Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell’s lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole-cell patch clamp results in a 50% increase in cell volume, whereas hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high-potassium solution causes volume increase up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and the concentrations of permeable ions in the cells surrounding. The model quantitatively predicts that the cell volume is directly proportional to the intracellular protein content.     

The use of fluorescent powders to track autocontamination of emerald ash borer (Coleoptera: Buprestidae) by the entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

We evaluated the use of fluorescent powders for tracking dispersal by the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), of Beauveria bassiana isolates from an autocontamination device. Neither of the two DayGlow powders tested (Arc Yellow and Aurora Pink) interfered with fungal germination or growth, nor did they affect survival of beetles in the laboratory, or affect virulence of the fungus. The powders persisted at least 10 days out-of-doors on dead beetles in sticky band traps, and at least 14 days on pouches inside autocontamination traps. During field trials of autocontamination traps with powder-dusted fungal pouches in southwestern Ontario, 8.0% of the 4010 beetles captured in green prism and sticky-band traps were positive for fluorescent powders. Only half (46.2–57.8%) of the powder-positive beetles actually carried viable fungal conidia, as determined by plating of beetle rinses, possibly as a result of patchy growth of fungal isolates and reduced conidia production on pouch surfaces during the 16-day trapping experiment. The presence of viable conidia (either one or both isolates) on about 10% of beetles that did not carry any visible powder particles may be an indication of horizontal transmission of the fungus by beetles that had visited the autocontamination traps.     

Effect of four antimicrobials against an Encephalitozoon sp. (Microsporidia) in a grasshopper host

Encephalitozoon spp. are the primary microsporidial pathogens of humans and domesticated animals. In this experiment, we test the efficacy of 4 commercial antimicrobials against an Encephalitozoon sp. infecting a grasshopper (Romalea microptera) host. Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts (93% and 88% respectively), whereas spore counts of grasshoppers fed quinine produced a non-significant 53% reduction in spores, and those fed streptomycin a non-significant 29% increase in spores, compared to the control. We observed a moderate dose-response effect for thiabendazole, whereby spore count decreased as drug consumption increased. No thiabendazole-treated animals died, whereas 27% of streptomycin-treated animals died, suggesting that thiabendazole was not toxic at the doses administered. The deaths among streptomycin-treated animals may have been caused by drug toxicity, parasite burden, or both. Although fumagillin and thiabendazole significantly reduced spore counts, in no individual was the pathogen totally eliminated. Our data confirm that microsporidia are difficult to control and that fumagillin and thiabendazole are partially effective antimicrobials against this group. Our study suggests that quinine and related alkaloids should be further examined for antimicrosporidial activity, and streptomycin should be examined as a possible enhancer of microsporidiosis.     

Nuclear membrane receptors for ET-1 in cardiovascular function

Plasma membrane endothelin type A (ET(A)) receptors are internalized and recycled to the plasma membrane, whereas endothelin type B (ET(B)) receptors undergo degradation and subsequent nuclear translocation. Recent studies show that G protein-coupled receptors (GPCRs) and ion transporters are also present and functional at the nuclear membranes of many cell types. Similarly to other GPCRs, ET(A) and ET(B) are present at both the plasma and nuclear membranes of several cardiovascular cell types, including human cardiac, vascular smooth muscle, endocardial endothelial, and vascular endothelial cells. The distribution and density of ET(A)Rs in the cytosol (including the cell membrane) and the nucleus (including the nuclear membranes) differ between these cell types. However, the localization and density of ET-1 and ET(B) receptors are similar in these cell types. The extracellular ET-1-induced increase in cytosolic ([Ca](c)) and nuclear ([Ca](n)) free Ca(2+) is associated with an increase of cytosolic and nuclear reactive oxygen species. The extracellular ET-1-induced increase of [Ca](c) and [Ca](n) as well as intracellular ET-1-induced increase of [Ca](n) are cell-type dependent. The type of ET-1 receptor mediating the extracellular ET-1-induced increase of [Ca](c) and [Ca](n) depends on the cell type. However, the cytosolic ET-1-induced increase of [Ca](n) does not depend on cell type. In conclusion, nuclear membranes' ET-1 receptors may play an important role in overall ET-1 action. These nuclear membrane ET-1 receptors could be targets for a new generation of antagonists.     

