Calcium waves induced by large voltage pulses in fish keratocytes.

Intracellular calcium waves in fish keratocytes are induced by the application of electric field pulses with amplitudes between 55 and 120 V/cm and full width at half-maximum of 65-100 ms. Calcium concentrations were imaged using two-photon excited fluorescence microscopy (Denk et al., 1990 Science. 248:73-76; Williams et al. 1994 FASEB J. 8:804-813) and the ratiometric calcium indicator indo-1. The applied electric field pulses induced waves with fast calcium rise times and slow decays, which nucleated in the lamellipodium at the hyperpolarized side of the cells and, less frequently, at the depolarized side. The effectiveness of wave generation was determined by the change induced in the membrane potential, which is about half the field strength times the cell width in the direction of the field. Stimulation of waves began at voltage drops across the cell above 150 mV and saturated at voltage drops above 300 mV, where almost all cells exhibited a wave. Waves were not induced in low-calcium media and were blocked by the nonselective calcium channel blockers cobalt chloride and verapamil, but not by specific organic antagonists of voltage-sensitive calcium channel conductance. Thapsigargin stopped wave propagation in the cell body, indicating that calcium release from intracellular stores is necessary. Thus a voltage pulse stimulates Ca2+ influx through calcium channels in the plasma membrane, and if the intracellular calcium concentration reaches a threshold, release from intracellular stores is induced, creating a propagating wave. These observations and the measured parameters (average velocity approximately 66 micron/s and average rise time approximately 68 ms) are consistent with a wave amplification model in which[equation, see text] determines the effective diffusivity of the propagating molecules, D approximately 300 micron2/s (Meyer, 1991. Cell. 64:675-678).     

Cyclic strain increases fibroblast proliferation, matrix accumulation, and elastic modulus of fibroblast-seeded polyurethane constructs

Rapid induction of matrix production and mechanical strengthening is essential to the development of bio-artificial constructs for repair and replacement of load-bearing connective tissues. Toward this end, we describe the development of a mechanical bioreactor and its application to investigate the influence of cyclic strain on fibroblast proliferation, matrix accumulation, and the mechanical properties of fibroblast-seeded polyurethane constructs (FSPC). Human fibroblasts were cultured in 10% serum-containing conditions within three-dimensional, porous elastomeric substrates under static conditions and a model regime of cyclic strain (10% strain, 0.25 Hz, 8 h/day), with and without ascorbic acid supplementation. After one week, the combination of cyclic strain and ascorbic acid resulted in significantly increased construct elastic modulus (>110%) relative to either condition alone. In contrast, cyclic strain alone was sufficient to stimulate significant increases in fibroblast proliferation. Mechanical strengthening of FSPCs was accompanied by increased type I collagen and fibronectin matrix accumulation and distribution, and significantly increased gene expression for type I collagen, TGFbeta-1, and CTGF. These results suggest that strain-induced conditioning in vitro leads to mechanical strengthening of fibroblast/material constructs, most likely resulting from increased collagen matrix deposition, secondary to strain-induced increases in cytokine production.     

Transduction of the IFN-gamma signal for HLA-DR expression in the promonocytic line THP-1 involves a late-acting PKC activity

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7. H7 when added up to 20 hr after the initial stimulation with IFN-gamma prevented the further expression of HLA-DR. The general kinase inhibitors H8, H9, and HA1004, all less potent PKC inhibitors than H7, did not block the IFN-gamma-induced expression of HLA-DR at the concentrations employed. W7, a calmodulin antagonist, but not a PKC inhibitor, was also unable to prevent the IFN-gamma-induced expression of HLA-DR. Treatment of THP-1 with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, alone or with Ca2+ ionophore A23187, was unable to induce HLA-DR expression. However, pretreatment with PMA for 24 hr prior to IFN-gamma stimulation decreased the IFN-gamma-induced expression of HLA-DR without decreasing IFN-gamma receptor levels. These results suggest that PKC plays a significant role in the IFN-gamma-induced signal transduction pathway leading to the expression of HLA-DR in cells of the mononuclear phagocytic lineage, and that PKC activity is required throughout the course of events leading to the actual expression of HLA-DR.     

