Marcília P. Costa

Molecular characterization of the Corynebacterium pseudotuberculosis hsp60-hsp10 operon, and evaluation of the immune response and protective efficacy induced by hsp60 DNA vaccination in mice

BackgroundHeat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. We identified and characterized the hsp60-hsp10 bicistronic operon of the animal pathogen Corynebacterium pseudotuberculosis, a Gram-positive bacterium of the class Actinobacteria, which causes caseous lymphadenitis (CLA) in small ruminants.FindingsTo construct the DNA vaccine, the hsp60 gene of C. pseudotuberculosis was cloned in a mammalian expression vector. BALB/c mice were immunized by intramuscular injection with the recombinant plasmid (pVAX1/hsp60).ConclusionThis vaccination induced significant anti-hsp60 IgG, IgG1 and IgG2a isotype production. However, immunization with this DNA vaccine did not confer protective immunity.

Preclinical anticancer effectiveness of a fraction from Casearia sylvestris and its component Casearin X : in vivo and ex vivo methods and microscopy examinations

ETHNOPHARMACOLOGICAL RELEVANCE Casearia sylvestris (Salicaceae) is found in South America and presents antiulcerogenic, cytotoxic, antimicrobial, anti-inflammatory and antihypertensive activities. AIM OF THE STUDY To assess the in vivo and ex vivo antitumor action of a fraction with casearins (FC) and its main component - Casearin X-isolated from C. sylvestris leaves. MATERIALS AND METHODS Firstly, Sarcoma 180 bearing Swiss mice were treated with FC and Cas X for 7 days. Secondly, BALB/c nude animals received hollow fibers with colon carcinoma (HCT-116) or glioblastoma (SF-295) cells and were treated with FC for 4 days. On 5th day, proliferation was determined by MTT assay. RESULTS FC 10 and 25mg/kg/day i.p. and 50mg/kg/day oral and Cas X 25mg/kg/day i.p. and 50mg/kg/day oral revealed tumor growth inhibition rates of 35.8, 86.2, 53.7, 90.0 and 65.5% and such tumors demonstrated rare mitoses and coagulation necrosis areas. Similarly, FC reduced multiplying of HCT-116 and SF-295 cells when evaluated by the Hollow Fiber Assay (2.5 and 5mg/kg/day i.p. and 25 and 50mg/kg/day oral), with cell growth inhibition rates ranging from 33.3 to 67.4% (p<0.05). Flow cytometry experiments revealed that FC reduced membrane integrity and induced DNA fragmentation and mitochondrial depolarization (p<0.05). CONCLUSIONS FC and Cas X were efficient antitumor substances against murine and human cancer cells and caused reversible morphological changes in liver, kidneys and spleens, emphasizing clerodane diterpenes as an emerging class of anticancer molecules.

Homology modeling, molecular dynamics and QM/MM study of the regulatory protein PhoP from Corynebacterium pseudotuberculosis

Corynebacterium pseudotuberculosis is a facultatively intracellular Gram-positive bacterium that causes caseous lymphadenitis, principally in sheep and goats, though sometimes in other species of animals, leading to considerable economic losses. This pathogen has a TCS known as PhoPR, which consists of a sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP). This system is involved in the regulation of proteins present in various processes, including virulence. The regulation is activated by PhoP protein phosphorylation, an event that requires a magnesium (Mg2+) ion. Here we describe the 3D structure of the regulatory response protein (PhoP) of C. pseudotuberculosis through molecular modeling by homology. The model generated provides the first structural information on a full-length member of the OmpR/PhoP subfamily. Classical molecular dynamics was used to investigate the stability of the model. In addition, we used quantum mechanical/molecular mechanical techniques to perform (internal, potential) energy optimizations to determine the interaction energy between the Mg2+ ion and the structure of the PhoP protein. Analysis of the interaction energy residue by residue shows that Asp-16 and Asp-59 play an important role in the protein–Mg2+ ion interactions. These results may be useful for the future development of a new vaccine against tuberculosis based on genetic attenuation via a point mutation that results in the polar residue Asp-16 and/or Asp-59 being replaced with a nonpolar residue in the DNA-binding domain of PhoP of C. pseudotuberculosis.

Controlled Release of Nor-β-lapachone by PLGA Microparticles: A Strategy for Improving Cytotoxicity against Prostate Cancer Cells.

Prostate cancer is one of the most common malignant tumors in males and it has become a major worldwide public health problem. This study characterizes the encapsulation of Nor-β-lapachone (NβL) in poly(d,l-lactide-co-glycolide) (PLGA) microcapsules and evaluates the cytotoxicity of the resulting drug-loaded system against metastatic prostate cancer cells. The microcapsules presented appropriate morphological features and the presence of drug molecules in the microcapsules was confirmed by different methods. Spherical microcapsules with a size range of 1.03 ± 0.46 μm were produced with an encapsulation efficiency of approximately 19%. Classical molecular dynamics calculations provided an estimate of the typical adsorption energies of NβL on PLGA. Finally, the cytotoxic activity of NβL against PC3M human prostate cancer cells was demonstrated to be significantly enhanced when delivered by PLGA microcapsules in comparison with the free drug.

