Functional evidence for physiological mechanisms to circumvent neurotoxicity of cardenolides in an adapted and a non-adapted hawk-moth species

Because cardenolides specifically inhibit the Na⁺K⁺-ATPase, insects feeding on cardenolide-containing plants need to circumvent this toxic effect. Some insects such as the monarch butterfly rely on target site insensitivity, yet other cardenolide-adapted lepidopterans such as the oleander hawk-moth, Daphnis nerii, possess highly sensitive Na⁺K⁺-ATPases. Nevertheless, larvae of this species and the related Manduca sexta are insensitive to injected cardenolides. By radioactive-binding assays with nerve cords of both species, we demonstrate that the perineurium surrounding the nervous tissue functions as a diffusion barrier for a polar cardenolide (ouabain). By contrast, for non-polar cardenolides such as digoxin an active efflux carrier limits the access to the nerve cord. This barrier can be abolished by metabolic inhibitors and by verapamil, a specific inhibitor of P-glycoproteins (PGPs). This supports that a PGP-like transporter is involved in the active cardenolide-barrier of the perineurium. Tissue specific RT-PCR demonstrated expression of three PGP-like genes in hornworm nerve cords, and immunohistochemistry further corroborated PGP expression in the perineurium. Our results thus suggest that the lepidopteran perineurium serves as a diffusion barrier for polar cardenolides and provides an active barrier for non-polar cardenolides. This may explain the high in vivo resistance to cardenolides observed in some lepidopteran larvae, despite their highly sensitive Na⁺K⁺-ATPases.     

Evidence that the product of the human X-linked CGD gene, gp91-phox, is a voltage-gated H(+) pathway

Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H(+) conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O(2).(2)). Suspensions of CHO91 cells exhibit arachidonate-activatable H(+) fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909-5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H(+). As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199-216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590-1598), a lowered external pH (pH(o)) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 microM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10-20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2'7' bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pH(i)) was approximately 6.9, as though pH(i) was largely determined by endogenous cellular regulation. Arachidonate (20 microM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl(-) concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH(2)-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H(+) permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H(+) selectivity. Mechanisms of H(+) permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319-1327) and the transfer of protons across an "H-X-X-X-H-X-X-X-H" motif lining a conducting pore.     

Extinction and ecological retreat in a community of primates

The lemurs of Madagascar represent a prodigious adaptive radiation. At least 17 species ranging from 11 to 160 kg have become extinct during the past 2000 years. The effect of this loss on contemporary lemurs is unknown. The concept of competitive release favours the expansion of living species into vacant niches. Alternatively, factors that triggered the extinction of some species could have also reduced community-wide niche breadth. Here, we use radiocarbon and stable isotope data to examine temporal shifts in the niches of extant lemur species following the extinction of eight large-bodied species. We focus on southwestern Madagascar and report profound isotopic shifts, both from the time when now-extinct lemurs abounded and from the time immediately following their decline to the present. Unexpectedly, the past environments exploited by lemurs were drier than the protected (albeit often degraded) riparian habitats assumed to be ideal for lemurs today. Neither competitive release nor niche contraction can explain these observed trends. We develop an alternative hypothesis: ecological retreat, which suggests that factors surrounding extinction may force surviving species into marginal or previously unfilled niches.     

The pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase

The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis. Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond. Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs. Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively. Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.     

2-Aminoperimidine, a specific inhibitor of bacterial NhaA Na(+)/H(+) antiporters

The diuretic drug amiloride and its numerous derivatives are competitive inhibitors of mammalian Na(+)/H(+) antiporters and other eukaryotic antiporters. Most prokaryotic antiporters, including the major NhaA family of enterobacteria, are resistant to these compounds. We show that 2-aminoperimidine (AP), a guanidine-containing naphthalene derivative with some similarity to amiloride, acts as a specific inhibitor of NhaA from Escherichia coli. Similar concentrations (IC(50) of 0.9 muM) inhibit the proton motive force dependent Na(+)(Li(+))/H(+) exchange reaction in inside-out sub-bacterial vesicles (at 10 mM NaCl, pH 8) as well as the initial rate of (22)Na(+)/Na(+) exchange mediated by pure NhaA in proteoliposomes. The inhibitor is specific to NhaA type antiporters, so AP is a new tool to study the mechanism and roles of NhaA antiporters of enterobacteria as well as the molecular basis of inhibition by an amiloride-like compound.     

