Chitosan as a Bioadhesive Agent Between Porphyrins and Phospholipids in a Biomembrane Model

Porphyrins are currently used in photodynamic therapy as photosensitizers. In this paper we studied the interaction of two charged porphyrins, 5, 10, 15, 20-mesotetrakis(N-metyl-4-pyridyl) porphyrin, (TMPyP/chloride salt) cationic, and 5, 10, 15, 20-meso-tetrakis(sulfonatophenyl) porphyrin, (TPPS(4)/sodium salt) anionic, nanoassembled in phospholipid Langmuir monolayers and Langmuir-Blodgett films. Furthermore, we used chitosan to mediate the interaction between the porphyrins and the model membrane, aiming to understand the role of the polysaccharide in a molecular level. The effect of the interaction of the photosensitizers on the fluidity of the lipid monolayer was investigated by using dilatational surface elasticity. We also used photoluminescence (PL) spectroscopy to identify the porphyrins adsorbed in the phospholipid films. We observed an expansion of the monolayer promoted by the adsorption of the porphyrins into the lipid-air interface which was more pronounced in the case of TMPyP, as a consequence of a strong electrostatic interaction with the anionic monolayer. The chitosan promoted a higher adsorption of the porphyrins on the phospholipid monolayers and enabled the porphyrin to stay in its monomeric form (as confirmed by PL spectroscopy), thus demonstrating that chitosan can be pointed out as a potential photosensitizer delivery system in photodynamic therapy.

Macromolecular assembly of polycystin-2 intracytosolic C-terminal domain

Mutations in PKD2 are responsible for approximately 15% of the autosomal dominant polycystic kidney disease cases. This gene encodes polycystin-2, a calcium-permeable cation channel whose C-terminal intracytosolic tail (PC2t) plays an important role in its interaction with a number of different proteins. In the present study, we have comprehensively evaluated the macromolecular assembly of PC2t homooligomer using a series of biophysical and biochemical analyses. Our studies, based on a new delimitation of PC2t, have revealed that it is capable of assembling as a homotetramer independently of any other portion of the molecule. Our data support this tetrameric arrangement in the presence and absence of calcium. Molecular dynamics simulations performed with a modified all-atoms structure-based model supported the PC2t tetrameric assembly, as well as how different populations are disposed in solution. The simulations demonstrated, indeed, that the best-scored structures are the ones compatible with a fourfold oligomeric state. These findings clarify the structural properties of PC2t domain and strongly support a homotetramer assembly of PC2.

ADAM23 Negatively Modulates alpha(v)beta(3) Integrin Activation during Metastasis

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation. [Cancer Res 2009;69(13):5546-52]

Induction of CRP3/MLP expression during vein arterialization is dependent on stretch rather than shear stress

Aims Cysteine- and glycine-rich protein 3/muscle LIM-domain protein (CRP3/MLP) mediates protein-protein interaction with actin filaments in the heart and is involved in muscle differentiation and vascular remodelling. Here, we assessed the induction of CRP3/MLP expression during arterialization in human and rat veins. Methods and results Vascular CRP3/MLP expression was mainly observed in arterial samples from both human and rat. Using quantitative real time RT-PCR, we demonstrated that the CRP3/MLP expression was 10 times higher in smooth muscle cells (SMCs) from human mammary artery (h-MA) vs. saphenous vein (h-SV). In endothelial cells (ECs), CRP3/MLP was scarcely detected in either h-MA or h-SV. Using an ex vivo flow through system that mimics arterial condition, we observed induction of CRP3/MLP expression in arterialized h-SV. Interestingly, the upregulation of CRP3/MLP was primarily dependent on stretch stimulus in SMCs, rather than shear stress in ECs. Finally, using a rat vein in vivo arterialization model, early (1-14 days) CRP3/MLP immunostaining was observed predominantly in the inner layer and later (28-90 days) it appeared more scattered in the vessel layers. Conclusion Here we provide evidence that CRP3/MLP is primarily expressed in arterial SMCs and that stretch is the main stimulus for CRP3/MLP induction in veins exposed to arterial haemodynamic conditions.

