The effect of rearing temperature on development, body size, energetics and fecundity of the diamondback moth.

Temperature is arguably the most important abiotic factor influencing the life history of ectotherms. It limits survival and affects all physiological and metabolic processes, including energy and nutrient procurement and processing, development and growth rates, locomotion ability and ultimately reproductive success. However, the influence of temperature on the energetic cost of development has not been thoroughly investigated. We show that in the diamondback moth [Plutella xylostella L. (Lepidoptera: Plutellidae)] rearing temperature (range 10-30°C) affected growth and development rates, the energetic cost of development and fecundity. Rearing at lower temperatures increased development times and slowed growth rate, but resulted in larger adult mass. Fecundity was lowest at 10°C, highest at 15°C and intermediate at temperatures of 20°C and above. At a given rearing temperature fecundity was correlated with pupal mass and most eggs were laid on the first day of oviposition, there was no correlation between total eggs laid and adult longevity. The highest production cost was incurred at 10°C; this decreased with increasing temperature, was minimized in the range 20-25°C, and then increased again at 30°C. These minimized production costs occurred at temperatures close to the intrinsic optimum temperature for this species and may reflect the rearing temperature for optimal fitness. Thus at sub-optimal temperatures greater food resources are required during the development period. Predicted increased temperatures at the margins of the current core distribution of P. xylostella could ameliorate current seasonal effects on fecundity, thereby increasing the probability of winter survival leading to more resilient range expansion and an increased probability of pest outbreaks.

Interaction of MnO and ZnO nanomaterials with biomedically important proteins and cells.

Zinc and manganese nanomaterials may have potential for biomedical nanotechnology. Here first generation Zn and Mn oxide nanomaterials were prepared as determined by XRD. Transmission electron microscopy confirmed their nanoscale in two dimensions and revealed a rod or belt-like morphology for MnO or ZnO respectively. Association of MnO and ZnO to three model biomedically important proteins (albumin, protamine and thrombin) has been characterized by ultra-violet and dynamic laser light spectroscopy, UVS and DLLS respectively. UVS demonstrated a concentration-dependent loss of protein from the supernatant upon sedimentation of MnO or ZnO. Shifts in the surface charge of the MnO or ZnO by DLLS confirmed the protein's adsorption to the surface. MnO and ZnO were incubated with live human cells in culture (HeLa, A375 or 1321N1). A marked difference was observed for the two nanomaterials behavior in cell culture where the MnO could be discerned associating at the cell surface whereas the ZnO caused the cells to exhibit a rounded up morphology. Trypan blue dye exclusion studies demonstrated cytotoxicity of the ZnO at high concentrations 62.5-31.5 microg/mL whereas surprisingly the MnO demonstrated no cytotoxicity at any of the concentrations tested.

An RGD sequence in the P2Y(2) receptor interacts with alpha(V)beta(3) integrins and is required for G(o)-mediated signal transduction.

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-alpha(V) integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with G(i/o) family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).

P2Y(2) nucleotide receptor signaling in human monocytic cells: activation, desensitization and coupling to mitogen-activated protein kinases.

Activation of P2Y(2) receptors by extracellular nucleotides has been shown to induce phenotypic differentiation of human promonocytic U937 cells that is associated with the inflammatory response. The P2Y(2) receptor agonist, UTP, induced the phosphorylation of the MAP kinases MEK1/2 and ERK1/2 in a sequential manner, since ERK1/2 phosphorylation was abolished by the MEK1/2 inhibitor PD 098059. Other results indicated that P2Y(2) receptors can couple to MAP kinases via phosphatidylinositol 3-kinase (PI3K) and c-src. Accordingly, ERK1/2 phosphorylation induced by UTP was inhibited by the PI3K inhibitors, wortmannin and LY294002, and the c-src inhibitors, radicicol and PP2, but not by inhibitors of protein kinase C (PKC). The phosphorylation of ERK1/2 was independent of the ability of P2Y(2) receptors to increase the concentration of intracellular free calcium, since chelation of intracellular calcium by BAPTA did not diminish the phosphorylation of ERK1/2 induced by UTP. A 5-minute treatment with UTP reduced U937 cell responsiveness to a subsequent UTP challenge. UTP-induced desensitization was characterized by an increase in the EC(50) for receptor activation (from 0.44 to 9.3 microM) and a dramatic ( approximately 75%) decrease in the maximal calcium mobilization induced by a supramaximal dose of UTP. Phorbol ester treatment also caused P2Y(2) receptor desensitization (EC(50) = 12.3 microM UTP and maximal calcium mobilization reduced by approximately 33%). The protein kinase C inhibitor GF 109203X failed to significantly inhibit the UTP-induced desensitization of the P2Y(2) receptor, whereas the protein phosphatase inhibitor okadaic acid blocked receptor resensitization. Recovery of receptor activity after UTP-induced desensitization was evident in cells treated with agonist for 5 or 30 min. However, P2Y(2) receptor activity remained partially desensitized 30 min after pretreatment of cells with UTP for 1 h or longer. This sustained desensitized state correlated with a decrease in P2Y(2) receptor mRNA levels. Desensitization of ERK1/2 phosphorylation was induced by a 5-minute pretreatment with UTP, and cell responsiveness did not return even after a 30-minute incubation of cells in the absence of an agonist. Results suggest that desensitization of the P2Y(2) receptor may involve covalent modifications (i.e., receptor phosphorylation) that functionally uncouple the receptor from the calcium signaling pathway, and that transcriptional regulation may play a role in long-term desensitization. Our results indicate that calcium mobilization and ERK1/2 phosphorylation induced by P2Y(2) receptor activation are independent events in U937 monocytes.

Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP.

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y2 receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y2 receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y2 receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y2 receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y2 receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y2 receptors. While ATP and UTP activated the murine and human P2Y2 receptors with similar potencies (EC50 values were 1.5-5.8 microM), ATP was approximately 10-fold less potent (IC50 = 9.1-21.2 microM) than UTP (IC50 = 0.7-2.9 microM) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor.

UTP activates P2Y, receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y, receptors with an EC50 of 0.2-1.0 microM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3-1.0 microM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50%, whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases-1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y, receptor agonists.

Desensitization of P2Y2 receptor-activated transepithelial anion secretion.

Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current (Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Isc with a potency profile of ATP = UTP > 2-methylthioATP >> alpha,beta-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2 receptor agonist UTP and increased in a concentration-dependent manner (IC50 approximately 10(-6) M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 x 10(-6) M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10(-5) M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor. Consequences of truncation of the C terminus.

Molecular determinants of P2Y2 receptor desensitization and sequestration have been investigated. Wild-type P2Y2 receptors and a series of five C-terminal truncation mutants of the receptor were epitope-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UTP-stimulated increases in intracellular calcium concentration, 2) homologous desensitization of the receptor, and 3) agonist-induced decreases in cell-surface density (receptor sequestration) of epitope-tagged receptors using fluorescence-activated cell sorting. The potency and efficacy of UTP were similar for the wild-type and all mutant P2Y2 receptors. Truncation of 18 or more amino acids from the C terminus increased by approximately 30-fold the concentration of UTP necessary to desensitize the receptor. Both the rate and magnitude of UTP-induced receptor sequestration were decreased with progressively larger truncations of the C terminus. Furthermore, the recovery from sequestration was slower for the most extensively truncated receptor. Complete desensitization was obtained with >50% of the original receptor complement remaining on the cell surface. Protein kinase C activation, which desensitizes the P2Y2 receptor, had no effect on sequestration, consistent with the ideas that desensitization and sequestration are discrete events and that agonist occupancy is required for receptor sequestration.

Changes in P2Y1 nucleotide receptor activity during the development of rat salivary glands.

Experiments that used dispersed salivary gland cells from 1-day-old rats indicated the presence of the P2Y nucleotide receptor subtype, P2Y1, based on the agonist potency profile for mobilization of intracellular free Ca2+ [2-methylthio-ATP > ADP > adenosine 5'-O-(2-thiodiphosphate) > ATP, with UTP ineffective] and sequence analysis of reverse transcription-polymerase chain reaction (RT-PCR) products obtained with P2Y1 receptor-specific primers. P2Y1 receptor activity appears to be developmentally regulated, since Ca2+ mobilization in response to the P2Y1-selective agonist, 2-methylthio-ATP, decreased as animal age increased, with the maximal response of 129 +/- 23 nM obtained in 1-day-old animals, decreasing to 30 +/- 3 nM in 4-wk-old animals. However, the abundance of P2Y1 receptor mRNA, assessed by semiquantitative RT-PCR, did not change over this time period, suggesting that receptor activity is regulated by some mechanism other than changes in steady-state levels of P2Y1 receptor mRNA. These findings indicate that functional P2Y1 nucleotide receptors are expressed in immature salivary glands and that receptor activity decreases as the glands mature, suggesting that P2Y1 receptors may have an important role during salivary gland development.

