Development of secondary palate requires strict regulation of ECM remodeling: sequential distribution of RECK, MMP-2, MMP-3, and MMP-9

We have evaluated RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), MMP-2 (matrix metalloproteinase-2), MMP-3, and MMP-9 involvement during palate development in mice by using various techniques. Immunohistochemical features revealed the distribution of RECK, MMP-2, and MMP-3 in the mesenchymal tissue and in the midline epithelial seam at embryonic day 13 (E13), MMPs-2, -3, and -9 being particularly expressed at E14 and E14.5. In contrast, RECK was weakly immunostained at these times. Involvement of MMPs was validated by measuring not only their protein expression, but also their activity (zymograms). In situ hybridization signal (ISH) for RECK transcript was distributed in mesenchymal and epithelial regions within palatal shelves at all periods evaluated. Importantly, the results from ISH analysis were in accord with those obtained by real-time polymerase chain reaction. The expression of RECK was found to be temporally regulated, which suggested possible roles in palatal ontogeny. Taken together, our results clearly show that remodeling of the extracellular matrix is finely modulated during secondary palate development and occurs in a sequential manner.

Characterization and Identification of Proteolytic Bacteria From the Gut of the Velvetbean Caterpillar (Lepidoptera: Noctuidae)

The characterization and identification of proteolytic bacteria from the gut of the velvetbean caterpillar (Anticarsia gemmatalis) were the objectives of this study. Twelve aerobic and anaerobic isolates of proteolytic bacteria were obtained from the caterpillar gut in calcium caseinate agar. The number of colony forming units (CFUs) of proteolytic bacteria was higher when the bacteria were extracted from caterpillars reared on artificial diet rather than on soybean leaves (1.73 +/- 0.35 X 10(3) and 0.55 +/- 0.22 X 10(3) CFU/mg gut, respectively). The isolated bacteria were divided into five distinct groups, according to their polymerase chain reaction restriction fragment-length polymorphism profiles. After molecular analysis, biochemical tests and fatty acid profile determination, the bacteria were identified as Bacillus subtilis, Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii, and Staphylococcus xylosus. Bacterial proteolytic activity was assessed through in vitro colorimetric assays for (general) proteases, serine proteases, and cysteine proteases. The isolated bacteria were able of hydrolyzing all tested substrates, except Staphylococcus xylosus, which did not exhibit serine protease activity. This study provides support for the hypothesis that gut proteases from velvetbean caterpillar are not exclusively secreted by the insect cells but also by their symbiotic gut bacteria. The proteolytic activity from gut symbionts of the velvetbean caterpillar is suggestive of their potential role minimizing the potentially harmful consequences of protease inhibitors from some of this insect host plants, such as soybean, with implications for the management of this insect pest species.

Integration of Ligand- and Target-Based Virtual Screening for the Discovery of Cruzain Inhibitors

A myriad of methods are available for virtual screening of small organic compound databases. In this study we have successfully applied a quantitative model of consensus measurements, using a combination of 3D similarity searches (ROCS and EON), Hologram Quantitative Structure Activity Relationships (HQSAR) and docking (FRED, FlexX, Glide and AutoDock Vina), to retrieve cruzain inhibitors from collected databases. All methods were assessed individually and then combined in a Ligand-Based Virtual Screening (LBVS) and Target-Based Virtual Screening (TBVS) consensus scoring, using Receiving Operating Characteristic (ROC) curves to evaluate their performance. Three consensus strategies were used: scaled-rank-by-number, rank-by-rank and rank-by-vote, with the most thriving the scaled-rank-by-number strategy, considering that the stiff ROC curve appeared to be satisfactory in every way to indicate a higher enrichment power at early retrieval of active compounds from the database. The ligand-based method provided access to a robust and predictive HQSAR model that was developed to show superior discrimination between active and inactive compounds, which was also better than ROCS and EON procedures. Overall, the integration of fast computational techniques based on ligand and target structures resulted in a more efficient retrieval of cruzain inhibitors with desired pharmacological profiles that may be useful to advance the discovery of new trypanocidal agents.

