Hyperpolarizing receptor potentials in lobster olfactory receptor cells: implications for transduction and mixture suppression

Physiological studies of olfactory receptor cells have focusedon excitatory responses, in part because the evidence for inhibitoryresponses from extracellular recordings, although long-standing,has been equivocal. Intracellular recording from the olfactorycells of two species of lobsters revealed that small but concentrationdependentand repeatable hyperpolarizing receptor potentials could beevoked by a mixture of L-arginine, L-cysteine and L-proline,as well as by histamine. Large, depolarizing receptor potentialswere evoked in the same cells by a complex odor mixture. Simultaneousapplication of depolarizing and hyperpolarizing stimuli reducedthe magnitude of the evoked depolarization. These results implythat multiple, opposing transduction mechanisms are presentin single lobster olfactory receptor cells and reveal a noncompetitivemechanism for peripheral mixture suppression.     

CTLA-4 in filarial infections: implications for a role in diminished T cell reactivity

To determine the role that CTLA-4 might play in mediating the diminished parasite Ag-specific T cell responsiveness that is characteristically seen in filaria-infected patients, several study populations and methods were used. First, quantitative assessment of mRNA expression determined that PBMC from uninfected adolescents exposed in utero to microfilarial (Mf) Ag demonstrated a strong up-regulation of CTLA-4 to the Mf stage of the parasite in contrast to that observed in cells from children born of uninfected mothers (p = 0.005). Next, the frequency of CTLA-4 expression was examined using flow cytometry in cells from filaria-infected and -uninfected individuals ex vivo. Individuals born in filarial endemic regions of the world (with long-standing infections) had greater percentages of CD4(+)CTLA-4(+) cells than did expatriate infected or uninfected individuals (p = 0.005 and 0.05, respectively); in addition, Mf(+) patients demonstrated higher frequencies of CD4(+)CTLA-4(+) and CD8(+)CTLA-4(+) cells (p = 0.027 and 0.037, respectively) than did Mf(-) infected individuals. Of interest, the greatest intensity of CTLA-4 expression occurred in CD4(+)CD25(+) cells, a population purported to include suppressor cells. Finally, in vitro blocking of CTLA-4 expression in PBMC from filaria-infected individuals induced a mean increase of 44% in IL-5 production to Mf Ag, whereas there was a concurrent mean decrease of 42% in IFN-gamma production, suggesting that CTLA-4 also acts to alter the Th1/Th2 balance in filaria-infected individuals. Together, these data indicate a significant role for CTLA-4 in regulating the host response to filarial infections and that factors such as length of exposure and patency are important codeterminants.     

Mitochondrial content is central to nuclear gene expression: Profound implications for human health

We review a recent paper in Genome Research by Guantes et al. showing that nuclear gene expression is influenced by the bioenergetic status of the mitochondria. The amount of energy that mitochondria make available for gene expression varies considerably. It depends on: the energetic demands of the tissue; the mitochondrial DNA (mtDNA) mutant load; the number of mitochondria; stressors present in the cell. Hence, when failing mitochondria place the cell in energy crisis there are major effects on gene expression affecting the risk of degenerative diseases, cancer and ageing. In 2015 the UK parliament approved a change in the regulation of IVF techniques, allowing “Mitochondrial replacement therapy” to become a reproductive choice for women at risk of transmitting mitochondrial disease to their children. This is the first time that this technique will be available. Therefore understanding the interaction between mitochondria and the nucleus has never been more important.     

G-CSF-mediated inhibition of JNK is a key mechanism for Lactobacillus rhamnosus-induced suppression of TNF production in macrophages