P2U purinoceptors: cDNA cloning, signal transduction mechanisms and structure-function analysis.

The cloning of a P2U purinoceptor cDNA has made it possible to use molecular biological approaches to investigate P2U purinoceptor function. Expression of recombinant P2U purinoceptors in mammalian cells lacking endogenous P2U purinoceptors has enabled us to characterize the receptor protein and its downstream effectors, and has allowed a partial analysis of the role of certain amino acid residues in ligand binding. These approaches have placed the pharmacological classification of the P2U purinoceptor on a firm molecular footing and have generated model systems that can be used to investigate receptor-ligand binding, regulation and signal transduction.

Cloning, expression, and chromosomal localization of the human uridine nucleotide receptor gene.

Extracellular ATP and ADP mediate diverse physiological responses in mammalian cells, in part through the activation of G protein-coupled P2 purinoceptors. The cloning and expression of cDNAs encoding several P2 purinoceptor subtypes have enabled rapid advances in our understanding of the structural and functional properties of these receptors. The current report describes the isolation of a gene from a human genomic library that encodes a protein with the greatest similarity to the human P2U purinoceptor, a subtype that is distinguished by its ability to be activated by uridine nucleotides as well as adenine nucleotides. When expressed in a mammalian cell line, this novel receptor is activated specifically by UTP and UDP but not by ATP and ADP. Activation of this uridine nucleotide receptor resulted in increased inositol phosphate formation and calcium mobilization. Fluorescence in situ hybridization revealed that the gene encoding the uridine nucleotide receptor is located in region q13 of the X chromosome. Dendrogram analysis of the G protein-coupled P2 purinoceptors and the uridine nucleotide receptor indicates that these receptors belong to a family that may be more aptly named nucleotide receptors.

Site-directed mutagenesis of P2U purinoceptors. Positively charged amino acids in transmembrane helices 6 and 7 affect agonist potency and specificity.

Two subtypes of G protein-coupled receptors for nucleotides (P2U and P2Y purinoreceptors) contain several conserved positively charged amino acids in the third, sixth, and seventh putative transmembrane helices (TMHs). Since the fully ionized form of nucleotides has been shown to be an activating ligand for both P2U and P2Y purinoceptors (P2UR and P2YR), we postulated that some of these positively charged amino acids are involved in binding of the negatively charged phosphate groups of nucleotides. To investigate the role of the conserved positively charged amino acids in purinoceptor function, a series of mutant P2UR cDNAs were constructed so that lysine 107 and arginine 110 in TMH 3, histidine 262 and arginine 265 in TMH 6, and arginine 292 in TMH 7 were changed to the neutral amino acid leucine or isoleucine. The mutated P2UR cDNAs were stably expressed in 1321N1 astrocytoma cells and receptor activity was monitored by quantitating changes in the concentration of intracellular Ca2+ upon stimulation with full (ATP, UTP) or partial (ADP, UDP) P2UR agonists. Neutralization of His262, Arg265, or Arg292 caused a 100-850-fold decrease in the potency of ATP and UTP relative to the unmutated P2UR and rendered ADP and UDP ineffective. In contrast, neutralization of Lys107 or Arg110 did not alter the agonist potency or specificity of the P2UR. Neutralization of Lys289 in the P2UR, which is expressed as a glutamine residue in the P2Y subtype, did not alter receptor activity; however, a conservative change from lysine to arginine at this position altered the rank order of agonist potency so that ADP and UDP were approximately 100-fold more potent than ATP and UTP. A three-dimensional model of the P2UR indicates the feasibility of His262, Arg265, and Arg292 interactions with the phosphate groups of nucleotides.

Molecular and functional analysis of the LYS1 gene of Candida albicans.

The LYS1 gene of Candida albicans has been localized to a 1.8-kb DNA fragment present on the plasmid YpBRG2. YpBRG2 has been shown to complement the saccharopine dehydrogenase mutant Stx4-4A of Saccharomyces cerevisiae. Transformants of S. cerevisiae Stx4-4A exhibited significant saccharopine dehydrogenase activity, and cells that had lost YpBRG2 after nonselective growth had no enzyme activity. The DNA sequence of the LYS1 gene has been determined. The LYS1 DNA contains typical yeast upstream regulatory sequences, including the GCN4 motif and candidate sequences responsible for transcription termination within the 3' noncoding region. The fragment contained an open reading frame of 1,146 nucleotides coding for a putative protein of 382 amino acids. The open reading frame has 60% identity at the nucleotide level and 71% similarity at the amino acid level to the LYS5 gene of Yarrowia lipolytica, which is believed to code for saccharopine dehydrogenase. A peptide of 11 amino acids has been found, which is present in S. cerevisiae, Y. lipolytica, and C. albicans. This peptide can be expanded to 16 amino acids when the sequences from Y. lipolytica and C. albicans are compared. A motif responsible for the binding of the adenosine residue of NADH has been described previously and is very similar to this peptide, which may be the site of NADH binding in the saccharopine dehydrogenase of C. albicans.