Following the Viterbi Path to Deduce Flagellar Actin‐Interacting Proteins of Leishmania spp.: Report on Cofilins and Twinfilins

For performing vital cellular processes, such as motility, eukaryotic cells rely on the actin cytoskeleton, whose structure and dynamics are tightly controlled by a large number of actin‐interacting (AIP) or actin‐related/regulating (ARP) proteins. Trypanosomatid protozoa, such as Leishmania, rely on their flagellum for motility and sensory reception, which are believed to allow parasite migration, adhesion, invasion and even persistence on mammalian host tissues to cause disease. Actin can determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes, while the identification and analyses of AIPs can help to improve understanding of their mechanical properties on physiological architectures, such as the present case regarding Leishmania flagellar apparatus. This work conveniently apply bioinformatics tools in some refined pattern recognition techniques (such as hidden Markov models (HMMs) through the Viterbi algorithm/path) in order to...

Hidden Markov models and the Viterbi algorithm applied to integrated bioinformatics analyses of putative flagellar actin-interacting proteins in Leishmania spp.

For performing vital cellular processes, such as motility, eukaryotic cells rely on the actin cytoskeleton, whose structure and dynamics are tightly controlled by a large number of actin-interacting proteins (AIPs). Actin can determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes, while the identification and analyses of AIPs can help to improve understanding of their mechanical properties on physiological/pathological architectures. In this work we employ bioinformatics tools in some refined pattern recognition techniques (such as hidden Markov models (HMMs) through the Viterbi algorithm/path) in order to improve the recognition of actin-binding/interacting activity through identification of AIPs in genomes, transcriptomes and proteomes of the flagellated protozoan Leishmania. We report our in silico analyses on cofilin and twinfilin, here predicted as flagellar proteins, a direct bioinformatics contribution in the secondary annotation of Leishmania genomes.

Actin-interacting and flagellar proteins in Leishmania spp: bioinformatics predictions to functional assignments in phagosome formation

Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented.

Actin-Interacting Proteins in flagellated pathogenic Leishmania spp.: a genome-based bioinformatics report on profilins, formins and katanins

In search for genomic and proteomic evidences of flagellar genes/proteins in Leishmania spp., we have used available databases and bioinformatics tools to distinguish Actin-Interacting Proteins (AIPs) that are flagellar and, also, probable virulence factors. Here we present results of sequence and structural analyses of profilins, formins and katanins, whose sequences were in silico selected for predicting viable roles on flagellum assembly, disassembly and dynamics in terms of intraflagellar mechanisms. Taken together, our results provide the first bioinformatics analyses of Leishmania profilin, katanin and formin genes and their gene products to contribute to a more detailed annotation of these important AIPs.

Flagellar proteins prediction after sequence-structure alignments of coronin and Arp2/3 complex in Leishmania spp.

Leishmania are uniflagellate protozoa, whose flagellum plays a key role in motility, being essential for parasite migration, invasion and persistence on host tissues. Actin is the major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin motor-associated proteins during both processes of motility and host cell entry might be key events for successful infection. In search for genomic and/or proteomic evidences of actin-interacting proteins (AIPs) involved in flagellar activities of Leishmania spp., we have applied computational tools (hidden Markov models, Viterbi algorithm and comparative modeling) to infer biological meaning through detailed sequence-structural-functional analyses on Leishmania AIPs, such as coronin and Arp2/3 complex proteins, two important elements on phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first integrated bioinformatics analyses of Leishmania coronin and Arp2/3 genes and their gene products, a direct contribution to genome annotation of these important actin-regulating proteins yet to be properly in vitro characterized in flagellate trypanosomatids towards their putative virulence.

Biopolymer from Adenanthera pavonina L. Seeds: Characterization, Photostability, Antioxidant Activity, and Biotoxicity Evaluation

Plant polysaccharides have been increasingly employed in the pharmaceutical, industrial, and food environments due to their versatile functional properties. In the present investigation, a heteropolysaccharide galactomannan (GAP) was extracted from Adenanthera pavonina L. seeds and characterized by physicochemical analyses to determine its thermal properties, photostability, antioxidant activity, and acute toxicity. GAP was characterized by FTIR, DSC, and TG. The photostability of GAP submitted to artificial UV irradiation was analyzed. Antioxidant activity was evaluated by the DPPH (2,2-diphenyl-1-1-picrylhydrazyl) free radical-scavenging method, while a bioassay method was carried out to study acute toxicity in Artemia salina L. Physical-chemical and functional characteristics of GAP support its potential role in the food and pharmaceutical industries. GAP was photostable under UV irradiation. In vitro GAP antioxidant evaluation showed that it bears free radical-scavenging activity for DPPH radicals. The median lethal concentration (LC50) of GAP was 239.4 mg∙mL−1, indicating that this biopolymer is nontoxic. Such results indicate that this biopolymer presents characteristics of neutrality, photostability, and nontoxicity that are commercially attractive.