First detection and quantification of N-monomethylarginine,a structural isomer of N-monomethylarginine,in humans using MS

The l-arginine metabolites methylated at the guanidino moiety, such as N^G-monomethyl-l-arginine (LNMMA), asymmetric N^G,N^G-dimethyl-l-arginine (ADMA), and symmetric N^G,N^G'-dimethyl-l-arginine (SDMA), are long known to be present in human plasma. Far less is known about the structural isomer of LNMMA, N^δ-monomethyl-l-arginine (δ-MMA). In prior work, it has been detected in yeast proteins, but it has not been investigated in mammalian plasma or cells. In this work, we present a method for the simultaneous and unambiguous quantification of LNMMA and δ-MMA in human plasma that is capable of detecting δ-MMA separately from LNMMA. The method comprises a simple protein precipitation sample preparation, hydrophilic interaction liquid chromatography (HILIC) gradient elution on an unmodified silica column, and triple stage mass spectrometric detection. Stable isotope-labeled D₆-SDMA was used as internal standard. The calibration ranges were 25–1000 nmol/L for LNMMA and 5–350 nmol/L for δ-MMA. The intra- and inter-batch precision determinations resulted in relative standard deviations of less than 12% for both compounds with accuracies of less than 6% deviation from the expected values. In a pilot study enrolling 10 healthy volunteers, mean concentrations of 48.0 ± 7.4 nmol/L for LNMMA and 27.4 ± 7.7 nmol/L for δ-MMA were found.     

Active-site alkylation destabilizes human O6-alkylguanine DNA alkyltransferase

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O(6)-alkylguanine and O(4)-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.     

Culture Age, Intracellular Ca2+ Concentration, and Protein Phosphorylation in Encystment-Induced Colpoda cucullus

In Colpoda cucullus, intracellular Ca²⁺ mediates the encystment induction and protein phosphorylation that occur just prior to morphogenetic transformation into the resting form. When rapidly growing cells were stimulated to encyst, encystment was not readily induced, and the protein phosphorylation level was lower. On the other hand, in post-growing cells stimulated to encyst, the encystment rate and protein phosphorylation level were elevated. These results suggest that protein phosphorylation is closely linked to encystment induction. Why, then, are the protein phosphorylation level and encystment rate difficult to elevate in the rapidly growing cells? Fura 2 ratiometry showed that the intracellular Ca²⁺ concentration (F₃₄₀/F₃₈₀ ratio) was raised in rapidly growing cells as well as in post-growing cells when the cells were stimulated to encyst. It is presumed that the Ca²⁺-mediated signal transduction pathways for protein phosphorylation and encystment may be triggered in rapidly growing cells, but downstream certain steps may be suppressed by certain intracellular components.     

Lessons from Plectocephalus (Compositae, Cardueae-Centaureinae): ITS disorientation in annuals and Beringian dispersal as revealed by molecular analyses

Background and Aims

The geographic distribution of the genus Plectocephalus comprises a single species in Ethiopia, two in North America and possibly four more in South America, in a striking disjunction that is exceptional for genera of the tribe Cardueae. The enormity of this disjunction cast doubts on the precise taxonomic delineation of the genus, which is not unanimously recognized as a natural entity. The aims of this study were to define the generic boundaries of Plectocephalus and to formulate a hypothesis that would explain its natural range.

Methods

A combined molecular approach, using nuclear internal transcribed spacers (ITS) and external transcribed spacers (ETS), and plastid trnL-trnL-F, rpl32-trnL^UAG and ndhF markers, was chosen for phylogenetic reconstruction by maximum parsimony and Bayesian inference.

Key Results

Phylogenetic analysis shows that Plectocephalus is a natural genus that includes the African species P. varians, together with all the native South American species, currently classified as Centaurea, C. cachinalensis, C. floccosa and C. tweediei. The recognition of Centaurodendron as an independent genus, which we consider appropriate, would make Plectocephalus paraphyletic. Affinities of Plectocephalus should lie with eastern representatives of Centaureinae. Geographic disjunction is explained as a consequence of dispersal via the Bering Land Bridge during the Miocene–Pliocene. The phylogeny of the basal grade of Centaureinae differs from previous phylogenies, and artefacts resulting from differences in mutation rates of annual and perennial taxa are confirmed. Sensitivity of ITS to these differences was the highest observed for all DNA regions used in this study.

