Elevated proline content in maize plants expressing a fragment of the proline dehydrogenase gene in antisense orientation

A fragment of the first exon of the proline dehydrogenase gene of arabidopsis (Arabidopsis thaliana L.) in antisense orientation was transferred to maize (Zea mays L.) genome by agrobacterial transformation in planta to elevate the content of free proline in maize cells. The presence of genetic structure carrying an antisense sequence of the fragment of the proline dehydrogenase gene (ASPG) from the genome of diploid maize seedlings was corroborated by PCR method. T-DNA insertions comprising ASPG were found in the genomes of 30 plants (1.2% of 2409 examined seedlings) belonging to the generation T0. A reliable 4.6-fold increase in proline content was registered in the leaves of transformed maize plants.     

Overexpression of the aspartic protease ASPG1 gene confers drought avoidance in Arabidopsis

Drought is one of the most severe environmental stresses affecting plant growth and limiting crop production. Although many genes involved in adaptation to drought stress have been disclosed, the relevant molecular mechanisms are far from understood. This study describes an Arabidopsis gene, ASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1), that may function in drought avoidance through abscisic acid (ABA) signalling in guard cells. Overexpression of the ASPG1 gene enhanced ABA sensitivity in guard cells and reduced water loss in ectopically overexpressing ASPG1 (ASPG1-OE) transgenic plants. In ASPG1-OE plants, some downstream targets in ABA and/or drought-signalling pathways were altered at various levels, suggesting the involvement of ASPG1 in ABA-dependent drought avoidance in Arabidopsis. By analysing the activities of several antioxidases including superoxide dismutase and catalase in ASPG1-OE plants, the existence was demonstrated of an effective detoxification system for drought avoidance in these plants. Analysis of ProASPG1-GUS lines showed a predominant guard cell expression pattern in various aerial tissues. Moreover, the protease activity of ASPG1 was characterized in vitro, and two aspartic acid sites, D180 and D379, were found to be key residues for ASPG1 aspartic protease activity in response to ABA. In summary, these findings suggest that functional ASPG1 may be involved in ABA-dependent responsiveness and that overexpression of the ASPG1 gene can confer drought avoidance in Arabidopsis.     

Differential Phenotypic and Functional Profiles of TcCA-2 -Specific Cytotoxic CD8+ T Cells in the Asymptomatic versus Cardiac Phase in Chagasic Patients

It has been reported that the immune response mediated by T CD8⁺ lymphocytes plays a critical role in the control of Trypanosoma cruzi infection and that the clinical symptoms of Chagas disease appear to be related to the competence of the CD8⁺ T immune response against the parasite. Herewith, in silico prediction and binding assays on TAP-deficient T2 cells were used to identify potential HLA-A*02:01 ligands in the T. cruzi TcCA-2 protein. The TcCA-2-specific CD8⁺ T cells were functionality evaluated by Granzyme B and cytokine production in peripheral blood mononuclear cells (PBMC) from Chagas disease patients stimulated with the identified HLA-A*02:01 peptides. The specific cells were phenotypically characterized by flow cytometry using several surface markers and HLA-A*02:01 APC-labeled dextramer loaded with the peptides. In the T. cruzi TcCA-2 protein four T CD8⁺ epitopes were identified which are processed and presented during Chagas disease. Interestingly, a differential cellular phenotypic profile could be correlated with the severity of the disease. The TcCA-2-specific T CD8⁺ cells from patients with cardiac symptoms are mainly effector memory cells (T_EM and T_EMRA) while, those present in the asymptomatic phase are predominantly naive cells (T_NAIVE). Moreover, in patients with cardiac symptoms the percentage of cells with senescence features is significantly higher than in patients at the asymptomatic phase of the disease. We consider that the identification of these new class I-restricted epitopes are helpful for designing biomarkers of sickness pathology as well as the development of immunotherapies against T. cruzi infection.     

Effects of Lactobacillus helveticus fermented milk on bone cells in vitro

Milk fermented with Lactobacillus helveticus (L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3-1.4 times with the 1 × 10⁻⁵, 1 × 10⁻⁴ and 1 × 10⁻³ solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.     