Human apolipoprotein A-I binds amyloid-beta and prevents A beta-induced neurotoxicity

Aggregates of the amyloid-P peptide (A beta) play a central role in the pathogenesis of Alzheimer`s disease (AD). Identification of proteins that physiologically bind A beta and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated A beta(1-42), We isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A-I (apoA-I). We show that purified human apoA-I and A beta form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by A beta. Significantly, A beta/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from A beta-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates A beta aggregation and A beta-induced neuronal damage and that the A beta-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of A beta toxicity. (C) 2009 Published by Elsevier Ltd.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B-1 and B-2 (kinin receptors) and AT(1) and AT(2) (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B, agonists Des-Arg(9)-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg(9)-BK or Des-Arg(10)-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT, agonist (Asp-Arg-Val-Tyr-lle-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B-1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT, receptor. To investigate this hypothesis, we replaced Arg(212) for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B, receptor by the 32 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg(212) in the TM V and a region of TM VI of rat B, receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII. (c) 2007 Elsevier B.V. All rights reserved.

Sildenafil inhibits duodenal contractility via activation of the NO-K+ channel pathway

Phosphodiesterase type-5 (PDE5) specifically cleaves cyclic guanosine monophosphate (cGMP), a key intracellular secondary messenger. The PDE5 inhibitor sildenafil is a well-known vasodilator that also has gastrointestinal myorelaxant properties. In the present study, we further investigated sildenafil-induced myorelaxation in rat isolated duodenum, assessing its interaction with nitric oxide (NO) synthase and K+ channel opening. The spontaneous contractions of duodenal strips were reversibly inhibited by sildenafil (0.1-300 mu M) in a concentration-dependent manner [mean (95% confidence interval); EC50 = 6.8 (2.7-17.3) mu M]. The sildenafil-induced myorelaxation was significantly decreased by the NO synthase inhibitor N-nitro-L-arginine methyl ester [increasing the EC50 value to 41.9 (26.1-67.3) mu M]. Sodium nitroprusside or forskolin pretreatments enhanced the sildenafil-induced myorelaxation. In isolated strips pretreated with BaCl2 (0.2 mM), 4-aminopyridine (4-AP, 3 mM), or glybenclamide (1 mu M), the sildenafil-induced EC50 value was significantly increased to 32.8 (19.1-56.4), 27.1 (15.2-48.3) and 20.1 (16.4-24.7) mu M, respectively. Minoxidil (50 mu M) or diazoxide (100 mu M) also significantly attenuated the sildenafil-induced potency. In conclusion, the NO synthase/cyclic nucleotide pathway activation is involved in sildenafil-induced inhibition of spontaneous duodenal contractions. Its pharmacological action seems to be influenced by K+ channel opening, especially the voltage-sensitive ones, being inhibited by 4-AP and K-ATP channels, sensitive to glybenclamide.

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume- activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of similar to 75 mOsm. These currents display the typical biophysical signature of volume- activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(-)>Cl(-)>F(-)). Staurosporine (200 nM) did not block the activation of I(Cl), swell. The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 mu M), SITS (200 mu M), ATP (500 mu M), pyridoxalphosphate-6- azophenyl-2`,4`-disulfonate (PPADS; 100 mu M), and Suramin (10 mu M) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(d)(0) and fractional distance of the binding site (delta) of 334 mu M and 47%, 880 mu M and 35%, 2,100 mu M and 49%, 188 mu M and 27%, and 66.5 mu M and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that ICl, swell in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling

