Application of plasma polymerized pyrrole nanoparticles to prevent or reduce de-differentiation of adult rat ventricular cardiomyocytes.

Cardiovascular diseases are the leading cause of death in the world, cell therapies have been shown to recover cardiac function in animal models. Biomaterials used as scaffolds can solve some of the problems that cell therapies currently have, plasma polymerized pyrrole (PPPy) is a biomaterial that has been shown to promote cell adhesion and survival. The present research aimed to study PPPy nanoparticles (PPPyN) interaction with adult rat ventricular cardiomyocytes (ARVC), to explore whether PPPyN could be employed as a nanoscaffold and develop cardiac microtissues. PPPyN with a mean diameter of 330 nm were obtained, the infrared spectrum showed that some pyrrole rings are fragmented and that some fragments of the ring can be dehydrogenated during plasma synthesis, it also showed the presence of amino groups in the structure of PPPyN. PPPyN had a significant impact on the ARVC´s shape, delaying dedifferentiation, necrosis, and apoptosis processes, moreover, the cardiomyocytes formed cell aggregates up to 1.12 mm2 with some aligned cardiomyocytes and generated fibers on its surface similar to cardiac extracellular matrix. PPPyN served as a scaffold for adult ARVC. Our results indicate that PPPyN-scaffold is a biomaterial that could have potential application in cardiac cell therapy (CCT).

Effect of a plasma synthesized polypyrrole coverage on polylactic acid/hydroxyapatite scaffolds for bone tissue engineering.

Composite biomaterials are solids that contain two or more different materials, combining the properties of their components to restore or improve the function of tissues. In this study, we report the generation of electrospun matrices with osteoconductive properties and porosity using the combination of a biodegradable polyester, polylactic acid (PLA), and hydroxyapatite (HA). Additionally, we report the effects of modifying these matrices through plasma polymerization of pyrrole on the growth and osteogenic differentiation of rabbit bone marrow stem cells. Cells were isolated, seeded and cultured on biomaterials for periods between 7 and 28 days. The matrices we obtained were formed by nano and microfibers containing up to 35.7 wt% HA, presenting a variety of apparent pore sizes to allow for the passage of nutrients to bone cells. Scanning electron microscopy showed that the fibers were coated with polypyrrole doped with iodine, and MTT assay demonstrated this increased cell proliferation and significantly improved cell viability due to the adhesive properties of the polymer. Our results show that PLA/HA/Pyrrole/Iodine matrices are favorable for bone tissue engineering.

Use of a Triple Prophylactic Strategy to Prevent Post-dural Puncture Headache: An Observational Study.

Objective Post-dural puncture headache (PDPH) after an accidental dural puncture is a very common complication of epidural analgesia/anesthesia. We observed the ability of a triple prophylactic method (epidural saline, morphine, and intravenous (IV) cosyntropin) to prevent PDPH and the need for a blood patch.  Methods We retrospectively evaluated the effect of the combination of epidural saline, IV cosyntropin, and epidural morphine in parturients who had an accidental dural puncture with regard to the PDPH rate and the need for an epidural blood patch. We report a case series of patients with accidental dural puncture who underwent triple prophylaxis and other methods. Results Thirty-one patients were included in the study. Fourteen cases received triple prophylaxis (45%). Three patients in this group developed PDPH (21%), with two of them requiring a blood patch (14%). Nine patients underwent preventive measures other than triple prophylaxis with a PDPH rate of 55% and one needing a blood patch (11%). Conservative management was used in eight patients with PDPH and blood patch rates of 100% and 62%, respectively. Conclusion The triple prophylactic regimen of epidural saline, IV cosyntropin, and epidural morphine used after accidental dural puncture exhibits great potential to reduce the incidence of PDPH and the need for blood patch in obstetric patients.

Correlative Light and Electron Microscopy to Study Microglial Interactions with ß-Amyloid Plaques.

A detailed protocol is provided here to identify amyloid Aß plaques in brain sections from Alzheimer's disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to ß-pleated sheets found in dense core Aß plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label ß-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for Aß (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing Aß plaques at the right location. Importantly, Aß plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

Malnutrition and the presence of intestinal parasites in children from the poorest municipalities of Mexico.

