Phisiology

The making and sealing of a tight junction (TJ) requires cell-cell contacts and Ca2−, and can be gauged through the development of transepithelial electrical resistance (TER) and the accumulation of ZO-1 peptide at the cell borders. We observe that pertussis toxin increases TER, while AIF3 and carbamil choline (carbachol) inhibit it, and 5-guanylylimidodiphosphate (GTPΓs) blocks the development of a cell border pattern of ZO-1, suggesting that G-proteins are involved. Phospholipase C (PLC) and protein kinase C (PKC) probably participate in these processes since (i) activation of PLC by thyrotropin-1 releasing hormone increases TER, and its inhibition by neomycin blocks the development of this resistance; (ii) 1,2-dioctanoylglycerol, an activator of PKC, stimulates TER development, while polymyxin B and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine dihydrochloride (H7), which inhibit this enzyme, abolish TER. Addition of 3-isobutyl-1-methyl-xanthine, dB-cAMP or forskolin do not enhance the value of TER, but have just the opposite effect. Trifluoperazine and calmidazoline inhibit TER development, suggesting that calmodulin (CaM) also plays a role in junction formation. These results indicate that junction formation may be controlled by a network of reactions where G-proteins, phospholipase C, adenylate cyclase, protein kinase C and CaM are involved.

Upon transferring confluent monolayers of Madin-Darby canine kidney (MDCK) cells from a low-Ca2+ médium (1-5  M) to one with 1.8 mM Ca2+ (Ca switch), tight junctions (TJs) assemble and seal, and transepithelial electrical resistance (TER) develops in 4-5 h, presumably through exocytotic fusion that incorporates junctional components to the surface membrane.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal survival rate. Persistent activation of pancreatic stellate cells (PSCs) can perturb the biomechanical homoeostasis of the tumour microenvironment to favour cancer cell invasion. Here we report that ATRA, an active metabolite of vitamin A, restores mechanical quiescence in PSCs via a mechanism involving a retinoic acid receptor beta (RAR-b)-dependent downregulation of actomyosin (MLC-2) contractility. We show that ATRA reduces the ability of PSCs to generate high traction forces and adapt to extracellular mechanical cues (mechanosensing), as well as suppresses force-mediated extracellular matrix remodelling to inhibit local cancer cell invasion in 3D organotypic models. Our findings implicate a RAR-b/MLC-2 pathway in peritumoural stromal remodelling and mechanosensory-driven activation of PSCs, and further suggest that mechanical reprogramming of PSCs with retinoic acid derivatives might be a viable alternative to stromal ablation strategies for the treatment of PDAC.

Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation of gene expression and cell proliferation. Here, we have studied the role of zona occludens (ZO)-2, a TJ peripheral protein, in the regulation of cyclin D1 transcription. We found that ZO-2 down-regulates cyclin D1 transcription in a dose-dependent manner. To understand how ZO-2 represses cyclin D1 promoter activity, we used deletion analyses and found that ZO-2 negatively regulates cyclin D1 transcription via an E box and that it diminishes cell proliferation. Because ZO-2 does not associate directly with DNA, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay were used to identify the transcription factors mediating the ZO-2–repressive effect. c-Myc was found to bind the E box present in the cyclin D1 promoter, and the overexpression of c-Myc augmented the inhibition generated by ZO-2 transfection. The presence of ZO-2 and c-Myc in the same complex was further demonstrated by immunoprecipitation. ChIP and reporter gene assays using histone deacetylases (HDACs) inhibitors demonstrated that HDACs are necessary for ZO-2 repression and that HDAC1 is recruited to the E box. We conclude that ZO-2 down-regulates cyclin D1 transcription by interacting with the c-Myc/E box element and by recruiting HDAC1.

The aim of this study was to identify cellular proteins that bind protein kinase C (PKC) and may influence its activity and its localization. A 32-kDa PKC-binding protein was purified to homogeneity from the Triton X-100-insoluble fraction obtained from hepatocytes homoge-nates. The protein was identified by NH 2-terminal amino acid sequencing as the previously described mature form of p32 (gC1qR). Recombinant p32 was expressed as a glutathione S-transferase fusion protein, affinity-purified, and tested for an in vitro interaction with PKC using an overlay assay approach. All PKC isoforms expressed in rat hepatocytes interacted in vitro with p32, but the binding dependence on PKC ac-tivators was different for each one. Whereas PKC only binds to p32 in the presence of PKC activators, PKC and PKC increase their binding when they are in the activated form. Other PKC isoforms such as , , and bind equally well to p32 regardless of the presence of PKC activators, and PKC binds even better in their absence. It was also found that p32 is not a substrate for any of the PKC isoforms tested, but interestingly, its presence had a stimulatory effect (2-fold for PKC) on PKC activity. We also observed in vivo interaction between PKC and p32 by immunofluorescence and confocal micros-copy. A time course of phorbol ester treatment of cultured rat hepatocytes (C9 cells) showed that PKC and p32 are constitutively associated in vivo, whereas PKC activation is required for its association with p32. Our data also showed that phorbol ester treatment induces a transient translocation of p32 from the cytoplasm to the cell nucleus. Together, these findings suggest that p32 may be a regulator of PKC location and function.