Conclusions

The natural status of the genus Plectocephalus is confirmed and several nomenclatural combinations are proposed. New evidence contributes to the debate concerning problems posed by the use of ITS in the phylogenetic reconstruction of groups that differ in terms of their life cycles. Dispersal from Caucasus and Anatolia along the Siberian route and then across the Bering Land Bridge follows a route previously proposed for other taxonomic groups.     

Correlating structure, dynamics, and function in transmembrane segment VII of the Na/H exchanger isoform 1

We place ¹⁵N nuclear magnetic resonance relaxation analysis and functional mutagenesis studies in the context of our previous structural and mutagenesis work to correlate structure, dynamics and function for the seventh transmembrane segment of the human Na⁺/H⁺ exchanger isoform 1. Although G261-S263 was previously identified as an interruption point in the helical structure of this isolated transmembrane peptide in dodecylphosphocholine micelles, and rapid conformational exchange was implicated in the NOE measurements, the six ¹⁵N labelled residues examined in this study all have similar dynamics on the ps-ns time scale. A mathematical model incorporating chemical exchange is the best fit for residues G261, L264, and A268. This implies that a segment of residues from G261 to A268 samples different conformations on the μs-ms time scale. Chemical exchange on an intermediate time scale is consistent with an alternating-access cycle where E262 is bent away from the cytosol during proton translocation by the exchanger. The functional importance of chemical exchange at G261-A268 is corroborated by the abrogated activity of the full-length exchanger with the bulky and restricting Ile substitutions F260I, G261I, E262I, S263I, and A268I.     

p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Are carbon and nitrogen exchange between fungi and the orchid Goodyera repens affected by irradiance?

Background and Aims The green orchid Goodyera repens has been shown to transfer carbon to its mycorrhizal partner, and this flux may therefore be affected by light availability. This study aimed to test whether the C and N exchange between plant and fungus is dependent on light availability, and in addition addressed the question of whether flowering and/or fruiting individuals of G. repens compensate for changes in leaf chlorophyll concentration with changes in C and N flows from fungus to plant.Methods The natural abundances of stable isotopes of plant C and N were used to infer changes in fluxes between orchid and fungus across natural gradients of irradiance at five sites. Mycorrhizal fungi in the roots of G. repens were identified by molecular analyses. Chlorophyll concentrations in the leaves of the orchid and of reference plants were measured directly in the field.Key Results Leaf δ¹³C values of G. repens responded to changes in light availability in a similar manner to autotrophic reference plants, and different mycorrhizal fungal associations also did not affect the isotope abundance patterns of the orchid. Flowering/fruiting individuals had lower leaf total N and chlorophyll concentrations, which is most probably explained by N investments to form flowers, seeds and shoot.Conclusions The results indicate that mycorrhizal physiology is relatively fixed in G. repens, and changes in the amount and direction of C flow between plant and fungus were not observed to depend on light availability. The orchid may instead react to low-light sites through increased clonal growth. The orchid does not compensate for low leaf total N and chlorophyll concentrations by using a ¹³C- and ¹⁵N-enriched fungal source.     

Sup35 methionine oxidation is a trigger for de novo [PSI+] prion formation

ABSTRACT. The molecular basis by which fungal and mammalian prions arise spontaneously is poorly understood. A number of different environmental stress conditions are known to increase the frequency of yeast [PSI⁺] prion formation in agreement with the idea that conditions which cause protein misfolding may promote the conversion of normally soluble proteins to their amyloid forms. A recent study from our laboratory has shown that the de novo formation of the [PSI⁺] prion is significantly increased in yeast mutants lacking key antioxidants suggesting that endogenous reactive oxygen species are sufficient to promote prion formation. Our findings strongly implicate oxidative damage of Sup35 as an important trigger for the formation of the heritable [PSI⁺] prion in yeast. This review discusses the mechanisms by which the direct oxidation of Sup35 might lead to structural transitions favoring conversion to the transmissible amyloid-like form. This is analogous to various environmental factors which have been proposed to trigger misfolding of the mammalian prion protein (PrP^C) into the aggregated scrapie form (PrP^Sc).     