Antimicrobial activity of the Naja atra cathelicidin and related small peptides

We have identified an 11-residue pattern (KR(F/A)KKFFKK(L/P)K), which we have named the ATRA motif, within the sequence of the Chinese cobra (Naja atra) cathelicidin. A series of 11-residue peptides (ATRA-1, -2, -1A and -1P) were designed to probe the significance of the conserved residues within the ATRA motif, and their contributions to antimicrobial performance. The antimicrobial activities of the peptides were assessed against Escherichia coli K12 strain and Aggregatibacter actinomycetemcomitans Y4. ATRA-1 and -1A, demonstrated potencies comparable to that of N. atra cathelicidin. Structural examination by circular dichroism of the four short peptides suggested the significance of specific amino acid positions within the motif by their contribution to helicity. The results of these studies indicate that short peptides derived from the repeated ATRA motif from the N. atra cathelicidin can demonstrate both low toxicity against host cells and high antimicrobial activity against the gram-negative bacteria used in this study. They constitute novel, effective antimicrobial peptides that are much shorter (and thus less expensive to produce) than the natural cathelicidins, and they may represent new templates for therapeutic drug development.     

Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli

Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H₂O₂ stimulation.     

The substrate specificity of the protein kinase induced in cells infected with herpesviruses: Studies with synthetic substatres indicate structural requirements distinct from other protein kinases

Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with α-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simples virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP-dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by K_m values) were found to be very different.     

Establishment, characterization and immortalization of a fibroblast cell line from the Chinese red belly toad Bombina maxima skin

The skin of the amphibian Bombina maxima is rich in biologically active proteins and peptides, most of which have mammalian analogues. The physiological functions of most of the mammalian analogues are still unknown. Thus, Bombina maxima skin may be a promising model to reveal the physiological role of these proteins and peptides because of their large capacity for secretion. To investigate the physiological role of these proteins and peptides in vitro, a fibroblast cell line was successfully established from Bombina maxima tadpole skin. The cell line grew to form a monolayer with cells of a uniform shape and abundant rough endoplasmic reticulum, which are typical characteristics of fibroblasts. Further identification at a molecular level revealed that they strongly expressed the fibroblast marker protein vimentin. The chromosome number of these cells is 2n = 28, and most of them were diploid. Growth property analysis showed that they grew well for 14 passages. However, cells showed decreased proliferative ability after passage 15. Thus, we tried to immortalize the cells through the overexpression of SV40 T antigen. After selecting by G418, cells stably expressed SV40 large T antigen and showed enhanced proliferative ability and increased telomerase activity. Signal transduction analysis revealed functional p42 mitogen-activated protein (MAP) kinase in immortalized Bombina maxima dermal fibroblasts. Primary fibroblast cells and the immortalized fibroblast cells from Bombina maxima cultured in the present study can be used to investigate the physiological role of Bombina maxima skin-secreted proteins and peptides. In addition, the methods for primary cell culturing and cell immortalization will be useful for culturing and immortalizing cells from other types of amphibians.     

Fine-tuning the physicochemical properties of peptide-based blood–brain barrier shuttles

N-methylation is a powerful method to modify the physicochemical properties of peptides. We previously found that a fully N-methylated tetrapeptide, Ac-(N-MePhe)₄-CONH₂, was more lipophilic than its non-methylated analog Ac-(Phe)₄-CONH₂. In addition, the former crossed artificial and cell membranes while the latter did not. Here we sought to optimize the physicochemical properties of peptides and address how the number and position of N-methylated amino acids affect these properties. To this end, 15 analogs of Ac-(Phe)₄-CONH₂ were designed and synthesized in solid-phase. The solubility of the peptides in water and their lipophilicity, as measured by ultra performance liquid chromatography (UPLC) retention times, were determined. To study the permeability of the peptides, the Parallel Artificial Membrane Permeability Assay (PAMPA) was used as an in vitro model of the blood–brain barrier (BBB). Contrary to the parent peptide, the 15 analogs crossed the artificial membrane, thereby showing that N-methylation improved permeability. We also found that N-methylation enhanced lipophilicity but decreased the water solubility of peptides. Our results showed that both the number and position of N-methylated residues are important factors governing the physicochemical properties of peptides. There was no correlation between the number of N-methylated amide bonds and any of the properties measured. However, for the peptides consecutively N-methylated from the N-terminus to the C-terminus (p1, p5, p11, p12 and p16), lipophilicity correlated well with the number of N-methylated amide bonds and the permeability of the peptides. Moreover, the peptides were non-toxic to HEK293T cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.     

