Cloning, mapping and identification of the Aeromonas salmonicida fstA, fepA and irpA genes, encoding the 86, 82 and 74 kDa iron-regulated outer membrane proteins, respectively

Three iromps (iron-regulated outer membrane proteins) of Aeromonas salmonicida were identified by the use of specific antibodies together with Southern hybridization analysis and limited nucleotide sequencing of their genes. The results of these experiments together with a search of the international database for homologous sequences led to their identification as follows: -86 kDa iromp (FstA) as a Vibrio anguillarum Fat A homologue -82 kDa iromp (FepA) as an Escherichia coli FepA homologue -74 kDa iromp (IrpA) as an Escherichia coli Cir homologue.

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.

Construção de bibliotecas de cDNAs subtraídos a partir das sementes de Mamona submetidas à estresse hídrico e análise in silico de transcritos potencialmente expressos sob condições de estresse: Metalotioneina, proteína reprimida por auxina de 12.5 kDa e Glutelina tipo-A3

Estresses ambientais abióticos são fatores que causam respostas ao nível molecular, fisiológico e morfológico em plantas, dependendo também de sua intensidade e duração. É visto que algumas espécies apresentam tolerância a condições estressantes e ao mesmo tempo são fontes naturais de matéria prima para indústria. Nesse contexto encontra-se a mamona (Ricinus comunnis L.), principal fonte de óleo de rícino valorizado por suas aplicações farmacêuticas e principalmente industriais, vem sendo usada como cultura em regiões onde a disponibilidade de água é reduzida, usada como fonte de renda para agricultura da região nordeste brasileira. Visto que pouco se sabe sobre as respostas moleculares que levam essa planta a tolerar regiões secas e como as sementes, principais foco de interesse, respondem a essa escassez, nesse trabalho foram construídas duas bibliotecas de cDNAs, onde a partir de uma abordagem subtrativa, continham RNAs diferencialmente expressos em sementes de plantas mamona submetidas ao estresse hídrico durante 5 dias (biblioteca L7), e a outra RNAs diferencialmente expressos em sementes controle (biblioteca L5). A biblioteca L7 apresentou a maior variedade de transcritos com um total de 182. A maior parte das funções estabelecidas pelo sistema Gene Ontology - GO, foram direcionadas aos “Processos Metabólicos” (526), em segundo “Respostas a estímulos” (57), o terceiro termo mais abundante foram referentes a “Desenvolvimento”(26). Já na biblioteca L5, foram encontrados 91 transcritos, com maior parte de suas funções referentes a “Processos Metabólicos”(413), em segundo “Respostas a estímulos” (8) e em terceiro Regulação (6). Alguns dos transcritos da biblioteca L7 foram escolhidos para análise por repetirem-se mais de 3x e não aparecerem na biblioteca L5, o que indica uma possível regulação positiva sobre estresse. As análises sobre Metalotioneína (4x), mostraram que a sequência de proteica apresentava os domínios conservados que a caracterizava como tipo II, onde são encontrados dois domínios funcionais ricos em cisteína com posições altamente conservadas, desempenhando a função de ligar-se a metais pesados, correlacionadas assim como a atividade de eliminação EROs e defesa contra o estresse oxidativo, além de apresentar homologia com a sequência de Bruguiera gymnorhiza, uma planta de mangue adaptada a ambientes salinos. Analisamos também os transcritos da referente a proteína AUXIN-REPRESSED 12.5 KDA (3x), apontada como sendo reprimida pelo hormônio auxina e associada ao processo de dormência da semente, é descrito em uma família gênica onde vários membros pertencem as vias de resposta ao estresse. Por último, analisamos a proteína GLUTELIN TYPE-A 3 (5x), uma importante proteína de armazenamento com caráter hidrofílico, possivelmente direcionada para o vacúolo. Em nosso trabalho foi possível observar um aumento de transcritos em relação a subtração controle, possivelmente reflexo do aumento do metabolismo da semente, tanto para resposta defensiva ao estresse hídrico quanto para o amadurecimento rápido da semente onde foram observados transcritos referentes a resposta oxidativa, controle hormonal, proteínas de reserva e produção de óleo.

Immobilized kidney 28 KDa endostatin-related (KES28KDa) fragment promotes endothelial cell survival

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Albumin Is Synthesized In Epididymis And Aggregates In A High Molecular Mass Glycoprotein Complex Involved In Sperm-egg Fertilization.

The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

Augmented β-cell Function And Mass In Glucocorticoid-treated Rodents Are Associated With Increased Islet Ir-β /akt/mtor And Decreased Ampk/acc And As160 Signaling.

Glucocorticoid (GC) therapies may adversely cause insulin resistance (IR) that lead to a compensatory hyperinsulinemia due to insulin hypersecretion. The increased β-cell function is associated with increased insulin signaling that has the protein kinase B (AKT) substrate with 160 kDa (AS160) as an important downstream AKT effector. In muscle, both insulin and AMP-activated protein kinase (AMPK) signaling phosphorylate and inactivate AS160, which favors the glucose transporter (GLUT)-4 translocation to plasma membrane. Whether AS160 phosphorylation is modulated in islets from GC-treated subjects is unknown. For this, two animal models, Swiss mice and Wistar rats, were treated with dexamethasone (DEX) (1 mg/kg body weight) for 5 consecutive days. DEX treatment induced IR, hyperinsulinemia, and dyslipidemia in both species, but glucose intolerance and hyperglycemia only in rats. DEX treatment caused increased insulin secretion in response to glucose and augmented β-cell mass in both species that were associated with increased islet content and increased phosphorylation of the AS160 protein. Protein AKT phosphorylation, but not AMPK phosphorylation, was found significantly enhanced in islets from DEX-treated animals. We conclude that the augmented β-cell function developed in response to the GC-induced IR involves inhibition of the islet AS160 protein activity.