2D QSAR and similarity studies on cruzain inhibitors aimed at improving selectivity over cathepsin L

Hologram quantitative structure-activity relationships (HQSAR) were applied to a data set of 41 cruzain inhibitors. The best HQSAR model (Q(2) = 0.77; R-2 = 0.90) employing Surflex-Sim, as training and test sets generator, was obtained using atoms, bonds, and connections as fragment distinctions and 4-7 as fragment size. This model was then used to predict the potencies of 12 test set compounds, giving satisfactory predictive R-2 value of 0,88. The contribution maps obtained from the best HQSAR model are in agreement with the biological activities of the study compounds. The Trypanosoma cruzi cruzain shares high similarity with the mammalian homolog cathepsin L. The selectivity toward cruzam was checked by a database of 123 compounds, which corresponds to the 41 cruzain inhibitors used in the HQSAR model development plus 82 cathepsin L inhibitors. We screened these compounds by ROCS (Rapid Overlay of Chemical Structures), a Gaussian-shape volume overlap filter that can rapidly identify shapes that match the query molecule. Remarkably, ROCS was able to rank the first 37 hits as being only cruzain inhibitors. In addition, the area under the curve (AUC) obtained with ROCS was 0.96, indicating that the method was very efficient to distinguishing between cruzain and cathepsin L inhibitors. (c) 2007 Elsevier Ltd. All rights reserved.

Experimental Chemotherapy against Trypanosoma cruzi Infection Using Ruthenium Nitric Oxide Donors

The ruthenium NO donors of the group trans-[Ru(NO)(NH(3))(4)L](n+), where the ligand (L) is N-heterocyclic H(2)O, SO(3)(2 -), or triethyl phosphite, are able to lyse Trypanosoma cruzi in vitro and in vivo. Using half-maximal (50%) inhibitory concentrations against bloodstream trypomastigotes (IC(50)(try)) and cytotoxicity data on mammalian V-79 cells (IC(50)(V79)), the in vitro therapeutic indices (TIs) (IC(50)(V79)/IC(50)(try)) for these compounds were calculated. Compounds that exhibited an in vitro TI of >= 10 and trypanocidal activity against both epimastigotes and trypomastigotes with an IC(50)(try/epi) of <= 100 mu M were assayed in a mouse model for acute Chagas` disease, using two different routes (intraperitoneal and oral) for drug administration. A dose-effect relationship was observed, and from that, the ideal dose of 400 nmol/kg of body weight for both trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) (isn, isonicotinamide) and trans-[Ru(NO)(NH3) 4imN](BF4) 3 (imN, imidazole) and median (50%) effective doses (ED50) of 86 and 190 nmol/kg, respectively, were then calculated. Since the 50% lethal doses (LD(50)) for both compounds are higher than 125 mu mol/kg, the in vivo TIs (LD(50)/ED(50)) of the compounds are 1,453 for trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) and 658 for trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3). Although these compounds exhibit a marked trypanocidal activity and are able to react with cysteine, they exhibit very low activity in T. cruzi -glycosomal glyceraldehyde-3-phosphate dehydrogenase tests, suggesting that this enzyme is not their target. The trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) and trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) compounds are able to eliminate amastigote nests in myocardium tissue at 400-nmol/kg doses and ensure the survival of all infected mice, thus opening a novel set of therapies to try against trypanosomatids.

Studies toward the structural optimization of novel thiazolylhydrazone-based potent antitrypanosomal agents

In previous studies, we identified promising anti-Trypanosoma cruzi cruzain inhibitors based on thiazolylhydrazones. To optimize this series, a number of medicinal chemistry directions were explored and new thiazolylhydrazones and thiosemicarbazones were thus synthesized. Potent cruzain inhibitors were identified, such as thiazolylhydrazones 3b and 3j, which exhibited IC(50) of 200-400 nM. Furthermore, molecular docking studies showed concordance with experimentally derived structure-activity relationships (SAR) data. In the course of this work, lead compounds exhibiting in vitro activity against both the epimastigote and trypomastigote forms of T. cruzi were identified and in vivo general toxicity analysis was subsequently performed. Novel SAR were documented, including the importance of the thiocarbonyl carbon attached to the thiazolyl ring and the direct comparison between thiosemicarbazones and thiazolylhydrazones. (C) 2010 Elsevier Ltd. All rights reserved.