BACKGROUND: For many years Chiapas, Mexico's poorest state, has had the highest rate of child mortality due to intestinal infections of unknown etiology in the country. To begin identifying the infectious agents, our work determined the prevalence of intestinal parasites as well as malnutrition in children from Chiapas's three most impoverished municipalities: Pantepec, Chanal, and Larrainzar. METHODOLOGY: In this cross-sectional study, conducted between January and November 2009, we assessed the prevalence of intestinal parasites by means of coproparasitological analysis in children <5 years of age (N=250) from three of the marginalized municipalities: Pantepec, Chanal and Larrainzar. The prevalence of malnutrition was then assessed using the Mexican official norm NOM-031-SSA2-1999 and WHO criteria. We evaluated the association between age (breast-fed and pre-school children) with parasites and nutritional status. RESULTS: Our analysis revealed the highest prevalence of intestinal parasites in children from Pantepec (62.8%), followed by Chanal (47.3%), and then Larrainzar (11.9 %). The nematode Ascaris lumbricoides was the most prevalent enteroparasite (33.6%). Anthropometric analysis revealed that >40% of children represented varying degrees of malnutrition and a marked constitutional delay in growth. A very high prevalence of stunting was also recorded in children from Chanal and Larrainzar (70% and 55%, respectively). An association between infection with intestinal parasites and malnutrition was observed in Pantepec. Preschool-age children were more likely to be infected with intestinal parasites. CONCLUSION: Our results indicate the urgent need for interventions in order to 1) improve the nutritional status of children and 2) reduce infection rates of enteric parasites.

Aversive reinforcement improves visual discrimination learning in free-flying honeybees.

BACKGROUND: Learning and perception of visual stimuli by free-flying honeybees has been shown to vary dramatically depending on the way insects are trained. Fine color discrimination is achieved when both a target and a distractor are present during training (differential conditioning), whilst if the same target is learnt in isolation (absolute conditioning), discrimination is coarse and limited to perceptually dissimilar alternatives. Another way to potentially enhance discrimination is to increase the penalty associated with the distractor. Here we studied whether coupling the distractor with a highly concentrated quinine solution improves color discrimination of both similar and dissimilar colors by free-flying honeybees. As we assumed that quinine acts as an aversive stimulus, we analyzed whether aversion, if any, is based on an aversive sensory input at the gustatory level or on a post-ingestional malaise following quinine feeding. METHODOLOGY/PRINCIPAL FINDINGS: We show that the presence of a highly concentrated quinine solution (60 mM) acts as an aversive reinforcer promoting rejection of the target associated with it, and improving discrimination of perceptually similar stimuli but not of dissimilar stimuli. Free-flying bees did not use remote cues to detect the presence of quinine solution; the aversive effect exerted by this substance was mediated via a gustatory input, i.e. via a distasteful sensory experience, rather than via a post-ingestional malaise. CONCLUSION: The present study supports the hypothesis that aversion conditioning is important for understanding how and what animals perceive and learn. By using this form of conditioning coupled with appetitive conditioning in the framework of a differential conditioning procedure, it is possible to uncover discrimination capabilities that may remain otherwise unsuspected. We show, therefore, that visual discrimination is not an absolute phenomenon but can be modulated by experience.

RNAi-mediated silencing of insulin receptor substrate-4 enhances actinomycin D- and tumor necrosis factor-alpha-induced cell death in hepatocarcinoma cancer cell lines.

Insulin receptor substrate-4 (IRS-4) transmits signals from the insulin-like growth factor receptor (IGF-IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS-4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS-4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS-4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor-alpha (TNF-alpha). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial-dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF-alpha amplifies the effect of Act D on HepG2 cell apoptosis increasing c-jun N-terminal kinase (JNK) activity, IkappaB-alpha proteolysis and glutathione depletion. IRS-4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS-4 action is independent of Akt, IkappaB kinase and JNK. IRS-4 down regulation, however, decreased gamma-glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF-alpha. These results suggest that IRS-4 protects HepG2 cells from oxidative stress induced by drug treatment.

Vasoactive intestinal peptide (VIP) induces c-fos expression in LNCaP prostate cancer cells through a mechanism that involves Ca2+ signalling. Implications in angiogenesis and neuroendocrine differentiation.