One of the main roles of epitelial membranes is to act as diffusion barriers between two compartments, one of them being the interstitium in free connection with bloodand the other representing the environment, even if this environment is sometimes reduced to the lumen of extremely small ...

Affective and cognitive factors play an important role in the activation and regulation of men’s sexual arousal. Barlow (1986) argued that initial affective reactions determine the allocation of attention to sexual stimuli. We applied Barlow’s model to our understanding of the role of sexual arousal in sexual orientation, where sexual arousal patterns have consistently been found to be congruent with self-reported orientation in men, but not in women. Visual attention of 28 heterosexual and 22 homosexual men to same- and opposite-sex erotic stimuli was assessed and experimentally-directed by means of a newly developed software application, while genital (penile rigidity) and affective responses (self-reported and physiological) were measured. In line with previous research, we found “category specificity” in men’s sexual arousal, in that sexual responses were strongest to orientation-congruent stimuli. Also, both homosexual and heterosexual men experienced stronger sexual responses to conditions in which their attention was directed to sexual versus nonsexual content of orientation-congruent stimuli. Only homosexual men manifested higher sexual responses when their visual attention was directed towards the sexual content of orientation-incongruent stimuli. Heterosexual men experienced weaker positive and stronger negative affective responses to orientation-incongruent content, suggestive of potential avoidance or inhibitory mechanisms.

Tight junctions seal the luminal border of epitelial cells and restrict the passage of substances across the paracellular route. The resistance offered by this path way is variable and depends on the number of strands, the length of the intercelular cleft, and the posible existence of compartmentalized flickering channels spanning the strands.

Giardia lamblia: Electrophysiology and ultrastructure of cytopathology in cultured epithe-lial cells. Experimental Parasitology 61, 379-389. The pathogenicity of Giardia lamblia is a subject of debate. Some studies of human biopsy material have mentioned the presence of trophozoites inside the intestinal mucosa, while in others, flagellates have only been found attached to the epithelium. To study the possible cytopathic effects of G. lamblia cultured under axenic conditions, trophozoites of the human l/Portland and WB strains were placed in contact with monolayers of Madin Derby Canine Kidney cells, a well characterized cell strain with morphological and functional properties similar to those of a transporting epithelium. After 24 and 48 hr of interaction, the effect of the parasite on epithelial cells was assessed by transmission, scanning, and freeze fracture electron microscopy. In addition , the possible action of living trophozoites and sonicates of G. lamblia on the trans-epithelial resistance of MDCK monolayers mounted in Ussing chambers was analyzed for periods varying up to 48 hr. The results demonstrate that G. lamblia trophozoites do not invade epithelial monolayers. Furthermore, the parasites fail to produce cytoplasmic changes on target cells and have no effect on transepithelial resistance as judged both elec-trophysiologically and by the failure to open the occluding junctions that bind together epithelial cells. Damage induced by the parasites to cultured cells was limited to focal distortion or depletion of microvilli at the site of adhesion, which may progress to leave circular areas devoid of microvilli, different from the adhesion marks reported by others for G. murk. Therefore, under the in vitro conditions described here, giardias showed no toxic or inVaSiVe &e&2.