Pharmacological characterization of TMEM16A currents

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC_inhA01 and T16A_inhA01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HEK 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC_inhA01 and T16A_inhA01 inhibited TMEM16A currents with IC₅₀ values of 12, 58, 1.7 and 1.5 µM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC_inhA01 and T16A_inhA01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC_inhA01 and T16A_inhA01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.     

Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases: IMPLICATIONS FOR THE REGULATION OF ADRENAL STEROIDOGENESIS*

Group X secretory phospholipase A₂ (GX sPLA₂) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA₂ is produced as a pro-enzyme (pro-GX sPLA₂) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA₂ activation have not been identified. We previously reported that GX sPLA₂ negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA₂ expression construct (FLAG-pro-GX sPLA₂), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA₂ processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA₂ processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA₂ processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA₂ to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA₂ processing and sPLA₂ activity in Y1 cells, and it significantly attenuated GX sPLA₂-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA₂ is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.     

Visual Cues Given by Humans Are Not Sufficient for Asian Elephants (Elephas maximus) to Find Hidden Food

Recent research suggests that domesticated species – due to artificial selection by humans for specific, preferred behavioral traits – are better than wild animals at responding to visual cues given by humans about the location of hidden food. \Although this seems to be supported by studies on a range of domesticated (including dogs, goats and horses) and wild (including wolves and chimpanzees) animals, there is also evidence that exposure to humans positively influences the ability of both wild and domesticated animals to follow these same cues. Here, we test the performance of Asian elephants (Elephas maximus) on an object choice task that provides them with visual-only cues given by humans about the location of hidden food. Captive elephants are interesting candidates for investigating how both domestication and human exposure may impact cue-following as they represent a non-domesticated species with almost constant human interaction. As a group, the elephants (n = 7) in our study were unable to follow pointing, body orientation or a combination of both as honest signals of food location. They were, however, able to follow vocal commands with which they were already familiar in a novel context, suggesting the elephants are able to follow cues if they are sufficiently salient. Although the elephants’ inability to follow the visual cues provides partial support for the domestication hypothesis, an alternative explanation is that elephants may rely more heavily on other sensory modalities, specifically olfaction and audition. Further research will be needed to rule out this alternative explanation.     

Colon cancer associated genes exhibit signatures of positive selection at functionally significant positions

ABSTRACT: BACKGROUND: Cancer, much like most human disease, is routinely studied by utilizing model organisms. Of these model organisms, mice are often dominant. However, our assumptions of functional equivalence fail to consider the opportunity for divergence conferred by ~180 Million Years (MY) of independent evolution between these species. For a given set of human disease related genes, it is therefore important to determine if functional equivalency has been retained between species. In this study we test the hypothesis that cancer associated genes have different patterns of substitution akin to adaptive evolution in different mammal lineages. RESULTS: Our analysis of the current literature and colon cancer databases identified 22 genes exhibiting colon cancer associated germline mutations. We identified orthologs for these 22 genes across a set of high coverage (>6X) vertebrate genomes. Analysis of these orthologous datasets revealed significant levels of positive selection. Evidence of lineage-specific positive selection was identified in 14 genes in both ancestral and extant lineages. Lineage-specific positive selection was detected in the ancestral Euarchontoglires and Hominidae lineages for STK11, in the ancestral primate lineage for CDH1, in the ancestral Murinae lineage for both SDHC and MSH6 genes and the ancestral Muridae lineage for TSC1. CONCLUSION: Identifying positive selection in the primate, Hominidae, Muridae and Murinae lineages suggest an ancestral functional shift in these genes between the rodent and primate lineages. Analyses such as this, combining evolutionary theory and predictions - along with medically relevant data, can thus provide us with important clues for modeling human diseases.     