The effect of vasoactive intestinal peptide (VIP) on intracellular Ca(2+) levels and its relationship with the expression of c-fos and vascular endothelial growth factor (VEGF) as well as with neuroendocrine (NE) differentiation were investigated in human prostate LNCaP cells. VIP induced the expression of c-fos mRNA as studied by reverse transcription polymerase chain reaction (RT-PCR). It was accompanied by VIP stimulation of c-fos protein synthesis, as measured by Western blot analysis. VIP enhanced intracellular Ca(2+) levels as evaluated using the calcium probe fura-2. VIP regulation of c-fos expression depended on [Ca(2+)](i) concentration since the intracellular calcium chelator BAPTA/AM decreased c-fos expression (both mRNA and protein) to basal levels. As shown by means of real-time RT-PCR, VIP stimulated VEGF mRNA expression: the effect was inhibited by 40% in the presence of curcumin (an inhibitor of AP-1 binding), and it was dependent on Ca(2+) since BAPTA/AM inhibited this VIP action by 43%. Similar observations were made on the effects of BAPTA/AM and curcumin on VIP stimulation of VEGF protein expression. Simultaneous treatment of cells with the protein kinase A inhibitor H89 and BAPTA/AM completely blocked this VIP effect, whereas each agent alone led only to a partial inhibition. In addition, the calcium chelator blocked by 37% the ability of VIP to induce NE cell differentiation as estimated by the observation of neurite development. These features support a VIP signalling pathway that could be mediated through both cAMP and [Ca(2+)](i) increase in prostate LNCaP cancer cells. Moreover, our data suggest the implication of c-Fos on the induction of the main angiogenic factor VEGF since the promoter region of the VEGF gene possesses AP-1 (i.e., c-Fos/c-Jun heterodimer) response elements. This feature represents a link between the nuclear oncogene c-fos, angiogenesis and NE differentiation by means of an initiating signal upon VIP receptors.

Expression of the transient receptor potential vanilloid 1 (TRPV1) in LNCaP and PC-3 prostate cancer cells and in human prostate tissue.

Vanilloid receptor subtype-1 (TRPV1), the founding member of the vanilloid receptor-like transient receptor potential channel family, is a non-selective cation channel that responds to noxious stimuli such as low pH, painful heat and irritants. In the present study, we show, as means of reverse transcriptase-polymerase chain reaction and Western blot analysis, that the vanilloid TRPV1 receptor is expressed in the prostate epithelial cell lines PC-3 and LNCaP as well as in human prostate tissue. The kinetic parameters inferred from [(125)I]-resiniferatoxin binding were in concordance with data of TRPV1 receptors expressed in other tissues. The contribution of the endogenously expressed TRPV1 channel to intracellular calcium concentration increase in the prostate cells was studied by measuring changes in Fura-2 fluorescence by fluorescence microscopy. Addition of capsaicin, (R)-methanandamide and resiniferatoxin to prostate cells induced a dose-dependent increase in the intracellular calcium concentration that was reversed by the vanilloid TRPV1 receptor antagonist capsazepine. These results indicate that the vanilloid TRPV1 receptor is expressed and functionally active in human prostate cells.

Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study.

The response of anandamide is terminated by a carrier-mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC-3 cells by a saturable and temperature-dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of KM=4.7+/-0.2 microm and Vmax=3.3+/-0.3 pmol min-1 (10(6) cells)-1. The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC-3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1-8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC-3 cells, with (9Z,12Z)-N-(fur-3-ylmethyl)octadeca-9,12-dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3+/-0.5 microM, respectively. In conclusion, PC-3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator. British Journal of Pharmacology (2004) 141, 457-467. doi:10.1038/sj.bjp.0705628

Enhancement of androgen receptor expression induced by (R)-methanandamide in prostate LNCaP cells.

It has been recently shown that cannabinoids may regulate the growth of many cell types. In the present work we examined the effect of the anandamide analogue (R)-methanandamide (MET) on androgen-dependent prostate LNCaP cell growth. We found that 0.1 microM MET had a mitogenic effect measured by [(3)H]thymidine incorporation into DNA. The effect exerted by MET was blocked by the cannabinoid receptor antagonists SR141716 (SR1) and SR144528 (SR2) as well as by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, suggesting an involvement of cannabinoid receptors and the PI3K pathway in the mechanism of MET action. MET treatment of LNCaP cells also induced an up-regulation of androgen receptor expression that was blocked by the two cannabinoid receptor antagonists SR1 and SR2. These results show for the first time that cannabinoids may modify androgen receptor expression in an androgen-dependent cell line and by this mechanism could regulate prostate cell growth.

Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction.

Cannabinoids exert a variety of physiological and pharmacological responses in humans through interaction with specific cannabinoid receptors. Cannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and are coupled through the inhibitory G(i) protein to adenylyl cyclase inhibition. However, downstream signal transduction mechanisms triggered by cannabinoids are poorly understood. We examined here the involvement of the phosphoinositide 3-kinase (PI3K)/PKB pathway in the mechanism of action of cannabinoids in human prostate epithelial PC-3 cells. Cannabinoid receptors CB(1) and CB(2) are expressed in these cells, as shown by RT-PCR, Western blot and immunofluorescence techniques. Treatment of PC-3 cells with either Delta(9)-tetrahydrocannabinol (THC), the major psychoactive ingredient of marijuana, or R-(+)-methanandamide (MET), an analogue of the endogenous cannabinoid anandamide, increased phosphorylation of PKB in Thr308 and Ser473. The stimulation of PKB induced by cannabinoids was blocked by the two cannabinoid receptor antagonists, SR 141716 and SR 144528, and by the PI3K inhibitor LY 294002. These results indicate that activation of cannabinoid receptors in PC-3 cells stimulate the PI3K/PKB pathway. We further investigated the involvement of Raf-1/Erk activation in the mechanism of action of cannabinoid receptors. THC and MET induced translocation of Raf-1 to the membrane and phosphorylation of p44/42 Erk kinase, which was reversed by cannabinoid receptor antagonists and PI3K inhibitor. These results point to a sequential connection between cannabinoid receptors/PI3K/PKB pathway and Raf-1/Erk in prostate PC-3 cells. We also show that this pathway is involved in the mechanism of NGF induction exerted by cannabinoids in PC-3 cells.

Expression of functionally active cannabinoid receptor CB1 in the human prostate gland.

BACKGROUND: Cannabinoids exert a wide spectrum of effects in men including alterations in the reproductive system. To date, two types of cannabinoid receptors have been cloned in humans, namely CB(1) and CB(2) belonging to the G protein-coupled receptor superfamily. Although cannabinoids have functional and morphologic effects in the prostate gland, the expression of cannabinoid receptors in this tissue has never been investigated. The aim of this study was to analyze the expression of cannabinoid receptors in the human prostate gland and their regulatory effects on adenylyl cyclase activity. METHODS: To investigate the existence of cannabinoid receptors in prostate, we used various methods, including reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. Adenylyl cyclase activity was analyzed by measuring the cAMP produced by means of a competitive assay by using PKA. RESULTS: Both mRNA for CB(1) and the corresponding protein are expressed in the human prostate gland at a level comparable with the receptor expressed in cerebellum. The molecular mass of the protein estimated from Western blot analysis was 58 kDa, which is in concordance with previous data for CB(1) in other tissues. Immunohistochemical studies show that CB(1) is preferentially expressed in the epithelia of the prostate. The cannabinoid receptor expressed in the prostate negatively regulates adenylyl cyclase activity through a pertussis toxin-sensitive protein.

Existence of a sex pheromone in Triatoma infestans (Hemiptera: Reduvidae): II. electrophysiological correlates

The stimulus provided by a copulating pair of Triatoma infestans significantly affects the electrical activity of the nervous system of Triatoma infestans. Electrophysiological recordings were perfomed on stationary adult males presented with stimuli of an air current carrying odors from males, females, non-copulating pairs and mating pairs. The electrophysiological response was characterized by the low frequency occurrence of biphasic compound impulses. A significant increase in the frequency of the impulses occurred in stationary males when exposed to air currents of mating pairs, when compared to that evoked by a clean air stream. Analysis of the time course of the assays, showed that the electrophisiological activity during the copula was higher than prior to or after copula. The electrophysiological evidence presented here strongly supports the existence of pheromone(s) released by one or both sexes during mating and which is perceived by male chemoreceptors located on the antennae.