Marcelo de Oliveira Santos

Biotechnological potential of antimicrobial peptides from flowers

Flowers represent a relatively unexplored source of antimicrobial peptides of biotechnological potential. This review focuses on flower-derived defense peptide classes with inhibitory activity towards plant pathogens. Small cationic peptides display diverse activities, including inhibition of digestive enzymes and bacterial and/or fungal inhibition. Considerable research is ongoing in this area, with natural crop plant defense potentially improved through the application of transgenic technologies. In this report, comparisons were made of peptide tertiary structures isolated from diverse flower species. A summary is provided of molecular interactions between flower peptides and pathogens, which include the role of membrane proteins and lipids. Research on these peptides is contributing to our understanding of pathogen resistance mechanisms, which will, given the perspectives for plant genetic modification, contribute long term to plant genetic improvement for increased resistance to diverse pathogens.

Strategies and molecular tools to fight antimicrobial resistance: resistome, transcriptome, and antimicrobial peptides

The increasing number of antibiotic resistant bacteria motivates prospective research toward discovery of new antimicrobial active substances. There are, however, controversies concerning the cost-effectiveness of such research with regards to the description of new substances with novel cellular interactions, or description of new uses of existing substances to overcome resistance. Although examination of bacteria isolated from remote locations with limited exposure to humans has revealed an absence of antibiotic resistance genes, it is accepted that these genes were both abundant and diverse in ancient living organisms, as detected in DNA recovered from Pleistocene deposits (30,000 years ago). Indeed, even before the first clinical use of antibiotics more than 60 years ago, resistant organisms had been isolated. Bacteria can exhibit different strategies for resistance against antibiotics. New genetic information may lead to the modification of protein structure affecting the antibiotic carriage into the cell, enzymatic inactivation of drugs, or even modification of cellular structure interfering in the drug-bacteria interaction. There are still plenty of new genes out there in the environment that can be appropriated by putative pathogenic bacteria to resist antimicrobial agents. On the other hand, there are several natural compounds with antibiotic activity that may be used to oppose them. Antimicrobial peptides (AMPs) are molecules which are wide-spread in all forms of life, from multi-cellular organisms to bacterial cells used to interfere with microbial growth. Several AMPs have been shown to be effective against multi-drug resistant bacteria and have low propensity to resistance development, probably due to their unique mode of action, different from well-known antimicrobial drugs. These substances may interact in different ways with bacterial cell membrane, protein synthesis, protein modulation, and protein folding. The analysis of bacterial transcriptome may contribute to the understanding of microbial strategies under different environmental stresses and allows the understanding of their interaction with novel AMPs.

Antimicrobial activity of recombinant Pg-AMP1, a glycine-rich peptide from guava seeds

Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168 bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98 kDa as a monomer and 13.96 kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases.

Toward Personalized Cell Therapies by Using Stem Cells: Seven Relevant Topics for Safety and Success in Stem Cell Therapy

Stem cells, both embryonic and adult, due to the potential for application in tissue regeneration have been the target of interest to the world scientific community. In fact, stem cells can be considered revolutionary in the field of medicine, especially in the treatment of a wide range of human diseases. However, caution is needed in the clinical application of such cells and this is an issue that demands more studies. This paper will discuss some controversial issues of importance for achieving cell therapy safety and success. Particularly, the following aspects of stem cell biology will be presented: methods for stem cells culture, teratogenic or tumorigenic potential, cellular dose, proliferation, senescence, karyotyping, and immunosuppressive activity.

Role of SERK genes in plant environmental response

In plants, cell signaling connects the environmental input to the intracellular responses in plants. Exogenous signals play an important role in cell metabolism leading to growth and defense responses. Some of these stimuli induce anatomical and physiological modifications that are generally modulated by gene expression. SERK belongs to a small family of genes that code for a transmembrane protein involved in signal transduction and that have been strongly associated with somatic embryogenesis and apomixis in a number of plant species. Recent studies corroborate its role in somatic embryogenesis and suggest a broader range of functions in plant response to biotic and abiotic stimuli. This mini-review aims to present new data on SERK and discuss its involvement in plant development as well as in response to environmental stress.

Identification of a Passiflora alata Curtis dimeric peptide showing identity with 2S albumins.

Antifungal proteins and peptides, essential compounds for plant defense, have been isolated from several tissues of various plants. These proteins could be used as a natural alternative to control phytopathogenic fungi. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptidic molecular masses of approximately 4500 Da and 7000 Da, in the presence of β-mercaptoethanol, while by removing the reducing agent a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimer mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporioides, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi.

