A novel antimicrobial peptide from Crotalaria pallida seeds with activity against human and phytopathogens.

An actual severe problem in agriculture consists of an expressive increase of economical losses caused by fungi and resistant bacteria toward antibiotics. In order to find a solution to this problem, several studies have been concentrating on the screening of novel plant defense peptides with antimicrobial activities. These peptides are commonly characterized by having low molecular masses and cationic charges. The present work reports the purification and characterization of a novel plant peptide with molecular mass of 5340 Da, named Cp-AMP, from seeds of C. pallida, a typical plant from Caatinga biome. Purification was achieved using a size exclusion S-200 column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Cp-AMP was able to inhibit the development of filamentous fungi Fusarium oxysporum as well as the gram-negative bacterium Proteus sp. The identification of Cp-AMP could contribute, in the near future, to the development of biotechnological products, such as transgenic plants with enhanced resistance to pathogenic fungi and/or of antibiotics production derived from plant sources in order to control bacterial infections.

Novel insights on the mechanism of action of alpha-amylase inhibitors from the plant defensin family.

Plant defensins are small cysteine-rich proteins commonly synthesized in plants, encoded by large multigene families. Most plant defensins that have been characterized to date show potent antifungal and/or bactericidal activities. This report describes VuD1, an unusual defensin that is able to inhibit insect-pest alpha-amylases. VuD1 was cloned from cowpea (Vigna unguiculata) seeds and expressed in a heterologous system. Inhibitory enzyme assays showed that VuD1 efficiently inhibits alpha-amylases from the weevils Acanthoscelides obtectus and Zabrotes subfasciatus, caused low inhibition toward mammalian enzymes and was unable to inhibit the alpha-amylases from Callosobruchus maculatus and Aspergillus fumigatus. To shed some light over the mechanism of action of VuD1, molecular modeling analyses were performed, revealing that the N-terminus of the molecule is responsible for binding with the active site of weevil enzymes. Moreover, models of VuD1 and mammalian enzymes were also generated to elucidate the specificity mechanisms. The data presented herein suggests that this defensin has potential application in the development of transgenic plants for insect pest control.

Identification of a novel storage glycine-rich peptide from guava (Psidium guajava) seeds with activity against Gram-negative bacteria.

Bacterial pathogens cause an expressive negative impact worldwide on human health, with ever increasing treatment costs. A significant rise in resistance to commercial antibiotics has been observed in pathogenic bacteria responsible for urinary and gastro-intestinal infections. Towards the development of novel approaches to control such common infections, a number of defense peptides with antibacterial activities have been characterized. In this report, the peptide Pg-AMP1 was isolated from guava seeds (Psidium guajava) and purified using a Red-Sepharose Cl-6B affinity column followed by a reversed-phase HPLC (Vydac C18-TP). Pg-AMP1 showed no inhibitory activity against fungi, but resulted in a clear growth reduction in Klebsiella sp. and Proteus sp., which are the principal pathogens involved in urinary and gastro-intestinal hospital infections. SDS-PAGE and mass spectrometry (MALDI-ToF) characterized Pg-AMP1 a monomer with a molecular mass of 6029.34Da and small quantities of a homodimer. Amino acid sequencing revealed clear identity to the plant glycine-rich protein family, with Pg-AMP1 the first such protein with activity towards Gram-negative bacteria. Furthermore, Pg-AMP1 showed a 3D structural homology to an enterotoxin from Escherichia coli, and other antibacterial proteins, revealing that it might act by formation of a dimer. Pg-AMP1 shows potential, in a near future, to contribute to development of novel antibiotics from natural sources.

Plant cyclotides: an unusual class of defense compounds.

Plant cyclotides are unusual peptides with low molecular masses and a three-dimensional structure characterized by the presence of a cyclic fold. Synthetic peptides can adopt this circular conformation, but it is not a common feature for most members of other peptide groups. Cyclotides present a wide range of functions, such as the ability to induce stronger contractions during childbirth and anti-tumor activity. Additionally, some cyclotides present anti-viral, insecticidal or proteinase inhibitory activity. In this paper, we describe the structural and functional characteristics of plant cyclotides, their most conserved features and the development of these peptides for human health and biotechnological applications.

Molecular identification of four different alpha-amylase inhibitors from baru (Dipteryx alata) seeds with activity toward insect enzymes.

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of alpha-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, alpha-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis alpha-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus alpha-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed alpha-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several alpha-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.

Anti-parasitic Peptides from Arthropods and their Application in Drug Therapy.

Africa, Asia, and Latin America are regions highly affected by endemic diseases, such as Leishmaniasis, Malaria, and Chagas' disease. They are responsible for the death of 1000s of patients every year, as there is not yet a cure for them and the drugs used are inefficient against the pathogenic parasites. During the life cycle of some parasitic protozoa, insects become the most important host and disseminator of the diseases triggered by these microorganisms. As infected insects do not develop nocive symptoms, they can carry the parasites for long time inside their body, enabling their multiplication and life cycle completion. Eventually, parasites infect human beings after insect's transmission through their saliva and/or feces. Hence, host insects and general arthropods, which developed a way to coexist with such parasites, are a promising source for the prospection of anti-parasitic compounds, as alternative methods for the treatment of protozoa-related diseases. Among the molecules already isolated and investigated, there are proteins and peptides with high activity against parasites, able to inhibit parasite activity in different stages of development. Although, studies are still taking their first steps, initial results show new perspectives on the treatment of parasitic diseases. Therefore, in this report, we describe about peptides from host insect sources with activity against the three most endemic parasites: Leishmania sp., Plasmodium sp., and Trypanosomes. Moreover, we discuss the future application insect peptides as anti-parasitic drugs and the use of non-hosts insect transcriptomes on the prospection of novel molecules for the treatment of parasitic neglected diseases.