Lactobacillus rhamnosus is a human commensal with known immunomodulatory properties. To date the mechanism of these immunomodulatory effects is not well understood. To unravel the immunomodulatory signalling mechanism, we investigated the effects of two strains of L. rhamnosus, L. rhamnosus GG and GR-1, in modulating production of tumour necrosis factor-alpha (TNF) in human monocytic cell line THP-1 and mouse macrophages. Live L. rhamnosus GG and GR-1 or their spent culture supernatant induced minuscule amounts of TNF production but large quantities of granulocyte-colony stimulating factor (G-CSF) in macrophages compared with those induced by pathogenic Escherichia coli GR-12 and Enterococcus faecalis. By using neutralizing antibodies and G-CSF receptor knockout mice, we demonstrated that G-CSF secreted from L. rhamnosus GG- and GR-1-exposed macrophages suppressed TNF production induced by E. coli- or lipopolysaccharide-activated macrophages through a paracrine route. The suppression of TNF production by G-CSF was mediated through activation of STAT3 and subsequent inhibition of c-Jun-N-terminal kinases (JNKs). The inhibition of JNK activation required STAT3alpha-mediated de novo protein synthesis. This demonstrates a novel role of G-CSF in L. rhamnosus-triggered anti-inflammatory effects and its mechanism in the suppression of TNF production in macrophages.     

Methaneseleninic acid is a substrate for truncated mammalian thioredoxin reductase: implications for the catalytic mechanism and redox signaling

Mammalian thioredoxin reductase is a homodimeric pyridine nucleotide disulfide oxidoreductase that contains the rare amino acid selenocysteine (Sec) on a C-terminal extension. We previously have shown that a truncated version of mouse mitochondrial thioredoxin reductase missing this C-terminal tail will catalyze the reduction of a number of small molecules. Here we show that the truncated thioredoxin reductase will catalyze the reduction of methaneseleninic acid. This reduction is fast at pH 6.1 and is only 4-fold slower than that of the full-length enzyme containing Sec. This finding suggested to us that if the C-terminal Sec residue in the holoenzyme became oxidized to the seleninic acid form (Sec-SeO(2)(-)) that it would be quickly reduced back to an active state by enzymic thiols and further suggested to us that the enzyme would be very resistant to irreversible inactivation by oxidation. We tested this hypothesis by reducing the enzyme with NADPH and subjecting it to high concentrations of H(2)O(2) (up to 50 mM). The results show that the enzyme strongly resisted inactivation by 50 mM H(2)O(2). To determine the redox state of the C-terminal Sec residue, we attempted to inhibit the enzyme with dimedone. Dimedone alkylates protein sulfenic acid residues and presumably will alkylate selenenic acid (Sec-SeOH) residues as well. The enzyme was not inhibited by dimedone even when a 150-fold excess was added to the reaction mixture containing the enzyme and H(2)O(2). We also tested the ability of the truncated enzyme to resist inactivation by oxidation as well and found that it also was resistant to high concentrations of H(2)O(2). One assumption for the use of Sec in enzymes is that it is catalytically superior to the use of cysteine. We and others have previously suggested that there are reasons for the use of Sec in enzymes that are unrelated to the conversion of substrate to product. The data presented here support this assertion. The results also imply that the redox signaling function of the thioredoxin system can remain active under oxidative stress.     

Bcr-Abl-mediated molecular mechanism for apoptotic suppression in multipotent haemopoietic cells: a role for PKCbetaII

Bcr-Abl protein tyrosine kinase (PTK) activity is a feature of chronic myeloid leukaemia and confers a survival advantage on haemopoietic progenitor cells. We have expressed conditional mutant of the Bcr-Abl PTK in the FDCP-Mix A4 multipotent haematopoietic cell line in order to examine the molecular mechanisms whereby Bcr-Abl PTK leads to enhanced cell survival under conditions in which normal cells die. Activation of Bcr-Abl PTK does not phosphorylate or activate either ERK-1/2 or JAK-2/STAT-5b, suggesting that these signal transduction pathways are not involved in Abl PTK-mediated suppression of apoptosis in FDCP-Mix cells. However, protein kinase C (PKC) does have a role to play. Inhibition of PKC results in a reversal of Bcr-Abl PTK-mediated survival in the absence of growth factor and Bcr-Abl stimulates translocation of the PKCbetaII isoform to the nucleus. Furthermore, expression of a constitutively activated PKCbetaII in haemopoietic progenitor FDCP-Mix cells stimulates enhanced cell survival when IL-3 is withdrawn. However, expression of this constitutively activated PKC isoform does not suppress cytotoxic drug-induced apoptosis. Thus Bcr-Abl PTK has pleiotropic effects which can suppress cell death induced by a number of stimuli.     