Evidence for a common role for the serine-type Plasmodium falciparum serine repeat antigen proteases : implications for vaccine and drug design

Serine repeat antigens (SERAs) are a family of secreted “cysteine-like” proteases of Plasmodium parasites. Several SERAs possess an atypical active-site serine residue in place of the canonical cysteine. The human malaria parasite Plasmodium falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. Here, we investigate the importance of the serine-type SERAs to blood-stage parasite development and examine the extent of functional redundancy among this group. We attempted to knock out the four P. falciparum serine-type SERA genes that have not been disrupted previously. SERA1, SERA4, and SERA9 knockout lines were generated, while only SERA5, the most strongly expressed member of the SERA family, remained refractory to genetic deletion. Interestingly, we discovered that while SERA4-null parasites completed the blood-stage cycle normally, they exhibited a twofold increase in the level of SERA5 mRNA. The inability to disrupt SERA5 and the apparent compensatory increase in SERA5 expression in response to the deletion of SERA4 provides evidence for an important blood-stage function for the serine-type SERAs and supports the notion of functional redundancy among this group. Such redundancy is consistent with our phylogenetic analysis, which reveals a monophyletic grouping of the serine-type SERAs across the genus Plasmodium and a predominance of postspeciation expansion. While SERA5 is to some extent further validated as a target for vaccine and drug development, our data suggest that the expression level of other serine-type SERAs is the only barrier to escape from anti-SERA5-specific interventions.

U18666A-mediated apoptosis in cultured murine cortical neurons : role of caspases, calpains and kinases

Studies have suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders such as Alzheimer's disease and Niemann–Pick disease type C. Previously, we have reported that U18666A, a cholesterol transport-inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we examined the effects of U18666A-mediated neuronal apoptosis, and found that chronic exposure to U18666A led to the activation of caspases and calpains and hyperphosphorylation of tau. Tau hyperphosphorylation is regulated by several kinases that phosphorylate specific sites of tau in vitro. Surprisingly, the kinase activity of cyclin-dependent kinase 5 decreased in U18666A-treated cortical neurons whereas its protein level remained unchanged. The amount of glycogen synthase kinase 3 and mitogen-activated protein kinases were found to decrease in their phosphorylated states by Western blot analysis. Gene transcription was further studied using microarray analysis. Genes encoding for kinases and phosphatases were differentially expressed with most up-regulated and some down-regulated in expression upon U18666A treatment. The activation of cysteine proteases and cholesterol accumulation with tauopathies may provide clues to the cellular mechanism of the inhibition of cholesterol transport-mediated cell death in neurodegenerative diseases.

Metallothionein 2A expression is associated with cell proliferation in breast cancer

Metallothioneins (MTs) belong to a family of cysteine-rich, metal-binding intracellular proteins, which have been linked with cell proliferation. In this study, expression levels of the 8 known MT-1 and MT-2 functional isoforms in human invasive ductal breast cancer specimens were determined by RT–PCR. The expression profiles of the MT protein and MT-2A mRNA were further evaluated in 79 cases of human invasive ductal breast carcinoma by immunohistochemistry and in situ hybridization, and correlated with cancer cell proliferation (determined by Ki-67 nuclear antigen immunolabeling). MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X and MT-2A but not MT-1B, were detected in breast cancer tissue samples. The MT-2A mRNA transcript was the highest among all the isoforms detected. A positive correlation was observed between MT-2A mRNA and MT protein expression with Ki-67 labeling (P = 0.0003 and P < 0.0001, respectively) but not with apoptosis (P = 0.1244 and P = 0.8189, respectively). Co-localization of the MT protein and Ki-67 nuclear antigen in breast cancer cells was demonstrated by double immunofluorescence staining. There was also significantly higher MT protein and MT-2A mRNA expression in histological grade 3 tumors than in histological grade 1 and 2 tumors. The finding that MT 2A appears to be the main isoform associated with cell proliferation in invasive ductal breast cancer tissues, may have therapeutic implications.

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70–86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

A conserved region in the EBL proteins Is implicated in microneme targeting of the malaria parasite Plasmodium falciparum

The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBAGFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.

N-Acetylcysteine infusion does not affect glucose disposal during prolonged moderate-intensity exercise in humans

