The inhibitor protein of the F1F0-ATP synthase is associated to the external surface of endothelial cells

The ATPase inhibitor protein (IP) of mitochondria was detected in the plasma membrane of living endothelial cells by flow cytometry, competition assays, and confocal microscopy of cells exposed to IP antibodies. The plasma membranes of endothelial cells also possess beta-subunits of the mitochondrial ATPase. Plasma membranes have the capacity to bind exogenous IP. TNF-alpha decreases the level of beta-subunits and increases the amount of IP, indicating that the ratio of IP to beta-subunit exhibits significant variations. Therefore, it is probable that the function of IP in the plasma membrane of endothelial cells is not limited to regulation of catalysis.     

PI 3 kinase-dependent Akt kinase and PKCepsilon independently regulate interferon-gamma-induced STAT1alpha serine phosphorylation to induce monocyte chemotactic protein-1 expression

Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-gamma (IFN-gamma) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-gamma stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-gamma-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-gamma-induced MCP-1 protein and mRNA expression. IFN-gamma increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1alpha, without any effect on its tyrosine phosphorylation, and decreased IFN-gamma-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCepsilon is a downstream target of PI 3 kinase in IFN-gamma signaling. Similar to dominant negative Akt kinase, dominant negative PKCepsilon also inhibited serine phosphorylation of STAT1alpha without any effect on tyrosine phosphorylation. Dominant negative PKCepsilon also abrogated MAPK activity, resulting in decrease in IFN-gamma-induced MCP-1 expression. Furthermore, Akt and PKCepsilon are present together in a signaling complex. IFN-gamma had no effect on this complex formation, but did increase PKCepsilon-associated Akt kinase activity. PKCepsilon did not regulate IFN-gamma-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-gamma-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCepsilon activation independently regulate MAPK activity and serine phosphorylation of STAT1alpha to increase expression of MCP-1.     

Citogenetic characterization of the tropical freshwater fish Parachromis managuensis (Pisces: Cichlidae)

To describe the cytogenetics of the jaguar cichlid fish Parachromis managuensis, we collected eight males and 13 females in Villahermosa, Tabasco, México. The specimens were processed with standard cytogenetic techniques (slightly modified), and high quality fields of chromosomes in mitosis and meiosis were obtained; 14 of these fields were analyzed by meristics and statistics methods. The specimens presented a diploid modal number of 2n = 48 chromosomes, which is similar to the number reported for others Central American cichlids; five pairs were submetacentric-metacentrics (biarmed) and 19 were subtelocentric-telocentric (uni-armed), giving a fundamental number (NF) of 58. The haploid number was confirmed by counting meiotic fields in metaphase I. There was not evidence of heteromorphism: sexual chromosomes were not identifiable.     

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 × 10⁶ to 3 × 10⁶ cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon “Haloquadra walsbyi,” although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the γ-proteobacterium “Pseudomonas halophila” DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the “P. halophila” cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

Functional and Structural Effects of Amyloid-β Aggregate on Xenopus laevis Oocytes

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of “spontaneous” blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca²⁺ was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca²⁺-dependent Cl⁻ currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T_out) and the serum-activated, oscillatory Cl⁻ currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca²⁺-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.     

Autocrine signaling involved in cell volume regulation: the role of released transmitters and plasma membrane receptors

Cell volume regulation is a basic homeostatic mechanism transcendental for the normal physiology and function of cells. It is mediated principally by the activation of osmolyte transport pathways that result in net changes in solute concentration that counteract cell volume challenges in its constancy. This process has been described to be regulated by a complex assortment of intracellular signal transduction cascades. Recently, several studies have demonstrated that alterations in cell volume induce the release of a wide variety of transmitters including hormones, ATP and neurotransmitters, which have been proposed to act as extracellular signals that regulate the activation of cell volume regulatory mechanisms. In addition, changes in cell volume have also been reported to activate plasma membrane receptors (including tyrosine kinase receptors, G-protein coupled receptors and integrins) that have been demonstrated to participate in the regulatory process of cell volume. In this review, we summarize recent studies about the role of changes in cell volume in the regulation of transmitter release as well as in the activation of plasma membrane receptors and their further implications in the regulation of the signaling machinery that regulates the activation of osmolyte flux pathways. We propose that the autocrine regulation of Ca2+-dependent and tyrosine phosphorylation-dependent signaling pathways by the activation of plasma membrane receptors and swelling-induced transmitter release is necessary for the activation/regulation of osmolyte efflux pathways and cell volume recovery. Furthermore, we emphasize the importance of studying these extrinsic signals because of their significance in the understanding of the physiology of cell volume regulation and its role in cell biology in vivo, where the constraint of the extracellular space might enhance the autocrine or even paracrine signaling induced by these released transmitters.