Severe dengue (SD) is a life-threatening complication of dengue that includes vascular permeability syndrome (VPS) and respiratory distress. Secondary infections are considered a risk factor for developing SD, presumably through a mechanism called antibody-dependent enhancement (ADE). Despite extensive studies, the molecular bases of how ADE contributes to SD and VPS are largely unknown. This work compares the cytokine responses of differentiated U937 human monocytic cells infected directly with dengue virus (DENV) or in the presence of enhancing concentrations of a humanized monoclonal antibody recognizing protein E (ADE-DENV infection). Using a cytometric bead assay, ADE-DENV-infected cells were found to produce significantly higher levels of the proinflammatory cytokines interleukin 6 (IL-6), IL-12p70, and tumor necrosis factor alpha (TNF-), as well as prostaglandin E 2 (PGE 2), than cells directly infected. The capacity of conditioned supernatants (conditioned medium [CM]) to disrupt tight junctions (TJs) in MDCK cell cultures was evaluated. Exposure of MDCK cell monolayers to CM collected from ADE-DENV-infected cells (ADE-CM) but not from cells infected directly led to a rapid loss of transepithelial electrical resistance (TER) and to delocalization and degradation of apical-junction complex proteins. Depletion of either TNF-, IL-6, or IL-12p70 from CM from ADE-DENV-infected cells fully reverted the disrupting effect on TJs. Remarkably, mice injected intraperitoneally with ADE-CM showed increased vascular permeability in sera and lungs, as indicated by an Evans blue quantification assay. These results indicate that the cytokine response of U937-derived macrophages to ADE-DENV infection shows an increased capacity to disturb TJs, while results obtained with the mouse model suggest that such a response may be related to the vascular plasma leakage characteristic of SD.

SUMMARY MDCK cells (epithelioid, derived from the kidney of a normal dog) cultured in monolayers on a permeable support, exhibit properties of natural transporting epithelia. Comparisons of the electrical resistance across the plasma membrane of MDCK cells (as studied with microelectrodes) and the resistance across the whole monolayer, (mounted as a flat sheet between two chambers) indicate that most of the current flows through an extracellular pathway. Scanning of the electrical field over the apical surface shows that this pathway is located at the intercellular space. Yet conductance is not evenly distributed along the intercellular space as in leaky epithelia, but is restricted to sites scattered irregularly along the intercellular space. Studies of freeze fracture electron microscopy indicate that the number of strands of the junctions is also distributed irregularly, varying from 1 to 10 in a few nanometers. This suggests that regions with few strands would correspond to spots with high conductance and vice versa. However, in this preparation the sealing property of the junction bears little relationship to its structure. Thus by changing the temperature from 37 to 3 °C and back, the electrical resistance increases reversibly by 306%, while the number and arrangement of the strands show no significant modification. The resistance of the monolayer varies also with the age of the cells, suggesting that sealing and ion-permeating components of the junction may be dynamic entities that are not permanently installed, but can be accommodated to the requirements of the tissue.

Objective: There is much interest in the role of dietary protein for weight control. However, there remains a need to characterize individual determinants of the thermogenic effects of protein. This study aimed to investigate the influence of menstrual cycle phase and the combined, monophasic oral contraceptive pill on the thermogenic response to a standardized high-protein (HP) versus normal-protein (NP) meal. Methods: Following an overnight fast, resting energy expenditure (EE) was measured in 16 healthy young women (8 taking and 8 not taking the pill) and 8 men for 30 minutes pre ingestion and 3 hours post ingestion of a NP (11%) or HP (24%) meal. Results: There was no effect of menstrual phase or contraceptive pill use on fasting EE or NP response. However, HP increased EE significantly more than NP in women not taking the oral contraceptive pill and in men, but not in women taking the pill. Conclusions: This study shows an absence of the greater thermic effect of HP versus NP in women taking the oral contraceptive pill and has important implications regarding the effectiveness of HP for body weight regulation in women. With current obesity treatment/prevention strategies remaining largely ineffective , understanding the relationship between oral contraceptive pill use and protein-induced thermo-genesis may enable the successful recalibration of existing dietary recommendations.

Cells from transporting epithelia have two basic properties: 1) a plasma membrane divided into an apical and a basolateral domain that confers vectoriality, and 2) tight junctions that transform epithelia into diffusion barriers. The expression of both properties depends on extracelular Ca2+.

Claudins are integral proteins of the TJ. Each epithelia in the organism expresses a unique set of claudins that determines the degree of sealing of the paracellular pathway and the ionic selectivity of the tissue. TJs are dynamic structures whose organization and composition change in response to alterations in the environment as well as under physiological and pathological conditions. Changes in claudin expression and subcellular distribution can be analyzed in western blot and immunofluorescence experiments, employing a wide array of available specific antibodies against claudins. In this chapter, we describe in detail protocols used for western blot and immunofluorescence detection of claudins in epithelial cell lines and in various tissue samples. Tight junctions (TJs) in epithelial cells localize at the most apical segment of the lateral membrane. TJ are integrated by a complex array of integral and peripheral proteins, among which claudins play a crucial role as back bone constituents of the filaments observed by freeze-fracture electron microscopy. The filaments that encircle epithelial cells below the apical microvilli are formed by lines of transmembrane particles with a 10 nm diameter that fuse when fixed with glutaraldehyde (1, 2). These particles are composed of claudin multimers, and their formation can be triggered upon claudin transfection, even in cells that lack TJs such as fibroblasts (3).

