The pathogen Moniliophthora perniciosa promotes differential proteomic modulation of cacao genotypes with contrasting resistance to witches´ broom disease.

BACKGROUND: Witches' broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction. RESULTS: A total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions. CONCLUSIONS: We present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype.

Study of small proline-rich proteins (SPRRs) in health and disease: a review of the literature.

Epithelial tissues are specialized to protect underlying tissues from environmental influences such as physical and chemical agents, infection by invasive microorganisms as well as water and heat loss. They are grouped into simple, transitional and stratified epithelia, which line the cavities and surfaces of structures throughout the body, and also form glands, separate compartments, regulate the exchange of molecules and act as sensory organs. Stratified epithelia such as the epidermis and the gingival and hard palate mucosa are in constant renewal, with cells proliferating in the lower layers, while the intermediate stratum and outermost layers undergo a tissue-specific process of differentiation to form a protective cornified barrier. This review focuses on a subclass of structural proteins, the small proline-rich proteins (SPRRs), which constitute cornified cell envelope precursors. Several studies have suggested that the SPRRs are related to increased epithelial proliferation and to malignant processes. Hence, we also review the literature for more extensive and in-depth profile of these proteins in cancer and other diseases. The understanding of SPRR functions has advanced in recent years, but many important questions about their role in pathophysiological processes remain unanswered, which stimulate new studies and approaches.

Proline rich-oligopeptides: diverse mechanisms for antihypertensive action.

Bradykinin-potentiating peptides from Bothrops jararaca (Bj) discovered in the early 1960s, were the first natural inhibitors of the angiotensin-converting enzyme (ACE). These peptides belong to a large family of snake venom proline-rich oligopeptides (PROs). One of these peptides, Bj-PRO-9a, was essential for defining ACE as effective drug target and development of captopril, an active site-directed inhibitor of ACE used worldwide for the treatment of human arterial hypertension. Recent experimental evidences demonstrated that cardiovascular effects exerted by different Bj-PROs are due to distinct mechanisms besides of ACE inhibition. In the present work, we have investigated the cardiovascular actions of four Bj-PROs, namely Bj-PRO-9a, -11e, -12b and -13a. Bj-PRO-9a acts upon ACE and BK activities to promote blood pressure reduction. Although the others Bj-PROs are also able to inhibit the ACE activity and to potentiate the BK effects, our results indicate that antihypertensive effect evoked by them involve new mechanisms. Bj-PRO-11e and Bj-PRO-12b involves induction of [Ca(2+)]i transients by so far unknown receptor proteins. Moreover, we have suggested argininosuccinate synthetase and M3 muscarinic receptor as targets for cardiovascular effects elicited by Bj-PRO-13a. In summary, the herein reported results provide evidence that Bj-PRO-mediated effects are not restricted to ACE inhibition or potentiation of BK-induced effects and suggest different actions for each peptide for promoting arterial pressure reduction. The present study reveals the complexity of the effects exerted by Bj-PROs for cardiovascular control, opening avenues for the better understanding of blood pressure regulation and for the development of novel therapeutic approaches.

Molecular characterization of Trypanosoma cruzi SAP proteins with host-cell lysosome exocytosis-inducing activity required for parasite invasion.

BACKGROUND: To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. METHODS AND FINDINGS: Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. CONCLUSIONS: This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization.

Antifreeze glycoprotein agents: structural requirements for activity.

Antifreeze glycoproteins (AFGPs) are considered to be the most efficient means to reduce ice damage to cell tissues since they are able to inhibit growth and crystallization of ice. The key element of antifreeze proteins is to act in a non-colligative manner which allows them to function at concentrations 300-500 times lowers than other dissolved solutes. During the past decade, AFGPs have demonstrated tremendous potential for many pharmaceutical and food applications. Presently, the only route to obtain AFGPs involves the time consuming and expensive process of isolation and purification from deep-sea polar fishes. Unfortunately, it is not amenable to mass production and commercial applications. The lack of understanding of the mechanism through which the AFGPs inhibit ice growth has also hampered the realization of industrial and biotechnological applications. Here we report the structural motifs that are essential for antifreeze activity of AFGPs, and propose a unified mechanism based on both recent studies of short alanine peptides and structure activity relationship of synthesized AFGPs.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B2and M1 muscarinic acetylcholine receptors.

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (

Enhancement of the citrulline-nitric oxide cycle in astroglioma cells by the proline-rich peptide-10c from Bothrops jararaca venom.