Isolation and characterization of NaCl resistant cell line of mulberry

The selected NaCl tolerant clones ofMorus alba L. cv. MR₂ grow better at higher concentration of NaCl than non-selected clones. With increasing NaCl concentration the Na⁺, Cl^? and proline content increased more and K⁺ and Ca²⁺ content decrease less in selected clones in comparison with non-selected ones.     

Enterotoxigenicity and frequency of Campylobacter jejuni, C. coli and C. laridis in human and animal stool isolates from different countries

Campylobacter jejuni and C. coli strains were collected during three different years from adult patients with enterocolitis in Sweden (n = 372) from 49 patients in Kuwait, and Campylobacter strains from hens from Mexico, Pakistan and Sweden (n = 107) and Swedish pigs (n = 47). C. jejuni was the predominant species in human and hen isolates, and C. coli in pigs C. coli was significantly more common in human isolates from Sweden, and more common in hen isolates from Pakistan, than in hens from Sweden and Mexico. C. laridis was only isolated from pigs (17%) and was in no case enterotoxigenic. Both in human and hen isolates, C. jejuni strains were more enterotoxigenic than C. coli strains. C. jejuni strains from Swedish hens were less enterotoxigenic than those from Pakistan and Mexico (P less than 0.001), and strains from pigs were less enterotoxigenic than those from hens (P less than 0.001). We conclude that C. jejuni are more often enterotoxigenic and possibly more virulent than c. coli and C. laridis. The relative frequency of C. jejuni and C. coli in humans and animals differs from one country to another.     

Isolation and characterisation of Isaria farinosa and Purpureocillium lilacinum associated with emerald ash borer,Agrilus planipennis in Canada

Entomopathogenic fungi of the genera Isaria and Purpureocillium were recovered from infestation sites of emerald ash borer (EAB) in Southern Ontario, Canada. Isolates were identified using morphological characters and by sequencing the ITS1-5.8S-ITS2 ribosomal DNA gene and partial β-tubulin gene. Phylogenetic analysis and constructed trees based on the ITS and β-tubulin gene explicitly confirm isolates L66B, SY17-a and LHY46-a as Isaria farinosa and B3A, B59A and SY45B-a as Purpureocillium lilacinum. Pathogenicity was tested in the laboratory against adult EAB using a single concentration (2.0×10⁷ conidia/ml) applied topically to adults. Controls included three commercial isolates: Isaria fumosorosea LRC176, Metarhizium brunneum LRC187 and Beauveria bassiana strain GHA. The native isolates I. farinosa L66B and P. lilacinum SY45B-a killed 75 and 50% of the beetles 14 days post-inoculation. Although these indigenous entomogenous fungi were less virulent compared with the commercial isolates, yearly isolation from EAB populations suggests they are one of the natural mortality factors of EAB in Canada.     

Brassinosteroids antagonize gibberellin- and salicylate-mediated root immunity in rice

