Development of films based on blends of gelatin and poly(vinyl alcohol) cross linked with glutaraldehyde

Both gelatin and poly(vinyl alcohol) (PVA) can be cross linked with glutaraldehyde (GLU). In the case of gelatin, the GLU reacts with each e-NH2 functional group of adjacent lysine residues, while for PVA, the GLU reacts with two adjacent hydroxyl groups, forming acetal bridges. Thus it can be considered possible to cross link adjacent macromolecules of gelatin and PVA using GLU. In this context, the aims of this work were the development of biodegradable films based on blends of gelatin and poly(vinyl alcohol) cross linked with GLU, and the characterization of some of their main physical and functional properties. All the films were produced from film-forming solutions (FFS) containing 2 g macromolecules (PVA + gelatin)/100 g FFS, 25 g glycerol/100 g macromolecules, and 4 g GLU (25% solution)/100 g FFS. The FFS were prepared with two concentrations of PVA (20 or 50 g PVA/100 g macromolecules) and two reaction temperatures: 90 or 55 degrees C, applied for 30 min. The films were obtained after drying (30 degrees C/24 h) and conditioning at 25 degrees C and 58% of relative humidity for 7 days, and were then characterized. The results for the color parameters, mechanical properties, phase transitions and infrared spectra showed that some chemical modifications occurred, principally for the gelatin. However, in general, all the characteristics of the films were either typical of films based on blends of these macromolecules without cross linking, or slightly higher. A greater improvement in the properties of this material was probably not observed due to the crystallinity of the PVA, which has a melting point above 90 degrees C. The presence of microcrystals in the polymer chain probably reduced macromolecular mobility, hindering the reaction. Thus more research is necessary to produce biodegradable films with improved properties. (C) 2011 Elsevier Ltd. All rights reserved.

Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins

In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, tip to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (similar to 3200 amu). A further three molecules (similar to similar to 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense. (C) 2008 Elsevier Inc. All rights reserved.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Mandibulofacial Syndrome With Growth and Mental Retardation, Microcephaly, Ear Anomalies With Skin Tags, and Cleft Palate in a Mother and Her Son: Autosomal Dominant or X-Linked Syndrome?

We report on a Brazilian mother and her son affected with mandibulofacial dysostosis, growth and mental retardation, microcephaly, first branchial arch anomalies, and cleft palate. To date only three males and one female, all sporadic cases, with a similar condition have been reported. This article describes the first familial case with this rare condition indicating autosomal dominant or X-linked inheritance. (C) 2009 Wiley-Liss, Inc.

The proliferative effect of synthetic N-POMC(1-28) peptides in rat adrenal cortex: A possible role for cyclin E

Modified synthetic N-POMC(1-28) without disulfide bridges has been shown to act as an adrenal mitogen. Cyclins and their inhibitors are the major cell cycle controls, but in the adrenal cortex the effect of ACTH and N-POMC on the expression of these proteins remains unclear. In this work, we evaluate the effect of different synthetic N-POMC peptides on the S-phase of the cell cycle. In addition, we examine the cyclin E expression in rat adrenal cortex. Rats treated with dexamethasone were injected with ACTH and/or synthetic modified N-POMC and/or synthetic N-POMC with disulfide bridges. DNA synthesis was determined by BrdU incorporation and protein expression was analyzed by immunoblotting and immunohistochemistry. The results showed that similarly to modified N-POMC without disulfide bridges, administration of synthetic N-POMC with disulfide bridges and the combination of ACTH and N-POMC promoted an increase of BrdU-positive nuclei in adrenal cortex. However, the proliferative effect of N-POMC was comparable to that of ACTH only in the zona glomerulosa. An increase in cyclin E expression was observed 6 h after N-POMC treatment in the outer fraction of the adrenal cortex, in agreement with immunohistochemical findings in the zona glomerulosa. In summary, the effect of synthetic N-POMC with disulfide bridges was similar to modified synthetic N-POMC, increasing proliferation in the adrenal cortex, confirming previous evidence that disulfide bridges are not essential to the N-POMC mitogenic effect. Moreover, cyclin E appears to be involved in the N-POMC- and ACTH-stimulated proliferation in the zona glomerulosa of the adrenal cortex. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

P>Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, similar to 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, similar to 1/3 showed no change, and similar to 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