Trk is a calmodulin-binding protein: implications for receptor processing

The tyrosine kinase receptors for the neurotrophins (Trk) are a family of transmembrane receptors that regulate the differentiation and survival of different neuronal populations. Neurotrophin binding to Trk leads to the activation of several signalling pathways including a rapid, but moderate, increase in intracellular calcium levels. We have previously described the role of calcium and its sensor protein, calmodulin, in Trk-activated intracellular pathways. Here we demonstrate that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST-fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C-terminal domain of TrkA in a Ca2+-dependent manner. We have also co-immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provide evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA-derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCgamma and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.     

Impaired methylation as a novel mechanism for proteasome suppression in liver cells

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.     

Antiprogestagenic inhibition of uterine prostaglandin inactivation: a permissive mechanism for uterine stimulation

The use of antiprogestins as abortifacients is more effective when antiprogestin priming is followed by the administration of a small dose of synthetic prostaglandin. This increased myometrial sensitivity towards PG has not been explained and experiments in the guinea-pig where no myometrial activity is observed after 48 h of antiprogestin administration together with measurements of PG metabolites in uterine vein blood have given rise to the suggestion that prostaglandin synthesis is inhibited by antiprogestins. We have treated groups of 50 day pregnant guinea-pigs with 10 mg RU486 or vehicle alone and examined the ability of homogenised uterine tissues (myometrium/decidua, cervix, chorion and amnion) to metabolize PGE when given a large excess of substrate and sufficient cofactors. In addition we have examined the ability of these homogenates to synthesis PG. Antiprogestin treatment in vivo resulted in a 9-fold reduction in metabolic activity in chorion (P less than 0.02) and a 4-fold reduction in myometrium/decidua (P less than 0.02). Reduction in activity seen in amnion and cervix was not significant. The maximum metabolism was seen in the chorion and minimal metabolism in the amnion. Maximum PG production was seen in the amnion and minimum in the chorion. These results show that the effect of antiprogestin in reducing prostaglandin catabolism would reduce the threshold above which PG production would cause contractions which would in turn stimulate PG production. Thus an explanation is provided of how low doses of exogenous PGs or transient synthesis of endogenous PG within an antiprogestin treated uterus can led to a self sustaining cycle of stimulation which will lead to abortion.     

Virtual electrodes in cardiac tissue: a common mechanism for anodal and cathodal stimulation.

Traditional cable analyses cannot explain complex patterns of excitation in cardiac tissue with unipolar, extracellular anodal, or cathodal stimuli. Epifluorescence imaging of the transmembrane potential during and after stimulation of both refractory and excitable tissue shows distinctive regions of simultaneous depolarization and hyperpolarization during stimulation that act as virtual cathodes and anodes. The results confirm bidomain model predictions that the onset (make) of a stimulus induces propagation from the virtual cathode, whereas stimulus termination (break) induces it from the virtual anode. In make stimulation, the virtual anode can delay activation of the underlying tissue, whereas in break stimulation this occurs under the virtual cathode. Thus make and break stimulations in cardiac tissue have a common mechanism that is the result of differences in the electrical anisotropy of the intracellular and extracellular spaces and provides clear proof of the validity of the bidomain model.     

Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.     

Habitat associations of wild goat in central Iran: implications for conservation

Wild goat (Capra aegagrus) is one of the highly valued but threatened ungulates of Iran. Seasonal habitat use of wild goat was studied from October 2006 to September 2008 in Haftad Gholleh Protected Area, central Iran. We developed averaged logistic regression models based on Akaike Information Criterion weights for each season. The obtained habitat suitability models showed high sensitivity (greater than 88%) for all evaluation datasets. Wild goat habitat use was positively related with steep slopes, areas near rocky substrates and water sources, west-facing slopes and areas far from roads in all seasons. Core zones of protected area covered nearly 70% of suitable habitats for wild goats, predicted by logistic regression models. Northern core zone covered more than 61% of predicted suitable habitats, which should receive more attention in management actions. The southern core zone boundaries should be considered to increase the encompassed suitable habitats. There was a substantial overlap between seasonal suitable habitats with the highest overlap observed between spring and summer and summer and fall.     