Plant gamma-thionins: novel insights on the mechanism of action of a multi-functional class of defense proteins.

This review focuses on the first plant defense protein class described in literature, with growth inhibition activity toward pathogens. These peptides were named gamma-thionins or defensins, which are small proteins that can be classified into four main subtypes according to their specific functions. Gamma-thionins are small cationic peptides with different and special abilities. They are able to inhibit digestive enzymes or act against bacteria and/or fungi. Current research in this area focuses particularly these two last targets, being the natural crop plant defenses improved through the use of transgenic technology. Here, we will compare primary and tertiary structures of gamma-thionins and also will analyze their similarities to scorpion toxins and insect defensins. This last comparison offers some hypothesis for gamma-thionins mechanisms of action against certain pathogens. This specific area has benefited from the recent determination of many gamma-thionin structures. Furthermore, we also summarize molecular interactions between plant gamma-thionins and fungi receptors, which include membrane proteins and lipids, shedding some light over pathogen resistance. Researches on gamma-thionins targets could help on plant genetic improvement for production of increased resistance toward pathogens. Thus, positive results recently obtained for transgenic plants and future prospects in the area are also approached. Finally, gamma-thionins activity has also been studied for future drug development, capable of inhibit tumor cell growth in human beings.

Recombinant glucagon: a differential biological activity.

In Brazil, there is a growing demand for specialised pharmaceuticals, and the high cost of their importation results in increasing costs, reaching US$ 1.34 billion in 2012 and US$ 1.61 billion in 2013. Worldwide expenses related to drugs could reach US$ 1.3 trillion in 2018, especially due to new treatments for hepatitis C and cancer. Specialised or high-cost pharmaceutical drugs used for the treatment of viral hepatitis, multiple sclerosis, HIV and diabetes are distributed free of charge by the Brazilian government. The glucagon peptide was included in this group of high-cost biopharmaceuticals in 2008. Although its main application is the treatment of hypoglycaemia in diabetic patients, it can also be used with patients in an alcoholic coma, for those patients with biliary tract pain, and as a bronchodilator. Therefore, in order to reduce biopharmaceutical production costs, the Brazilian government passed laws focusing on the development and increase of a National Pharmaceutical Industrial Centre, including the demand for the national production of glucagon. For that reason and given the importance and high cost of recombinant glucagon, the purpose of this study was to develop methods to improve production, purification and performance of the biological activity of recombinant glucagon. Glucagon was recombined into a plasmid vector containing a Glutathione S-transferase tag, and the peptide was expressed in a heterologous Escherichia coli system. After purification procedures and molecular analyses, the biological activity of this recombinant glucagon was examined using in vivo assays and showed a highly significant (p < 0.00001) and prolonged effect on glucose levels when compared with the standard glucagon. The experimental procedure described here facilitates the high level production of recombinant glucagon with an extended biological activity.

Molecular approaches to improve the insecticidal activity of Bacillus thuringiensis Cry toxins.

Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.

Proteomic evaluation of coffee zygotic embryos in two different stages of seed development.

Coffee seed development is accompanied by severe modifications in water soluble proteins, several of these being associated to a specific developmental stage. For this reason, a proteomic approach has been used to describe spatial-temporal proteome modifications in zygotic embryos at different stages of seed development. Embryos from Coffea arabica seeds were harvested in two different developmental stages: stage 1 at 210 days after anthesis and stage 2 at 255 days. Total proteins were extracted and submitted to 2-DE. From these gels, several spots were identified by mass spectrometry including kinases, MYB transcription factor and enzymes involved in metabolic pathways. All proteins identified seem to affect coffee development in different ways, being directly involved in plant growth or used as an intermediate in some metabolic pathway that, indirectly, will influence coffee development. This is the first work using two-dimensional electrophoresis followed by mass spectrometry analyses that evaluates the expression of proteins during coffee zygotic embryos development. Data here reported supply some light over coffee development and could be used in a near future to improve coffee plants' growth and development by molecular strategies.

Structure and enzyme properties of Zabrotes subfasciatus alpha-amylase.

Digestive alpha-amylases play an essential role in insect carbohydrate metabolism. These enzymes belong to an endo-type group. They catalyse starch hydrolysis, and are involved in energy production. Larvae of Zabrotes subfasciatus, the Mexican bean weevil, are able to infest stored common beans Phaseolus vulgaris, causing severe crop losses in Latin America and Africa. Their alpha-amylase (ZSA) is a well-studied but not completely understood enzyme, having specific characteristics when compared to other insect alpha-amylases. This report provides more knowledge about its chemical nature, including a description of its optimum pH (6.0 to 7.0) and temperature (20-30 degrees C). Furthermore, ion effects on ZSA activity were also determined, showing that three divalent ions (Mn2+, Ca2+, and Ba2+) were able to enhance starch hydrolysis. Fe2+ appeared to decrease alpha-amylase activity by half. ZSA kinetic parameters were also determined and compared to other insect alpha-amylases. A three-dimensional model is proposed in order to indicate probable residues involved in catalysis (Asp204, Glu240, and Asp305) as well other important residues related to starch binding (His118, Ala206, Lys207, and His304).