AF199, capaz de quebrar a resistência propiciada pelos genes mo1¹ e mo1²), com base na virulência em cultivares diferenciadoras. Análise comparativa das seqüências de nucleotídeos de isolados provenientes da Europa, América do Norte, Oriente Médio e os dois isolados brasileiros não permitiu sua separação em estirpes, pois as porcentagens de homologia foram sempre superiores a 95%. Entretanto, análise filogenética dos isolados sugere uma origem comum entre o isolado AF-198 e os isolados LMV-R e LMV-0 (patótipo II, provenientes dos Estados Unidos e da França, respectivamente). O isolado AF199 apresentou uma alta homologia de seqüência com os isolados LMV-Aud e LMV-13, ambos provenientes da França. Esses isolados também são relacionados a isolados provenientes do Chile, embora uma origem comum não seja proposta. Eventos independentes de mutação podem estar ocorrendo em diferentes partes do mundo, propiciando o surgimento de novas estirpes de LMV capazes de quebrar a resistência conferida pelos genes mo1¹ e mo1².

Molecular characterization of two Brazilian isolates of Lettuce mosaic virus with distinct biological properties

Efetuou-se a clonagem e seqüenciamento do gene que codifica a proteína capsidial de dois isolados do vírus do mosaico da alface (Lettuce mosaic virus, LMV) provenientes do estado de São Paulo, previamente caracterizados como pertencentes aos patótipos II (AF198, incapaz de infetar cultivares com os genes de resistência mo1¹ ou mo1²) e IV (AF199, capaz de quebrar a resistência propiciada pelos genes mo1¹ e mo1²), com base na virulência em cultivares diferenciadoras. Análise comparativa das seqüências de nucleotídeos de isolados provenientes da Europa, América do Norte, Oriente Médio e os dois isolados brasileiros não permitiu sua separação em estirpes, pois as porcentagens de homologia foram sempre superiores a 95%. Entretanto, análise filogenética dos isolados sugere uma origem comum entre o isolado AF-198 e os isolados LMV-R e LMV-0 (patótipo II, provenientes dos Estados Unidos e da França, respectivamente). O isolado AF199 apresentou uma alta homologia de seqüência com os isolados LMV-Aud e LMV-13, ambos provenientes da França. Esses isolados também são relacionados a isolados provenientes do Chile, embora uma origem comum não seja proposta. Eventos independentes de mutação podem estar ocorrendo em diferentes partes do mundo, propiciando o surgimento de novas estirpes de LMV capazes de quebrar a resistência conferida pelos genes mo1¹ e mo1².

The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.

p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

Characterization of polyamine oxidase from the aquatic nitrogen-fixing fern Azolla imbricata

Biochemical properties of a polyamine oxidase (PAO; EC 1.5.3.3) purified from the aquatic nitrogen-fixing fern Azolla imbricata (Roxb.) Nak. were studied. The native molecular mass of the enzyme estimated by Sephadex G 200 get filtration was 66.2 kDa. SDS-PAGE gave a single protein band corresponding to a molecular mass of 65.5 kDa. The light yellow enzyme had absorption maxima at 278, 372 and 454 nm with 1 mol FAD per mole enzyme molecule as its cofactor. The PAO was active on both the triamine Spd and the tetraamine Spm as substrates. However, it was inactive on the diamines Put and Cad. It had a pH optimum of 6.5 for both Spd and Spm. The K-m(S) for Spd and Spm were 6.71 x 10(-2) and 1.13 x 10(-1) nM, respectively. Pre-incubation with 10 mM of K+ (KCl), Ca2(+) (CaCl2) or Mg2+ (MgCl2) had no effect on PAO activity. However, 10 mM Cu2+ (CuCl2), Mn2+ (MnCl2) and Fe2+ (FeSO4) inhibited enzyme activity by 37%, 43% and 58%, respectively. The metal chelator EDTA (10 mM), the carbonyl reagent hydroxylamine (0.5 mM) and the sulfhydryl reagent p-chloro-mercuribenzoate (0.5 mM) had no effect on PAO activity. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Molecular cloning reveals that the p160 myb-binding protein is a novel, predominantly nucleolar protein which may play a role in transactivation by Myb

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.

Functional expression cloning reveals proapoptotic role for protein phosphatase 4

Functional expression cloning strategies are highly suitable for the analysis of the molecular control of apoptosis. This approach has two critical advantages. Firstly, it eliminates prior assumptions about the properties of the proteins involved, and, secondly, it selectively targets proteins that are causally involved in apoptosis control and which affect the crucial cellular decision between survival and death. The application of this strategy to the isolation of cDNAs conferring resistance to dexamethasone and gamma-irradiation resulted in the isolation of a partial cDNA for the catalytic subunit of protein phosphatase 4 (PP4). Cells transfected with this partial cDNA in an expression vector downregulated PP4 and were resistant to both dexamethasone and UV radiation, as demonstrated by both membrane integrity and colony-forming assays. These observations suggest that PP4 plays an important proapoptotic role in T lymphocytes.

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation. (C) 2008 Elsevier Inc. All rights reserved.