[Abstract] Dengue virus infection usually courses as a benign self-limited fever, called dengue fever. However, on occasions it can progress to a life-threatening complication known as severe dengue (SD). A hallmark of SD is a sharp increase in vascular permeability. Secondary infections are considered a risk factor to develop SD, presumably through a mechanism called Antibody-Dependent Enhancement (ADE) of infection in cells with the capacity to bind antigen-antibody complexes, such as macrophages, and to trigger a subsequent aberrant cytokine response. The massive release of cytokine from macrophages has been postulated to cause changes in vascular permeability. The vascular permeability assay presented in this protocol is designed to assess whether any compound or cell-secreted product or soluble factor present in sera from patients may induce plasma leakage in mice. This test was used in the laboratory to determine whether cytokines and soluble factors produced in vitro by macrophages infected with dengue virus or dengue virus in the presence of facilitating antibodies are able to induce plasma leakage in vivo. Macrophages were infected with dengue virus or dengue virus in the presence of facilitating antibodies for 48 hours. After this time, the conditioned supernatant containing cytokines and soluble factors released by the macrophages were collected and inoculated intraperitoneally into CD-1 mice. Twenty four hours after the first inoculation, mice were reinoculated with a second dose with Evans blue dye. After another 24 h, mice were euthanized and the amount of Evans blue present in the blood and lung was determined by spectrophotometric analysis. The assay was able to show differences in the capacity of the conditioned media to induce vascular permeability changes in the inoculated animals (Puerta-Guardo et al., 2013).

Testosterone is a steroid hormone involved in the expression of many morphological, physiological, and behavioral traits that arguably affect reproductive success. The evidence for that link is, however, incomplete or absent in the research on crocodilian species. Testosterone levels are also known to change throughout the breeding season, often on an hourly basis, which may further complicate studying their relationship with breeding success. We tested here whether baseline testosterone levels, measured out of the breeding season, are correlated with morphometry and reproductive success in Spectacled Caiman (Caiman crocodilus (Linnaeus, 1758)). Paternity tests, based on the amplification and genotyping of eight fluorochrome labeled microsatellites, failed to support a continuous relationship between these variables. Although adult males of all sizes contribute to reproduction, paternity was overrepresented in a few males with high values of maleness index (bigger males), supporting a despotic or pyramidal hierarchy among males. Maternity assignments supported the existence of multiple paternity, a phenomenon previously attributed in this species to the lack of large males caused by human hunting. The idea of larger males having more offspring is widespread in crocodilians, but to our knowledge, this is the first investigation that prove this dogma.

Background Acute and chronic effects of nitric oxide on oxidative stress and neuronal demyelination have been reported in human and animal models. Objective Oxidative stress is involved in the pathogenesis of demyelinated animals; thus, this study investigated the effects of nitric oxide system and ethidium bromide on oxidative stress in the hippocampal formation. Methods This experimental study was performed on eight groups of male rats, as follows: control, control, ethidium bromide, L-Arginine, L-NAME, ethidium bromide + L-Arginine, ethidium bromide + L-NAME, and ethidium bromide + L-Arginine + L-NAME. Three days after the injection of drugs into the hippocampal CA1 area, hippocampal biopsy was performed, and oxidative stress parameters were measured in this area. One-way analysis of variance (ANOVA) and posthoc tests were used for data analysis. Findings Injection of 3 μL ethidium bromide into the CA1 region increased oxidative stress parameters (P<0.01). Injection of 15.3 μg/rat L-Arginine in this region stopped the response of ethidium bromide (P<0.05, P<0.01); while, the injection of 15.1 μg/rat L-NAME failed to return the effects of the ethidium bromide. Moreover, the combination of 15.3 μg/rat L-Arginine and 15.1 μg/rat L-NAME only returned lipid peroxidation caused by the injection of 3 μL of ethidium bromide to normal (P<0.05); this process failed to improve antioxidant enzymes. Conclusion The obtained results suggested the excitatory effect of the nitric oxide system on alleviating oxidative stress induced by ethidium bromide in the CA1 region of male rats.