Rapidly increasing methyl mercury in endangered ivory gull (Pagophila eburnea) feathers over a 130 year record

Mercury (Hg) is increasing in marine food webs, especially at high latitudes. The bioaccumulation and biomagnification of methyl mercury (MeHg) has serious effects on wildlife, and is most evident in apex predators. The MeHg body burden in birds is the balance of ingestion and excretion, and MeHg in feathers is an effective indicator of overall MeHg burden. Ivory gulls (Pagophila eburnea), which consume ice-associated prey and scavenge marine mammal carcasses, have the highest egg Hg concentrations of any Arctic bird, and the species has declined by more than 80% since the 1980s in Canada. We used feathers from museum specimens from the Canadian Arctic and western Greenland to assess whether exposure to MeHg by ivory gulls increased from 1877 to 2007. Based on constant feather stable-isotope (δ¹³C, δ¹⁵N) values, there was no significant change in ivory gulls'' diet over this period, but feather MeHg concentrations increased 45× (from 0.09 to 4.11 µg g⁻¹ in adults). This dramatic change in the absence of a dietary shift is clear evidence of the impact of anthropogenic Hg on this high-latitude threatened species. Bioavailable Hg is expected to increase in the Arctic, raising concern for continued population declines in high-latitude species that are far from sources of environmental contaminants.     

GITR expression on T-cell receptor-stimulated human CD8 T cell in a JNK-dependent pathway

Glucocorticoid-induced tumor necrosis factor receptor (TNFR) (GITR) family-related gene is a member of the TNFR super family. GITR works as one of the immunoregulatory molecule on CD4(+) regulatory T cells and has an important role on cell survival or cell death in CD4(+) T cells. Little is known about the expression of GITR on human CD8(+) T cells on antigen-specific and non-specific activation. Here, we report that expression of GITR on human CD8(+) T cells on T-cell receptor (TCR) (anti-CD3)-mediated stimulation is dependent on the JNK pathway. The activation of CD8(+) T cells was measured by the expression of IL-2 receptor-α (CD25), GITR and by IFN-γ production upon re-stimulation with anti-CD3 antibody. We studied the signaling pathway of such inducible expression of GITR on CD8(+) T cells. We found that a known JNK-specific inhibitor, SP600125, significantly down-regulates GITR expression on anti-CD3 antibody-mediated activated CD8(+) T cells by limiting JNK phosphorylation. Subsequently, after stimulation of the CD8(+) cells, we tested for the production of IFN-γ by the activated cells following restimulation with the same stimulus. It appears that the expression of GITR on activated human CD8(+) T cells might also be regulated through the JNK pathway when the activation is through TCR stimulation. Therefore, GITR serves as an activation marker on activated CD8(+) cells and interference with JNK phosphorylation, partially or completely, by varying the doses of SP600125 might have implications in CD8(+) cytotoxic T cell response in translational research.     

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization

In a previous study, we identified Xenopus egg uroplakin III (xUPIII), a single-transmembrane protein that localized to lipid/membrane rafts and was tyrosine-phosphorylated upon fertilization. An antibody against the xUPIII extracellular domain abolishes fertilization, suggesting that xUPIII acts not only as tyrosine kinase substrate but also as a receptor for sperm. Previously, it has been shown that the protease cathepsin B can promote a transient Ca2+ release and egg activation as seen in fertilized eggs (Mizote, A., Okamoto, S., Iwao, Y., 1999. Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization. Dev. Biol. 208, 79-92). Here, we show that activation of Xenopus eggs by cathepsin B is accompanied by tyrosine phosphorylation of egg-raft-associated Src, phospholipase Cgamma, and xUPIII. Cathepsin B also promotes a partial digestion of xUPIII both in vitro and in vivo. A synthetic xUPIII-GRR peptide, which contains a potential proteolytic site, inhibits the cathepsin-B-mediated proteolysis and tyrosine phosphorylation of xUPIII and egg activation. Importantly, this peptide also inhibits sperm-induced tyrosine phosphorylation of xUPIII and egg activation. Protease activity that digests xUPIII in an xUPIII-GRR peptide-sensitive manner is present in Xenopus sperm. Several protease inhibitors, which have been identified to be inhibitory toward Xenopus fertilization, are shown to inhibit sperm-induced tyrosine phosphorylation of xUPIII. Uroplakin Ib, a tetraspanin UP member, is found to be associated with xUPIII in egg rafts. Our results highlight novel mechanisms of fertilization signaling by which xUPIII serves as a potential target for sperm protease essential for fertilization.