Differential gene expression associated with flower development of mango (Mangifera indica L.) varieties with different shelf-life

Mango (Mangifera indica L.) is one of the most important commercial fruit crop grown in many parts of the world. Major challenges affecting mango trade are short shelf-life, high susceptibility to chilling injury, post-harvest diseases and consumer demand for improved fruit quality. The objective of the present study was to reveal the key regulators present in bud and flower tissues during flower development stage, associated with fruit development and affect the shelf-life of the mango fruit. RNA-sequencing of contrasting genotypes having short and long shelf-life, was carried out. Comparative differential expression pathway studies of long shelf-life (Totapuri) and short shelf-life (Bombay Green) mango genotypes revealed a total of 177 highly differentially expressed genes. Out of 177 total genes, 101 genes from endoplasmic reticulum pathway and very few from gibberellins (3) and jasmonic acid (1) pathway were identified. Genes from endoplasmic reticulum pathway like hsp 90, SRC2, DFRA, CHS, BG3 and ASPG1 mainly up regulated in Bombay Green. Uniprotein B9R8D3 also shows up regulation in Bombay Green. Ethylene insensitive pathway gene EIL1 up regulated in Bombay Green. Gene CAD1 from phenylpropanoid pathway mainly up regulated in Bombay Green. A total of 4 SSRs and 227 SNPs were mined from these pathways specific to the shelf-life. Molecular studies of endoplasmic reticulum, phenylpropanoid, ethylene, polygalacturonase and hormone pathways at the time of bud and flower formation revealed key regulators that determine the shelf-life of mango fruit.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Exploratory Study on Th1 Epitope-Induced Protective Immunity against Coxiella burnetii Infection

Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. In the present study, 131 candidate peptides were selected from the major immunodominant proteins (MIPs) of C. burnetii due to their high-affinity binding capacity for the MHC class II molecule H2 I-A^b based on bioinformatic analyses. Twenty-two of the candidate peptides with distinct MIP epitopes were well recognized by the IFN-γ recall responses of CD4⁺ T cells from mice immunized with parental proteins in an ELISPOT assay. In addition, 7 of the 22 peptides could efficiently induce CD4⁺ T cells from mice immunized with C. burnetii to rapidly proliferate and significantly increase IFN-γ production. Significantly higher levels of IL-2, IL-12p70, IFN-γ, and TNF-α were also detected in serum from mice immunized with a pool of the 7 peptides. Immunization with the pool of 7 peptides, but not the individual peptides, conferred a significant protection against C. burnetii infection in mice, suggesting that these Th1 peptides could work together to efficiently activate CD4⁺ T cells to produce the Th1-type immune response against C. burnetii infection. These observations could contribute to the rational design of molecular vaccines for Q fever.     

Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells

Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.     

The Antimicrobial Domains of Wheat Puroindolines Are Cell-Penetrating Peptides with Possible Intracellular Mechanisms of Action

The puroindoline proteins (PINA and PINB) of wheat display lipid-binding properties which affect the grain texture, a critical parameter for wheat quality. Interestingly, the same proteins also display antibacterial and antifungal properties, attributed mainly to their Tryptophan-rich domain (TRD). Synthetic peptides based on this domain also display selectivity towards bacterial and fungal cells and do not cause haemolysis of mammalian cells. However, the mechanisms of these activities are unclear, thus limiting our understanding of the in vivo roles of PINs and development of novel applications. This study investigated the mechanisms of antimicrobial activities of synthetic peptides based on the TRD of the PINA and PINB proteins. Calcein dye leakage tests and transmission electron microscopy showed that the peptides PuroA, Pina-M and Pina-W→F selectively permeabilised the large unilamellar vesicles (LUVs) made with negatively charged phospholipids mimicking bacterial membranes, but were ineffective against LUVs made with zwitterionic phospholipids mimicking eukaryotic membranes. Propidium iodide fluorescence tests of yeast (Saccharomyces cerevisiae) cells showed the peptides were able to cause loss of membrane integrity, PuroA and Pina-M being more efficient. Scanning electron micrographs of PINA-based peptide treated yeast cells showed the formation of pits or pores in cell membranes and release of cellular contents. Gel retardation assays indicated the peptides were able to bind to DNA in vitro, and the induction of filamental growth of E. coli cells indicated in vivo inhibition of DNA synthesis. Together, the results strongly suggest that the PIN-based peptides exert their antimicrobial effects by pore formation in the cell membrane, likely by a carpet-like mechanism, followed by intracellular mechanisms of activity.