Brassinosteroids (BRs) are a unique class of plant steroid hormones that orchestrate myriad growth and developmental processes. Although BRs have long been known to protect plants from a suite of biotic and abiotic stresses, our understanding of the underlying molecular mechanisms is still rudimentary. Aiming to further decipher the molecular logic of BR-modulated immunity, we have examined the dynamics and impact of BRs during infection of rice (Oryza sativa) with the root oomycete Pythium graminicola. Challenging the prevailing view that BRs positively regulate plant innate immunity, we show that P. graminicola exploits BRs as virulence factors and hijacks the rice BR machinery to inflict disease. Moreover, we demonstrate that this immune-suppressive effect of BRs is due, at least in part, to negative cross talk with salicylic acid (SA) and gibberellic acid (GA) pathways. BR-mediated suppression of SA defenses occurred downstream of SA biosynthesis, but upstream of the master defense regulators NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 and OsWRKY45. In contrast, BR alleviated GA-directed immune responses by interfering at multiple levels with GA metabolism, resulting in indirect stabilization of the DELLA protein and central GA repressor SLENDER RICE1 (SLR1). Collectively, these data favor a model whereby P. graminicola coopts the plant BR pathway as a decoy to antagonize effectual SA- and GA-mediated defenses. Our results highlight the importance of BRs in modulating plant immunity and uncover pathogen-mediated manipulation of plant steroid homeostasis as a core virulence strategy.     

G-protein-coupled receptors, channels, and Na+-H+ exchanger in nuclear membranes of heart, hepatic, vascular endothelial, and smooth muscle cells

The action of several peptides and drugs is thought to be primarily dependent on their interactions with specific cell surface G-protein-coupled receptors and ionic transporters such as channels and exchangers. Recent development of 3-D confocal microscopy allowed several laboratories, including ours, to identify and study the localization of receptors, channels, and exchangers at the transcellular level of several cell types. Using this technique, we demonstrated in the nuclei of several types of cells the presence of Ca(2+) channels as well as Na(+)-H(+) exchanger and receptors such as endothelin-1 and angiotensin II receptors. Stimulation of these nuclear membrane G-protein-coupled receptors induced an increase of nuclear Ca(2+). Our results suggest that, similar to the plasma membrane, nuclear membranes possess channels, exchangers and receptors such as those for endothelin-1 and angiotensin II, and that the nucleus seems to be a cell within a cell. This article will emphasize these findings.     

Co-Inhibition of BCL-W and BCL2 Restores Antiestrogen Sensitivity through BECN1 and Promotes an Autophagy-Associated Necrosis

BCL2 family members affect cell fate decisions in breast cancer but the role of BCL-W (BCL2L2) is unknown. We now show the integrated roles of the antiapoptotic BCL-W and BCL2 in affecting responsiveness to the antiestrogen ICI 182,780 (ICI; Fulvestrant Faslodex), using both molecular (siRNA; shRNA) and pharmacologic (YC137) approaches in three breast cancer variants; MCF-7/LCC1 (ICI sensitive), MCF-7/LCC9 (ICI resistant), and LY2 (ICI resistant). YC137 inhibits BCL-W and BCL2 and restores ICI sensitivity in resistant cells. Co-inhibition of BCL-W and BCL2 is both necessary and sufficient to restore sensitivity to ICI, and explains mechanistically the action of YC137. These data implicate functional cooperation and/or redundancy in signaling between BCL-W and BCL2, and suggest that broad BCL2 family member inhibitors will have greater therapeutic value than targeting only individual proteins. Whereas ICI sensitive MCF-7/LCC1 cells undergo increased apoptosis in response to ICI following BCL-W±BCL2 co-inhibition, the consequent resensitization of resistant MCF-7/LCC9 and LY2 cells reflects increases in autophagy (LC3 cleavage; p62/SQSTM1 expression) and necrosis but not apoptosis or cell cycle arrest. Thus, de novo sensitive cells and resensitized resistant cells die through different mechanisms. Following BCL-W+BCL2 co-inhibition, suppression of functional autophagy by 3-methyladenine or BECN1 shRNA reduces ICI-induced necrosis but restores the ability of resistant cells to die through apoptosis. These data demonstrate the plasticity of cell fate mechanisms in breast cancer cells in the context of antiestrogen responsiveness. Restoration of ICI sensitivity in resistant cells appears to occur through an increase in autophagy-associated necrosis. BCL-W, BCL2, and BECN1 integrate important functions in determining antiestrogen responsiveness, and the presence of functional autophagy may influence the balance between apoptosis and necrosis.