Trichophyton rubrum is a dermatophyte that infects human skin and nails. Its growth on keratin as its carbon source shifts the ambient pH from acidic to alkaline, which may be an efficient strategy for its successful infection and maintenance in the host. In this study, we used suppression subtractive hybridization to identify genes preferentially expressed in T rubrum incubated at either pH 5.0 or pH 8.0. The functional grouping of the 341 overexpressed unigenes indicated proteins putatively involved in diverse cellular processes, such as membrane remodeling, cellular transport, metabolism, cellular protection, fungal pathogenesis, gene regulation, interaction with the environment, and iron uptake. Although the basic metabolic machinery identified under both growth conditions seems to be functionally similar, distinct genes are upregulated at acidic or alkaline pHs. We also isolated a large number of genes of unknown function, probably unique to T rubrum or dermatophytes. Interestingly, the transcriptional profiling of several genes in a pacC mutant suggests that, in T rubrum, the transcription factor PacC has a diversity of metabolic functions, in response to either acidic or alkaline ambient pH. (C) 2009 Elsevier Ltd. All rights reserved.

Focal CO2 dialysis in raphe obscurus does not stimulate ventilation but enhances the response to focal CO2 dialysis in the retrotrapezoid nucleus

Simultaneous inhibition of the retrotrapezoid nucleus (RTN) and raphe obscurus (ROb) decreased the systemic CO2 response by 51%, an effect greater than inhibition of RTN (- 24%) or ROb (0%) alone, suggesting that ROb modulates chemoreception by interaction with the RTN (19). We investigated this interaction further by simultaneous dialysis of artificial cerebrospinal fluid equilibrated with 25% CO2 in two probes located in or adjacent to the RTN and ROb in conscious adult male rats. Ventilation was measured in a whole body plethysmograph at 30 C. There were four groups (n = 5): 1) probes correctly placed in both RTN and ROb (RTN-ROb); 2) one probe correctly placed in RTN and one incorrectly placed in areas adjacent to ROb (RTN-peri-ROb); 3) one probe correctly placed in ROb and one probe incorrectly placed in areas adjacent to RTN (peri-RTN-ROb); and 4) neither probe correctly placed (peri-RTN-peri-ROb). Focal simultaneous acidification of RTN-ROb significantly increased ventilation ((V) over dot E) up to 22% compared with baseline, with significant increases in both breathing frequency and tidal volume. Focal acidification of RTN-peri-ROb increased (V) over dot E significantly by up to 15% compared with baseline. Focal acidification of ROb and peri-RTN had no significant effect. The simultaneous acidification of regions just outside the RTN and ROb actually decreased (V) over dot E by up to 11%. These results support a modulatory role for the ROb with respect to central chemoreception at the RTN.

Isolation and partial characterization of Brazilian samples of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. FIV has extensive sequence variation, a typical feature of lentiviruses. Sequence analysis showed that diversity was not evenly distributed throughout the genome, but was greatest in the envelope gene, env. The virus enters host cells via a sequential interaction, initiated by the envelope glycoprotein (env) binding the primary receptor molecule CD134 and followed by a subsequent interaction with chemokine co-receptor CXCR4. The purpose of this study was to isolate and characterize isolates of FIV from an open shelter in Sao Paulo, Brazil. The separated PBMC from 11 positive cats were co-cultured with MYA-1 cells. Full-length viral env glycoprotein genes were amplified and determined. Chimeric feline x human CD134 receptors were used to investigate the receptor utilization of 17 clones from Brazilian isolates of Fly. Analyses of the sequence present of molecular clones showed that all clones grouped within subtype B. In contrast to the virulent primary isolate FIV-GL8, expression of the first cysteine-rich domain (CRD1) of feline CD134 in the context of human CD134 was sufficient for optimal receptor function for all Brazilian FIV isolates tested. (C) 2011 Elsevier B.V. All rights reserved.