There is evidence that reactive oxygen species (ROS) signalling is required for normal increases in glucose uptake during contraction of isolated mouse skeletal muscle, and that AMP-activated protein kinase (AMPK) is involved. The aim of this study was to determine whether ROS signalling is involved in the regulation of glucose disposal and AMPK activation during moderate-intensity exercise in humans. Nine healthy males completed 80 min of cycle ergometry at 62 ± 1 of peak oxygen consumption ( . A 6,6-2H-glucose tracer was infused at rest and during exercise, and in a double-blind randomised cross-over design, N-acetylcysteine (NAC) or saline (CON) was co-infused. NAC was infused at 125 mg kg?1h?1for 15 min and then at 25 mg kg?1h?1for 20 min before and throughout exercise. NAC infusion elevated plasma NAC and cysteine, and muscle NAC and cysteine concentrations during exercise. Although neither NAC infusion nor exercise significantly affected muscle reduced or oxidised glutathione (GSH or GSSG) concentration (P> 0.05), S-glutathionylation (an indicator of oxidative stress) of a protein band of ?270 kDa was increased ?3-fold with contraction and this increase was prevented by NAC infusion. Despite this, exercised-induced increases in tracer determined glucose disposal, plasma lactate, plasma non-esterified fatty acids (NEFAs), and decreases in plasma insulin were not affected by NAC infusion. In addition, skeletal muscle AMPK? and acetyl-CoA carboxylase-? (ACC?) phosphorylation increased during exercise by ?3- and ?6-fold (P< 0.05), respectively, and this was not affected by NAC infusion. Unlike findings in mouse muscle ex vivo, NAC does not attenuate skeletal muscle glucose disposal or AMPK activation during moderate-intensity exercise in humans.

Redox voltammetry of sub-parts per billion levels of Cu2+ at polyaspartate-modified gold electrodes

An electrochemical sensor for the detection of Cu2+ is reported which incorporates poly-l-aspartic acid (PLAsp) with 32–96 aspartate units as a selective ligand for the metal ion. PLAsp is covalently attached to a gold electrode modified with a monolayer of 3-mercaptopropionic acid using carbodiimide coupling via an N-hydroxysuccinimide (NHS) ester intermediate. The acid side groups and deprotonated peptide nitrogens on two aspartate moieties are thought to be primarily responsible for chelation of Cu2+, which remains bound when reduced to Cu+. A consequence of the multiple binding points that are available with a polypeptide is the low detection limit. The lowest concentration detected was 3 nM (0.2 ppb) achieved with Osteryoung square wave voltammetry. This detection limit compares favourably with that of ICP-OES and previously reported cysteine-modified electrodes. Analysis of tap and lake water samples using the PLAsp-modified electrode agreed well with ICP-OES analysis of the same samples.

Electrochemical metal ion sensors. Exploiting amino acids and peptides as recognition elements

Amino acids and peptides are known to bind metal ions, in some cases very strongly. There are only a few examples of exploiting this binding in sensors. The review covers the current literature on the interaction of peptides and metals and the electrochemistry of bound metal ions. Peptides may be covalently attached to surfaces. Of particular interest is the attachment to gold via sulfur linkages. Sulfur-containing peptides (eg cysteine) may be adsorbed directly, while any amino group can be covalently attached to a carboxylic acid-terminated thiol. Once at a surface, the possibility for using the attached peptide as a sensor for metal ions becomes realised. Results from the authors’ laboratory and elsewhere have shown the potential for selective monitoring of metal ions at ppt levels. Examples of the use of poly-aspartic acid and the copper binding peptide Gly-Gly-His for detecting copper ions are given.

A role for glutathione in the pathophysiology of bipolar disorder and schizophrenia? Animal models and relevance to clinical practice.

The tripeptide, glutathione (glutamylcysteinylglycine) is the primary endogenous free radical scavenger in the human body. When glutathione (GSH) levels are reduced there is an increased potential for cellular oxidative stress, characterised by an increase and accruement of reactive oxygen species (ROS). Oxidative stress has been implicated in the pathology of schizophrenia and bipolar disorder. This could partly be caused by alterations in dopaminergic and glutamatergic activity that are implicated in these illnesses. Glutamate and dopamine are highly redox reactive molecules and produce ROS during normal neurotransmission. Alterations to these neurotransmitter pathways may therefore increase the oxidative burden in the brain. Furthermore, mitochondrial dysfunction, as a source of oxidative stress, has been documented in both schizophrenia and bipolar disorder. The combination of altered neurotransmission and this mitochondrial dysfunction leading to oxidative damage may ultimately contribute to illness symptoms. Animal models have been established to investigate the involvement of glutathione depletion in aspects of schizophrenia and bipolar disorder to further characterise the role of oxidative stress in psychopathology. Stemming from preclinical evidence, clinical studies have recently shown antioxidant precursor treatment to be effective in schizophrenia and bipolar disorder, providing a novel clinical angle to augment often suboptimal conventional treatments.