Indirect oxidation of Co(II) in the presence of the marine Mn(II)-oxidizing bacterium Bacillus sp. strain SG-1

Cobalt(II) oxidation in aquatic environments has been shown to be linked to Mn(II) oxidation, a process primarily mediated by bacteria. This work examines the oxidation of Co(II) by the spore-forming marine Mn(II)-oxidizing bacterium Bacillus sp. strain SG-1, which enzymatically catalyzes the formation of reactive nanoparticulate Mn(IV) oxides. Preparations of these spores were incubated with radiotracers and various amounts of Co(II) and Mn(II), and the rates of Mn(II) and Co(II) oxidation were measured. Inhibition of Mn(II) oxidation by Co(II) and inhibition of Co(II) oxidation by Mn(II) were both found to be competitive. However, from both radiotracer experiments and X-ray spectroscopic measurements, no Co(II) oxidation occurred in the complete absence of Mn(II), suggesting that the Co(II) oxidation observed in these cultures is indirect and that a previous report of enzymatic Co(II) oxidation may have been due to very low levels of contaminating Mn. Our results indicate that the mechanism by which SG-1 oxidizes Co(II) is through the production of the reactive nanoparticulate Mn oxide.     

Genetic variation in the 6p22.3 gene DTNBP1, the human ortholog of the mouse dysbindin gene,is associated with schizophrenia

Prior evidence has supported the existence of multiple susceptibility genes for schizophrenia. Multipoint linkage analysis of the 270 Irish high-density pedigrees that we have studied, as well as results from several other samples, suggest that at least one such gene is located in region 6p24-21. In the present study, family-based association analysis of 36 simple sequence-length-polymorphism markers and of 17 SNP markers implicated two regions, separated by approximately 7 Mb. The first region, and the focus of this report, is 6p22.3. In this region, single-nucleotide polymorphisms within the 140-kb gene DTNBP1 (dystrobrevin-binding protein 1, or dysbindin) are strongly associated with schizophrenia. Uncorrected, empirical P values produced by the program TRANSMIT were significant (P<.01) for a number of individual SNP markers, and most remained significant when the data were restricted to include only one affected offspring per nuclear family per extended pedigree; multiple three-marker haplotypes were highly significant (P=.008-.0001) under the restricted conditions. The pattern of linkage disequilibrium is consistent with the presence of more than one susceptibility allele, but this important issue is unresolved. The number of markers tested in the adjacent genes, all of which are negative, is not sufficient to rule out the possibility that the dysbindin gene is not the actual susceptibility gene, but this possibility appears to be very unlikely. We conclude that further investigation of dysbindin is warranted.     

Seasonal timing of inundation affects riparian plant growth and flowering: implications for riparian vegetation composition

Changes to the timing of peak river flows caused by flow regulation affect riparian vegetation composition, but the mechanisms driving such vegetation changes are not well understood. We investigated experimentally the effects of timing of inundation on riparian plant growth and flowering. We collected 168 sods from 14 sites across five lowland rivers in south-eastern Australia. Plant cover and flowering within the sods were surveyed each season for a year. During this period, sods were inundated for 6 weeks in either early spring or in summer. Terrestrial plant taxa (which included most exotic species) senesced in response to inundation, regardless of its timing. In contrast, native amphibious species (particularly amphibious forbs) responded favourably to inundation in spring, but were unaffected by inundation in summer. Native and exotic emergent macrophytes responded favourably to inundation regardless of timing, and flowered frequently in both the spring- and the summer-inundation treatments. In contrast, many native annuals flowered only in the spring-inundation treatment, while more exotic grasses flowered in the summer-inundation treatment. In temperate climates, inundation in early spring followed by non-flooded conditions is likely to be important for promoting the growth of amphibious forbs and the recruitment and flowering of riparian annuals. Without inundation in spring, many terrestrial exotic weeds may flourish and set seed prior to any subsequent inundation (e.g. in summer). We contend that natural seasonal timing (i.e. winter-early spring) of flow peaks is important for the maintenance of native riverbank vegetation and reducing the extent of terrestrial exotic species within the riparian zone.