APOBEC2 is a monomer in solution: implications for APOBEC3G models

Although the physiological role of APOBEC2 is still largely unknown, a crystal structure of a truncated variant of this protein was determined several years ago [Prochnow, C. (2007) Nature445, 447-451]. This APOBEC2 structure had considerable impact in the HIV field because it was considered a good model for the structure of APOBEC3G, an important HIV restriction factor that abrogates HIV infectivity in the absence of the viral accessory protein Vif. The quaternary structure and the arrangement of the monomers of APOBEC2 in the crystal were taken as being representative for APOBEC3G and exploited in explaining its enzymatic and anti-HIV activity. Here we show, unambiguously, that in contrast to the findings for the crystal, APOBEC2 is monomeric in solution. The nuclear magnetic resonance solution structure of full-length APOBEC2 reveals that the N-terminal tail that was removed for crystallization resides close to strand β2, the dimer interface in the crystal structure, and shields this region of the protein from engaging in intermolecular contacts. In addition, the presence of the N-terminal region drastically alters the aggregation propensity of APOBEC2, rendering the full-length protein highly soluble and not prone to precipitation. In summary, our results cast doubt on all previous structure-function predictions for APOBEC3G that were based on the crystal structure of APOBEC2.     

Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules

We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. The displacement in switch 1 resembles that of the G-protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide-binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice-embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central beta-tubulin monomer and the two flanking alpha-tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G-proteins suggests that the rate-limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity.     

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: implications for interpreting phase behavior

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (l(d)) domains on the surface of GUVs composed of 40:60 mol% DOPC/DPPC, over a wide range of temperatures (14-25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of Lissamine(TM) rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.     

Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases

Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids. There are more than 19 different isoforms of PLA2 in the mammalian system, but recent studies have focused on three major groups, namely, the group IV cytosolic PLA2, the group II secretory PLA2 (sPLA2), and the group VI Ca(2+)-independent PLA2. These PLA2s are involved in a complex network of signaling pathways that link receptor agonists, oxidative agents, and proinflammatory cytokines to the release of arachidonic acid (AA) and the synthesis of eicosanoids. PLA2s acting on membrane phospholipids have been implicated in intracellular membrane trafficking, differentiation, proliferation, and apoptotic processes. All major groups of PLA2 are present in the central nervous system (CNS). Therefore, this review is focused on PLA2 and AA release in neural cells, especially in astrocytes and neurons. In addition, because many neurodegenerative diseases are associated with increased oxidative and inflammatory responses, an attempt was made to include studies on PLA2 in cerebral ischemia, Alzheimer's disease, and neuronal injury due to excitotoxic agents. Information from these studies has provided clear evidence for the important role of PLA2 in regulating physiological and pathological functions in the CNS.     

Antibody-induced suppression and postsuppression stimulation of complement in vitro. III. Long-term C4 suppression is actively maintained by a soluble suppressor factor (FsC4)

Previous work in our laboratory established that individual complement components can be regulated in vivo by administration of specific antibody or immunocompetent cells to newborns and in vitro by administration of specific antibody to cultured peritoneal macrophages or splenic fragments. Antibody-induced suppression of C4 was much longer lasting in cultured guinea pig splenic fragments than in cultured guinea pig peritoneal macrophages, suggesting that splenic fragments contained elements necessary for long-term suppression that were not present in the macrophage monolayers. This publication presents data in support of this concept. Antibody-treated splenic fragments from normal guinea pigs--but not from C4-deficient guinea pigs--elaborated a soluble factor (FsC4) that suppressed C4 production in previously untreated splenic fragments. FsC4 activity was most potent in splenic fragment culture supernatants at those times when intracellular and secreted C4 hemolytic activity and C4 antigen were at their lowest. C4 itself or a fragment of C4 was therefore unlikely to mediate suppression in this system. Residual anti-C4 antibody was ruled out as a mediator of FsC4 activity since it was shown by two independent methods that the amount of anti-C4 antibody carried over with the supernatant was orders of magnitude less than the amount necessary to cause suppression or to neutralize fluid phase C4 in fresh splenic fragment cultures. Preliminary data revealed that FsC4 activity may be mediated by two or more distinct molecular species or may be mediated by a single molecule that exhibits secondary size and charge heterogeneity. The identification of factors that are capable of regulating C4 suggests that, as with immunoglobulins, complement components may be regulated by complex networks of immunocompetent cells and their soluble products.