Emperipolesis is considered a physiological phenomena often present in various pathophysiological conditions, but its etiology is still unknown. In this study, we analyzed the number of megakaryocytes and the percentage of emperipoletic cells in the sternal and femoral bone marrow of Wistar rats. Five types in the thrombopoiesis lineage (megakaryoblasts, promegakaryocytes and megakaryocytes-acidophilic, basophilic and thrombocytogenic) were determined. Except for basophilic megakaryocytes, significant differences were found for number of thrombopoietic cells in the sternal and femoral bone marrow. A larger number of thrombocytogenic megakaryocytes were present in the sternal bone marrow. Emperipoletic cells were significantly present in the femoral compared to the sternal bone marrow. Emperipolesis was typical for lymphocytes and neutrophils individually, while emperipolesis with two or more cells within thrombopoietic cell was also present (1-7 %) and significant differences between the sternal and femoral bone marrow were detected. Emperipolesis was found in all analysed rats and it most commonly occured within mature megakaryocytes and rarely megakaryoblasts, while it was not recorded in the promegakaryocytes. The high incidence of megakaryocytes with emperopolesis in rats could be a consequence of "normal" cell retention in the cytoplasm of megakaryocytes while passing blood cells to circulation or related to haematopoietic response due to high incidence of inbreeding.

Se analiza la relación de la teleología platónica y la fisiología médica de Alcmeón de Crotona y del  papel hígado en  el Timeo de Platón, su  función  en  el  cuerpo  y  su  relación con el alma apetitiva. A continuación, se explora la relación del hígado y el alma apetitiva  con el hombre tirano y la tiranía tal y como son caracterizados  en  la  República,  el  Timeo  y  las  Leyes, y la correspondencia entre fisiología, psicología  y  política.

In the context of the great philosophical synthesis of Francis Suárez, between XVI and XVII centuries, his commentary to De Anima manifest the originality of his thought, between continuity and innovation in front of the big issues that Aristotle’s work had left open. His reflection on the problems of knowledge will open the path to a modern way of thinking, not exempt of some critical aspects. As he recognizes the active role of sensitive powers and he analyzes their physiological dimension, he will reduce inner sensitivity to only one power. The characterization of this only inner sense requires an adequate distinction of it as regards both external sensitivity and intellectual power, and implies some localization in the brain. Surarez’s dialogue with the physiology of his time places him at the historical beginning of neuroscience.

Continuous inability of Mozambique's National Assembly to timely respond to issues of common national interest presented by the opposition parties has prompted me to equate the FRELIMO party's Members of Parliament (MPs) in Mozambique to a Grouplink. This paper briefly describes how the behavior of FRELIMO caucus has caused Mozambicans to miss many opportunities to prevent a return to the now re-emerging civil war, and to avoid the ongoing embarrassment caused by hidden loans, characterized by withdrawal of financial support by the Bretton Wood's Financial Institutions, and by some of Mozambique's most assiduous bilateral cooperation partners known as G-19, followed by the United States of America and Canada. It further criticizes the inability of FRELIMO MPs to co-exist with their peers from other political parties and pins this behavior down to groupthink mentality and the phenomenon's 8 symptoms. Deputados com Pensamento Grupal A bancada parlamentar da FRELIMO na Assembleia da República nunca conseguiu tomar uma decisão inteligente em prôl do bem-estar do povo moçambicano e da Nação. O exemplo disso é a multiplicação de projectos que bem vistos, tinham um certo mérito e, por isso, deviam ter sido encarados com mais seriedade, o que não aconteceu devido à leviandade desta bancada parlamentar. Num esforço de garantirem o reconhecimento do seu trabalho pelo partido FRELIMO, os membros da bancada parlamentar da FRELIMO invariavelmente tomaram, e continuam a tomar as suas decisões de votação sem uma análise crítica profunda, limitando-se apenas a guiar-se por um orgulho desmedido corporizado na reprovação sistemática de tudo que vem da oposição. Algumas iniciativas mais críticas para a vida nacional da iniciativa da oposição que careciam de um parecer nacionalista favorável da bancada parlamentar da FRELIMO mas que foram reprovadas pela mesma devido ao seu pensamento grupal incluem ass seguintes: • A Revisão e alteração dos símbolos nacionais; • Autarcisação das províncias • Revisão da Constituição da República, para acomodar a autarcisação das províncias; • Pedido de comparência do governo na Assembleia da República, para explicar os contornos da dívida da EMATUM.

Anticarsia gemmatalis and Spodoptera frugiperda (Lepidoptera: Noctuidae) are serious defoliators of a number of crops. In order to better understand the digestion process of these pests and considering that the midgut is one of the most important and attractive targets for biological pesticides, this report shows the evaluation of midgut proteins from A. gemmatalis and S. frugiperda by proteomic analyses.