Lysine biosynthesis in selected pathogenic fungi: characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans.

The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. Until now, this unique metabolic pathway has never been investigated in the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. Five of the eight enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase) of the alpha-aminoadipate pathway and glucose-6-phosphate dehydrogenase, a glycolytic enzyme used as a control, were demonstrated in wild-type cells of these organisms. All enzymes were present in Saccharomyces cerevisiae and the pathogenic organisms except C. neoformans 32608 serotype C, which exhibited no saccharopine reductase activity. The levels of enzyme activity varied considerably from strain to strain. Variation among organisms was also observed for the control enzyme. Among the pathogens, C. albicans exhibited much higher homocitrate synthase, homoisocitrate dehydrogenase, and alpha-aminoadipate reductase activities. Seven lysine auxotrophs of C. albicans and one of Candida tropicalis were characterized biochemically to determine the biochemical blocks and gene-enzyme relationships. Growth responses to alpha-aminoadipate- and lysine-supplemented media, accumulation of alpha-aminoadipate semialdehyde, and the lack of enzyme activity revealed that five of the mutants (WA104, WA153, WC7-1-3, WD1-31-2, and A5155) were blocked at the alpha-aminoadipate reductase step, two (STN57 and WD1-3-6) were blocked at the saccharopine dehydrogenase step, and the C. tropicalis mutant (X-16) was blocked at the saccharopine reductase step. The cloned LYS1 gene of C. albicans in the recombinant plasmid YpB1078 complemented saccharopine dehydrogenase (lys1) mutants of S. cerevisiae and C. albicans. The Lys1+ transformed strains exhibited significant saccharopine dehydrogenase activity in comparison with untransformed mutants. The cloned LYS1 gene has been localized on a 1.8-kb HindIII DNA insert of the recombinant plasmid YpB1041RG1. These results established the gene-enzyme relationship in the second half of the alpha-aminoadipate pathway. The presence of this unique pathway in the pathogenic fungi could be useful for their rapid detection and control.

Use of alpha-aminoadipate and lysine as sole nitrogen source by Schizosaccharomyces pombe and selected pathogenic fungi.

alpha-Aminodipate, an intermediate of the lysine biosynthetic pathway of fungi, or lysine when used as the sole nitrogen source in the medium was growth inhibitory and toxic to Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe and pathogenic fungi Candida albicans, Filobasidiella neoformans and Aspergillus fumigatus grew in the medium containing alpha-aminoadipate as the sole nitrogen source. C. albicans, A. fumigatus, and one of the strains of F. neoformans also grew in the medium containing lysine as the sole nitrogen source. When grown in the alpha-aminoadipate medium, only S. pombe accumulated a significant amount of alpha-ketoadipate in the culture supernatant. Also, 14C-alpha-aminoadipate was converted to 14C-alpha-ketoadipate in vivo. In the ammonium sulfate medium, S. pombe cells converted 14C-alpha-aminoadipate to lysine. The levels of glutamate-alpha-ketoadipate transaminase, an enzyme responsible for the conversion of alpha-aminoadipate to alpha-ketoadipate, and alpha-aminoadipate reductase, an enzyme required for the conversion of alpha-aminoadipate to lysine, were similar in S. pombe cells grown in the alpha-aminoadipate or ammonium sulfate medium. However, the level of homoisocitrate dehydrogenase, an enzyme before the alpha-ketoadipate step, was twelvefold lower in S. pombe cells grown in the alpha-aminoadipate medium compared to the level in cells grown in the ammonium sulfate medium. Pathogenic fungi used in this study did not accumulate alpha-ketoadipate and alpha-aminoadipate-delta-semialdehyde when grown in medium containing alpha-aminoadipate and lysine, respectively, as sole nitrogen source. However, only pathogenic fungi used both lysine and alpha-aminoadipate as sole nitrogen source. This unique metabolic property could be useful for the identification of these pathogens.