Hemopressins and other hemoglobin-derived peptides in mouse brain: comparison between brain, blood, and heart peptidome and regulation in Cpefat/fat mice

P>Many hemoglobin-derived peptides are present in mouse brain, and several of these have bioactive properties including the hemopressins, a related series of peptides that bind to cannabinoid CB1 receptors. Although hemoglobin is a major component of red blood cells, it is also present in neurons and glia. To examine whether the hemoglobin-derived peptides in brain are similar to those present in blood and heart, we used a peptidomics approach involving mass spectrometry. Many hemoglobin-derived peptides are found only in brain and not in blood, whereas all hemoglobin-derived peptides found in heart were also seen in blood. Thus, it is likely that the majority of the hemoglobin-derived peptides detected in brain are produced from brain hemoglobin and not erythrocytes. We also examined if the hemopressins and other major hemoglobin-derived peptides were regulated in the Cpefat/fat mouse; previously these mice were reported to have elevated levels of several hemoglobin-derived peptides. Many, but not all of the hemoglobin-derived peptides were elevated in several brain regions of the Cpefat/fat mouse. Taken together, these findings suggest that the post-translational processing of alpha and beta hemoglobin into the hemopressins, as well as other peptides, is up-regulated in some but not all Cpefat/fat mouse brain regions.

Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease

Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This `experiment of nature` indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.

Comparative effect of FGF2, synthetic peptides 1-28 N-POMC and ACTH on proliferation in rat adrenal cell primary cultures

There is evidence that pro-opiomelanocortin (POMC)-derived peptides other than adrenocorticotropic hormone (ACTH) have a role in adrenal cell proliferation. We compared the activity of synthetic rat N-terminal POMC fragment 1-28 with disulfide bridges (N-POMC(w)) and without disulfide bridges (N-POMC(w/o)), with the activity of fibroblast growth factor (FGF2), a widely studied adrenal growth factor, and ACTH, in well-characterized pure cultures of both isolated adrenal Glomerulosa (G) and Fasciculata/Reticularis (F/R) cells. Three days of FGF2-treatment had a proliferative effect similar to serum, and synthetic peptide N-POMC(w) induced proliferation more efficiently than N-POMC(w/o). Moreover, both induced proliferation via the ERK1/2 pathway. In contrast, sustained ACTH treatment decreased proliferation and viability through apoptosis induction, but not necrosis, and independently of PKA and PKC pathways. Further elucidation of 1-28 POMC signal transduction is of interest, and primary cultures of adrenal cells were found to be useful for examining the trophic activity of this peptide.

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations. (C) 2010 Elsevier Inc. All rights reserved.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

Background: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results: An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1`. Conclusions: BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

TICK INNATE IMMUNITY

Ticks are blood feeding parasites transmitting a wide variety of pathogens to their vertebrate hosts. The vector competence of ticks is tightly linked with their immune system. Despite its importance, our knowledge of tick innate immunity is still inadequate and the limited number of sufficiently characterized immune molecules and cellular reactions are dispersed across numerous tick species. The phagocytosis of microbes by tick hemocytes seems to be coupled with a primitive complement-like system, which possibly involves self/nonself recognition by fibrinogen-related lectins and the action of thioester-containing proteins. Ticks do not seem to possess a pro-phenoloxidase system leading to melanization and also coagulation of tick hemolymph has not been experimentally proven. They are capable of defending themselves against microbial infection with a variety of antimicrobial peptides comprising lysozymes, defensins and molecules not found in other invertebrates. Virtually nothing is known about the signaling cascades involved in the regulation of tick antimicrobial immune responses. Midgut immunity is apparently the decisive factor of tick vector competence. The gut content is a hostile environment for ingested microbes, which is mainly due to the antimicrobial activity of hemoglobin fragments generated by the digestion of the host blood as well as other antimicrobial peptides. Reactive oxygen species possibly also play an important role in the tick-pathogen interaction. The recent release of the Ixodes scapularis genome and the feasibility of RNA interference in ticks promise imminent and substantial progress in tick innate immunity research.

Differential effects of alpha-helical and beta-hairpin antimicrobial peptides against Acanthamoeba castellanii

In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic alpha-helical peptides P5 and a beta-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells, P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.

Intracellular peptides as natural regulators of cell signaling

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 mu M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R-CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmume diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. (C) 2009 Elsevier Inc. All rights reserved.