The biological activity of the proline-rich decapeptide Bj-PRO-10c, a processing product of the C-type natriuretic peptide precursor protein, expressed in the brain and the venom gland of the pit viper Bothrops jararaca, was originally attributed to the inhibition of the somatic angiotensin-converting enzyme activity with subsequent anti-hypertensive effect. However, recent results suggest broader biological activity may also be involved in the cardiovascular effects of this peptide. Here we show that Bj-PRO-10c enhances and sustains the generation of nitric oxide (NO) by regulating argininosuccinate synthase activity and thereby velocity of the citrulline-NO cycle. Bj-PRO-10c-mediated effects not restricted to the cardiovascular system, since NO production was also induced in cells of astroglial origin. Bj-PRO-10c was internalized by C6 astroglioma cells where it induces NO production and upregulation of the citrulline-NO cycle cells in a dose-dependent fashion. In view of that, astroglial cells function as L-arginine pool for NO production in neighboring neurons, we suggest a regulatory function for Bj-PRO-10c on the metabolism of this gaseous neurotransmitter in the CNS. Moreover, proliferation of astroglial cells was reduced in the presence of Bj-PRO-10c; however, cell death was not induced. Since NO donors have been studied for the treatment of solid cancers, Bj-PRO-10c may serve as structural model for developing drugs to improve the effects of cancer therapy based on the peptide's ability to augment NO production.

The central nervous system as target for antihypertensive actions of a proline-rich peptide from Bothrops jararaca venom.

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (

Molecular characterization of serine-, alanine-, and proline-rich proteins of Trypanosoma cruzi and their possible role in host cell infection.

We previously reported the isolation of a novel protein gene family, termed SAP (serine-, alanine-, and proline-rich protein), from Trypanosoma cruzi. Aided by the availability of the completed genome sequence of T. cruzi, we have now identified 39 full-length sequences of SAP, six pseudogenes and four partial genes. SAPs share a central domain of about 55 amino acids and can be divided into four groups based on their amino (N)- and carboxy (C)-terminal sequences. Some SAPs have conserved N- and C-terminal domains encoding a signal peptide and a glycosylphosphatidylinositol anchor addition site, respectively. Analysis of the expression of SAPs in metacyclic trypomastigotes by two-dimensional electrophoresis and immunoblotting revealed that they are likely to be posttranslationally modified in vivo. We have also demonstrated that some SAPs are shed into the extracellular medium. The recombinant SAP exhibited an adhesive capacity toward mammalian cells, where binding was dose dependent and saturable, indicating a possible ligand-receptor interaction. SAP triggered the host cell Ca2+ response required for parasite internalization. A cell invasion assay performed in the presence of SAP showed inhibition of internalization of the metacyclic forms of the CL strain. Taken together, these results show that SAP is involved in the invasion of mammalian cells by metacyclic trypomastigotes, and they confirm the hypothesis that infective trypomastigotes exploit an arsenal of surface glycoproteins and shed proteins to induce signaling events required for their internalization.

Differential expression of isoproterenol-induced salivary polypeptides in two mouse strains that are congenic for the H-2 histocompatibility gene complex.

Two inbred mouse strains, A/Snell and A.Swiss, which were produced as congenic with regard to the H-2 histocompatibility gene complex, are homozygous for two different groups of isoproterenol-induced salivary polypeptides (IISP). These polypeptides, which have been considered as markers of the hypertrophic growth of the parotid acinar cells, are members of the complex family of salivary proline-rich proteins (PRP) on the basis of both their massive accumulation in the parotid acinar cells in response to chronic isoproterenol, secretory character, high solubility in trichloroacetic acid and metachromatic staining by Coomassie blue. IISP expressed in both mouse strains were identified by unidimensional SDS-polyacrylamide electrophoresis and Coomassie blue staining both in parotid gland homogenates and in whole salivas obtained from mice repeatedly stimulated at 24-h intervals with isoproterenol. Parotid glands from 40 mice (20 A/Snell and 20 A.Swiss) and salivas from 270 mice (200 A/Snell and 70 A.Swiss) were analyzed. One of the congenic strains (A/Snell) expressed five IISP (Mr 65, 61, 51.5, 38, and 37 kDa) and the other strain (A.Swiss) expressed six IISP (Mr 59, 57, 54.5, 46, 36, and 34 kDa). No inter-individual intra-strain variations were observed, thus defining strain-associated patterns of IISP (PRP).

Electrophoretic analysis of whole saliva and prevalence of dental caries. A study in Mexican dental students.

BACKGROUND: Variability in salivary proteins and their posttranslational modifications may play an important role in determining their protective features against dental caries. Knowledge of molecular content of saliva in different populations is important for a better understanding of protective properties of this biological fluid. Aims of this study were to analyze electrophoretic pattern and protein composition in resting human whole saliva (HWS) of a Mexican population and to correlate these data with decayed, missing, and filled teeth (DMFT) index in these subjects. METHODS: Resting human whole saliva samples were collected from 120 healthy Mexican dental students. Salivary flow rate, protein concentration, and electrophoretic profile analyzed qualitatively by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were correlated with DMFT index. Gels were successively triple-stained with Coomassie brilliant blue R250, periodic acid Schiff (PAS), silver stain, and salivary molecules were scored as absent (-), present (+/-), and high intensity and size (+). RESULTS: These showed no substantial differences in number of bands between males and females; however, a slight correlation between total protein concentration and sex was found (p