ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

A new scenario of bioprospecting of Hymenoptera venoms through proteomic approach

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

Immunomodulatory effects of tick saliva

Ticks are bloodsucking ectoparasites that cause great damage to host organisms, so these ectoparasites are of great importance in medicine and veterinary medicine. All the biological success achieved by ticks is due to the action of bioactive components present in their saliva, which are synthesized by the salivary glands. These components have great diversity of functions such as enabling feeding and the permanence of ectoparasites on hosts, since they modulate immune system acting as complement inhibitors, immunosuppressors, cytokine expression modulator and chemokine binders of hosts. In addition, these components are an important source of protective antigens. In this sense, salivary glands/saliva are considered a potential source of multifunctional molecules. In this context, many studies have been conducted aiming at searching to establish a better understanding on the biology and morphophysiology of some organs such as salivary glands, as well as elucidate the complex relationship of these ectoparasites with their hosts. Such studies are conducted with the main objective of developing new immunobiological products aimed at the alternative control of ticks, as well as for the identification and isolation of bioactive molecules with pharmacological properties and with great therapeutic potential in the search for treatments for some diseases.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

Background: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli ( Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.Results: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside ( IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.Conclusion: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

The phylogenetic relationships of the charismatic poster frogs, Phyllomedusinae (Anura, Hylidae)

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

Computer-aided design of a novel ligand for retinoic acid receptor in cancer chemotherapy

The isotypes of RAR and RXR are retinoic acid and retinoid X acid receptors, respectively, whose ligand-binding domain contains the ligand-dependent activation function, with distinct pharmacological targets for retinoids, involved in the treatment of various cancers and skin diseases. Due to the major challenge which cancer treatment and cure still imposes after many decades to the international scientific community, there is actually considerable interest in new ligands with increased bioactivity. We have focused on the retinoid acid receptor, which is considered an interesting target for drug design. In this work, we carried out density functional geometry optimizations, and different docking procedures. We performed screening in a large database (hundreds of thousands of molecules which we optimized at the AM1 level) yielding a set of potential bioactive ligands. A new ligand was selected and optimized at the B3LYP/6-31G* level. A flexible docking program was used to investigate the interactions between the receptor and the new ligand. The result of this work is compared with several crystallographic ligands of RAR. Our theoretically more bioactive new-ligand indicates stronger and more hydrogen bonds as well as hydrophobic interactions with the receptor. (c) 2005 Wiley Periodicals, Inc.

New sources of oilseeds from Latin American native fruits

Three Latin American oilseeds obtained from native fruits: nopal (tuna) (Opuntia ficus-indica), cherimoya (chirimoya) (Annona cherimola), and papaya, Chilean variety (Carica pubescens or C. candamarcensis) were studied for their fatty acid composition and bioactive compounds, such as tocols and phytosterols, looking for new sources of special oilseeds for this region. The results indicated that each species represents an interesting possibility. Nopal oilseed is a good source of linoleic acid (62%), with a good balance between SFA and MUFA (1: 1.3). Cherimoya oilseed presents quite a different composition, with 24% SFA, 43% MUFA and 33% PUFA. Palmitic and stearic acids (15% and 7.6%, respectively) are the main SFA. A good balance between oleic acid (42.7%) and linoleic acid (31%) was observed. Papaya oilseed is a highly MUFA oil (72% with 71% oleic acid), with a very interesting composition, according to the new nutritional and technological recommendations.With respect to bioactive compounds, the main tocol in these three oilseeds was gamma-tocopherol, with 136, 300 and 317 mg/kg for cherimoya, papaya and nopal oilseeds, respectively. According to the total tocol content, papaya oilseed presented the highest value with 384 mg/kg. The total amount and distribution of phytosterols was different, with values of 3092, 3554 and 5474 mg/kg for nopal, cherimoya and papaya oilseeds, respectively, with beta-sitosterol forming 47.6%, 65.0 % and 78.7% of the total phytosterol fractions, respectively. From the results obtained, Chilean papaya oilseed can be considered as a very promising new source of special plant oil for different applications, followed by cherimoya and nopal oilseeds.

A multi-screening approach for marine-derived fungal metabolites and the isolation of cyclodepsipeptides from Beauveria felina

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

Desenvolvimento e caracterização de suportes porosos de polietileno de ultra alto peso molecular (PEUAPM) para utilização como biomaterial para reposição e regeneração óssea

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

Dihydroisocoumarins produced by Xylaria sp and Penicillium sp., endophytic fungi associated with Piper aduncum and Alibertia macrophylla

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

Cytotoxic Clerodane Diterpenoids from Casearia obliqua

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

Natural products from Brazilian biodiversity as a source of new models for medicinal chemistry

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

Geranylation of benzoic acid derivatives by enzymatic extracts from Piper crassinervium (Piperaceae)

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

Folk uses and pharmacological properties of Casearia sylvestris: a medicinal review

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