Evaluation of three different adhesive systems using a bacterial method to develop secondary caries in vitro

Purpose: To assess the effects of three different dental adhesive systems on the formation of secondary root caries, in vitro, with a standardized interfacial gap in a filled cavity model. Methods: 40 sound human molars were selected and randomly assigned to four experimental groups: Clearfil SE Bond (CSEB), Xeno III (X-III), Scotchbond Multi-Purpose Plus (SBMP) and negative control (NC) without an adhesive system. After the standardized Class V cavity preparations on the buccal and lingual surfaces, restorations were placed with resin composite (Filtek Z250) using a standardized interfacial gap, using a 3 x 2 mm piece of 50 mu m metal matrix. The teeth were sterilized with gamma irradiation and exposed to a cariogenic challenge using a bacterial system with Streptococcus mutans. Depth and extension of wall lesions formed and the depth of outer lesions were measured by software coupled with light microscopy. Results: For wall lesion extension the ANOVA test showed differences between groups except between X-HI and SBMP (P= 0.294). The Tukey`s test of confidence intervals indicated smaller values for the CSEB group than for the others. For wall lesion depth the CSEB group also presented the smallest mean values of wall lesion depth when compared to the others (P< 0.0001) for all comparisons using Tukey`s test. Regarding outer lesion depth, all adhesives showed statistically similar behavior. SEM evaluation of the morphologic appearance of caries lesions confirmed the statistical results showing small caries lesion development for cavities restored with CSEB adhesive system, which may suggest that this adhesive system interdiffusion zone promoted a good interaction with subjacent dentin protecting the dental tissues from recurrent caries. (Am J Dent 2010;23:93-97).

Different air-water spray regulations affect the healing of Er,Cr:YSGG laser incisions

Surgeries performed with high-intensity laser devices may be improved with accurate protocols, including the air-water spray regulation. Thus, this study sought to investigate the healing process of wounds made on the dorsum of rat tongues using an Er,Cr:YSGG laser device with different air-water spray regulations. The incisions were made on the dorsum of Wistar rat tongues using an Er,Cr:YSGG laser with three different air-water spray regulations (100/0%, 50/50%, 11/7%). Scalpel incisions functioned as controls. The sacrifices occurred between 0 and 14 days after surgery. Morphological, histological, and immunohistochemical (fibronectin and type III collagen) analysis of the wounds were performed. The air-water spray regulation influenced wound healing and the inflammatory response, especially in the earlier stages. Incisions performed using the 100/0% air/water spray regulation had the worst results, expressing a greater amount of fibronectin and type III collagen. The 50/50% air/water spray regulation brought in a non-clear surgical field and poor laser interaction with the tissue. The 11/7% air/water spray regulation showed the best clinical results and less pronounced histological events. According to the results encountered, the air-water spray should be regulated to improve surgery.

Understanding Contradictory Data in Contraction Stress Tests

The literature shows contradictory results regarding the role of composite shrinkage and elastic modulus as determinants of polymerization stress. The present study aimed at a better understanding of the test mechanics that could explain such divergences among studies. The hypothesis was that the effects of composite shrinkage and elastic modulus on stress depend upon the compliance of the testing system. A commonly used test apparatus was simulated by finite element analysis, with different compliance levels defined by the bonding substrate (steel, glass, composite, or acrylic). Composites with moduli between 1 and 12 GPa and shrinkage values between 0.5% and 6% were modeled. Shrinkage was simulated by thermal analogy. The hypothesis was confirmed. When shrinkage and modulus increased simultaneously, stress increased regardless of the substrate. However, if shrinkage and modulus were inversely related, their magnitudes and interaction with rod material determined the stress response.

Association between sleep bruxism and temporomandibular disorders: A polysomnographic pilot study

The aim of this study was to verify the association between sleep bruxism (SB) and temporomandibular disorders (TMD) in a sample of 14 TMD patients and 12 healthy control subjects. All participants were evaluated using a clinical questionnaire, visual analog scale (VAS) for TMJ/muscle palpation, and by functional examination. The experimental group was divided into three TMD subgroups: joint sounds and pain, muscular tenderness, and mixed diagnosis. All participants underwent polysomnographic recording (PSG). A second clinical examination was then carried out to verify the relationship between rhythmic masticatory muscle activity and pain/tenderness on the following morning. e experimental and control groups presented VAS mean scores of 36.85 +/- 23.73 mm and 0 mm, respectively. The presence of SB was neither associated with TMD (p>0.05) nor with pain on palpation (p>0.05). Further research with a more representative sample of each TMD subgroup is necessary to elucidate its interaction with SB.