IN VITRO PROPAGATION OF ENDANGERED LIPPIA FILIFOLIA MART. AND SCHAUER EX SCHAUER

SummaryThis work describes an efficient micropropagation protocol of Lippia filifolia. Nodal segments cultivation in MS medium supplemented with 6-benzylaminopurine (4.5 μM)/α-naphthaleneacetic acid (NAA; 54nM) induced multiple shoots (in average 27 shoots per explant). Elongated shoots were rooted with NAA (0.11 μM) and they maintained ploidy level of the in vitro produced explants. The basic chromosome number were 2n=2x=24. Regenerated rooted shoots were successfully acclimatized under shading house conditions. This is the first report involving the establishment of a protocol for shoot multiplication and rooting for endangered L. filifolia, contributing for germplasm preservation of this species.

Occurrence of a conserved domain in ATP diphosphohydrolases from pathogenic organisms associated to antigenicity in human parasitic diseases

A polypeptide (r78-117) belonging to the potato apyrase was identified as a conserved domain shared with apyrase-like proteins from distinct pathogenic organisms, and was obtained as a 6xHis tag polypeptide (r-Domain B). By ELISA, high IgG, and IgG1 and IgG2a subtypes levels were detected in BALB/c mice pre-inoculated with r-Domain B. In Schistosoma mansoni adult worm or Leishmania (V.) braziliensis promastigote preparation, anti-r-Domain B antibodies inhibit 22-72% of the phosphohydrolytic activities and when immobilized on Protein A-Sepharose immunoprecipitate 42-91% of them. Western blots of the immunoprecipitated resin-antibody-antigen complexes identified bands of mw similar to those predicted for parasite proteins. Total IgG and subclasses of patients with leishmaniasis or schistosomiasis exhibited cross-immunoreactivity with r-Domain B. Therefore, the domain B within both S. mansoni SmATPDase 2 (r156-195) and L. (V.) braziliensis NDPase (r83-122) are potentially involved in the host immune response, and also seem to be conserved during host and parasites co-evolution.

Oil production at different stages of leaf development in Lippia alba

The aim of this work was to analyze terpene oil production and terpene synthases (TPS) gene expression from leaves at different developmental stages of different chemotypes of Lippia alba (Mill.) N.E. Br. ex Britton & P. Wilson, Verbenaceae. Hydro-distilled essential oil were used for chemical analysis and gene expression of three monoterpene synthase genes called LaTPS12, LaTPS23 and LaTPS25 were used for analyses of gene expression associated to oil production. The putative genes were associated to TPS-b gene class. Semi-quantitative PCR and quantitative PCR (qPCR) analysis were used to investigate the expression profile of those three putative genes in different leaf stages and different chemotypes. Additionally, total oil production and gene expression of putative TPS genes cloned from L. alba chemotype linalool were evaluated at different stages of leaf development. The expression level of those three genes was higher when the highest oil production was observed, mainly in young leaves at the fourth nodal segment for all evaluated chemotypes. Total oil production was higher at leaves that had unopened trichomes. We also observed that the 1mM of MeJA treatment increased the gene expression in all chemotypes after 24 h elicitation.

Embriogênese somática em híbridos de Pennisetum sp. e avaliação de estabilidade genômica por citometria

The objectives of this study were to establish an efficient protocol for somatic embryogenesis in triploid hybrids between napiergrass (Pennisetum purpureum Schumach.) and pearl millet (P. glaucum (L.) R. Br.), and to assess the genomic stability by flow cytometry of the plants obtained in vitro. Somatic embryogenesis and plant regeneration were successfully established from mature zygotic embryos of napiergrass and pearl millet hybrids. Four treatments with 2,4 dichlorophenoxyacetic acid (2,4-D) at 0, 1, 2 e 3 mg L-1 were tested for embryogenic calli induction and two treatments with inositol at 1 e 2 g L-1 were tested for plant regeneration. The treatments were arranged in a completely randomized design. The optimum hormone combinations were 2 mg L-1 of 2,4 D for embryogenic callus induction, and 1 g L-1 of inositol for embryos conversion and plant regeneration. The analysis of DNA content by flow cytometry of the regenerated plantlets indicated that no ploidy changes had been induced during somatic embryogenesis and plant regeneration. The nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous in comparison to those of the control plants. There was no occurrence of karyological instability in the regeneration system utilized for Pennisetum hybrid.