Association Between Cationic Liposomes And Low Molecular Weight Hyaluronic Acid.

This work presents a study of the association between low molecular weight hyaluronic acid (16 kDa HA) and cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The cationic liposome/HA complexes were evaluated to determine their mesoscopic structure, average size, zeta potential, and morphology as a function of the amount of HA in the system. Small angle X-ray scattering results revealed that neighboring cationic liposomes either stick together after a partial coating of low concentration HA or disperse completely in excess of HA, but they never assemble as multilamellar vesicles. Cryo-transmission electron microscopy images confirm the existence of unilamellar vesicles and large aggregates of unilamellar vesicles for HA fractions up to 80% (w/w). High concentrations of HA (> 20% w/w) proved to be efficient for coating extruded liposomes, leading to particle complexes with sizes in the nanoscale range and a negative zeta potential.

Low Molecular Weight Protein Tyrosine Phosphatase (lmwptp) Upregulation Mediates Malignant Potential In Colorectal Cancer.

Phosphatases have long been regarded as tumor suppressors, however there is emerging evidence for a tumor initiating role for some phosphatases in several forms of cancer. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP; acid phosphatase 1 [ACP1]) is an 18 kDa enzyme that influences the phosphorylation of signaling pathway mediators involved in cancer and is thus postulated to be a tumor-promoting enzyme, but neither unequivocal clinical evidence nor convincing mechanistic actions for a role of LMWPTP have been identified. In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia. Chemical inhibition of LMWPTP significantly reduces CRC growth. Furthermore, downregulation of LMWPTP in CRC leads to a reduced migration ability in both 2D- and 3D-migration assays, and sensitizes tumor cells to the chemotherapeutic agent 5-FU. In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.

Induction of a gloverin-like antimicrobial polypeptide in the sugarcane borer Diatraea saccharalis challenged by septic injury

Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80% identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest.

Influência das características das substâncias húmicas aquáticas na eficiência da coagulação com o cloreto férrico

The aim of this study was to verify the influence of the apparent molecular size of aquatic humic substances on the effectiveness of coagulation with ferric chloride. Coagulation-filtration tests using jar test and bench-scale sand filters were carried out on samples of water with true color of approximately 100 Hazen units, prepared with aquatic humic substances of different molecular sizes (F1: < 0.45 µm, F2: 100 kDa - 0.45 µm, F3: 30 - 100 kDa and F4': < 30 kDa). For the water samples with lower apparent molecular size fractions, greater dosages of coagulant was needed to remove the color around 5.0 Hanzen units, mainly because these water samples contain higher concentrations of fulvic acids, which exhibited a larger number of negatively-charged groups.

Evaluation of capillaries with different inner coatings for DNA analysis using dilute polymer solutions by capillary electrophoresis

In this work three capillary columns, one with uncoated inner wall and two with covalently-bound internal coatings - poly(vinyl alcohol) (PVA) and poly(dimethylacrylamide) (PDMA) - both covalently covered - were used to separate DNA fragments and compared to DNA separation using replaceable polymer solutions. The separations were performed using hydroxyethylcellulose (HEC) (90-105 kDa) in concentrations ranging from 0.00 to 2.00% m/v. The results indicated that the separation efficiency was higher in the PVA capillary than in the PDMA in all evaluated concentrations of HEC. In addition, higher resolution was also observed in PVA-coated capillary since in PDMA the shape of the peaks was not reproducible when subsequent runs were performed. Contrary to what has previously been reported in the literature, no reasonable separation was possible in bare fused silica.

An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.

Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection

Background: It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins. Results: Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis. Conclusion: Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

An insight into the sialotranscriptome of the brown dog tick, Rhipicephalus sanguineus

Background: Rhipicephalus sanguineus, known as the brown dog tick, is a common ectoparasite of domestic dogs and can be found worldwide. R. sanguineus is recognized as the primary vector of the etiological agent of canine monocytic ehrlichiosis and canine babesiosis. Here we present the first description of a R. sanguineus salivary gland transcriptome by the production and analysis of 2,034 expressed sequence tags (EST) from two cDNA libraries, one consctructed using mRNA from dissected salivary glands from female ticks fed for 3-5 days (early to mid library, RsSGL1) and the another from ticks fed for 5 days (mid library, RsSGL2), identifying 1,024 clusters of related sequences. Results: Based on sequence similarities to nine different databases, we identified transcripts of genes that were further categorized according to function. The category of putative housekeeping genes contained similar to 56% of the sequences and had on average 2.49 ESTs per cluster, the secreted protein category contained 26.6% of the ESTs and had 2.47 EST's/clusters, while 15.3% of the ESTs, mostly singletons, were not classifiable, and were annotated as ""unknown function"". The secreted category included genes that coded for lipocalins, proteases inhibitors, disintegrins, metalloproteases, immunomodulatory and antiinflammatory proteins, as Evasins and Da-p36, as well as basic-tail and 18.3 kDa proteins, cement proteins, mucins, defensins and antimicrobial peptides. Comparison of the abundance of ESTs from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes, such as genes coding for Evasins and a thrombin inhibitor, which were over expressed in the RsSGL1 (early to mid library) versus RsSGL2 (mid library), indicating their role in inhibition of inflammation at the tick feeding site from the very beginning of the blood meal. Conversely, sequences related to cement (64P), which function has been correlated with tick attachment, was largely expressed in the mid library. Conclusions: Our survey provided an insight into the R. sanguineus sialotranscriptome, which can assist the discovery of new targets for anti-tick vaccines, as well as help to identify pharmacologically active proteins.

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8 +/- 25.2 nM and 167.39 +/- 60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4 +/- 15.9 nM to laminin and of 290.8 +/- 11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.