OpenUCT :: Browsing by Subject "Animals"

Browsing by Subject "Animals"

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Open Access
Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans
(2009) Lindemans, M; Liu, F; Janssen, T; Husson, S J; Mertens, I; Gäde, G; Schoofs, L
In mammals, hypothalamic gonadotropin-releasing hormone (GnRH) is a neuropeptide that stimulates the release of gonadotropins from the anterior pituitary. The existence of a putative functional equivalent of this reproduction axis in protostomian invertebrates has been a matter of debate. In this study, the ligand for the GnRH receptor in the nematode Caenorhabditis elegans (Ce-GnRHR) was found using a bioinformatics approach. The peptide and its precursor are reminiscent of both insect adipokinetic hormones and GnRH-preprohormone precursors from tunicates and higher vertebrates. We cloned the AKH-GnRH-like preprohormone and the Ce-GnRHR and expressed the GPCR in HEK293T cells. The GnRHR was activated by the C. elegans AKH-GnRH-like peptide (EC50 = 150 nM) and by Drosophila AKH and other nematode AKH-GnRHs that we found in EST databases. Analogous to both insect AKH receptor and vertebrate GnRH receptor signaling, Ce-AKH-GnRH activated its receptor through a Gαq protein with Ca2+ as a second messenger. Gene silencing of Ce-GnRHR, Ce-AKH-GnRH, or both resulted in a delay in the egg-laying process, comparable to a delay in puberty in mammals lacking a normal dose of GnRH peptide or with a mutated GnRH precursor or receptor gene. The present data support the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution and that its role in reproduction might have been developed before the divergence of protostomians and deuterostomians.
Open Access
Apparent Absorption Efficiencies of Nectar Sugars in the Cape Sugarbird, with a Comparison of Methods
(1998) Jackson, Susan; Nicolson, Susan W; van Wyk, Ben‐Erik
Nectarivore sugar preferences and nectar composition in the Cape Floristic Kingdom (southern Africa) differ from trends reported for analogous systems in America and Europe in that sugarbirds and sunbirds show no aversion to sucrose, which is the dominant nectar sugar in many of their food plants. To elucidate the physiological bases (if any) of nectarivore sugar preferences, we determined apparent sugar absorption efficiencies in a passerine endemic to this region, the Cape sugarbird Promerops cafer. Apparent absorption efficiencies for the three major nectar sugars, sucrose, glucose, and fructose, were extremely high (> 99%), as in other specialized avian nectarivores. Xylose, a pentose sugar recently reported in the nectar of some Proteaceae, was absorbed and/or metabolized inefficiently, with a mean of 47.1% of ingested sugar recovered in cloacal fluid. We did not measure the proportions of xylose that were absorbed and/or metabolized. We also compared three methods of estimating absorption efficiency: (1) measurements of total sugar in cloacal fluid with refractometry, without correction for differences between volumes of ingesta and excreta; (2) the same measurements combined with correction for volume differences; and (3) HPLC analyses quantifying individual sugars in cloacal fluid, with correction for volume differences. Refractometry has been frequently used in previous studies. For all sugars except xylose, method 1 yielded results similar to those obtained with method 2, but the convergence was artifactual, and we do not recommend use of this method. Apparent absorption efficiencies calculated with method 2 underestimated true absorption efficiency, because refractometry measures nonsugar solutes, but this error is biologically significant only when efficiencies are low.
Open Access
Asparagine 706 and Glutamate 183 at the Catalytic Site of Sarcoplasmic Reticulum Ca 2+ -ATPase Play Critical but Distinct Roles in E 2 States
(2006) Clausen, Johannes D; McIntosh, David B; Woolley, David G; Anthonisen, Anne Nyholm; Vilsen, Bente; Andersen, Jens Peter
Mutants with alteration to Asn(706) of the highly conserved (701)TGDGVND(707) motif in domain P of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed for changes in transport cycle kinetics and binding of the inhibitors vanadate, BeF, AlF, and MgF. The fluorides likely mimic the phosphoryl group/P(i) in the respective ground, transition, and product states of phosphoenzyme hydrolysis (Danko, S., Yamasaki, K., Daiho, T., and Suzuki, H. (2004) J. Biol. Chem. 279, 14991-14998). Binding of BeF, AlF, and MgF was also studied for mutant Glu(183) --> Ala, where the glutamate of the (181)TGES(184) motif in domain A is replaced. Mutations of Asn(706) and Glu(183) have in common that they dramatically impede the function of the enzyme in E2 states, but have little effect in E1. Contrary to the Glu(183) mutant, in which E2P slowly accumulates (Clausen, J. D., Vilsen, B., McIntosh, D. B., Einholm, A. P., and Andersen, J. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2776-2781), E2P formation was not detectable with the Asn(706) mutants. Differential sensitivities of the mutants to inhibition by AlF, MgF, and BeF made it possible to distinguish different roles of Asn(706) and Glu(183). Hence, Asn(706) is less important than Glu(183) for gaining the transition state during E2P hydrolysis but plays critical roles in stabilization of E2P ground and E2.P(i) product states and in the major conformational changes associated with the Ca(2)E1P --> E2P and E2 --> Ca(2)E1 transitions, which seem to be facilitated by interaction of Asn(706) with domain A.
Open Access
Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase
(2008) Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter
ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.
Open Access
Development of plant-produced protein body vaccine candidates for bluetongue virus
(2017) van Zyl, Albertha R; Meyers, Ann E; Rybicki, Edward P
BACKGROUND: Bluetongue is a disease of domestic and wild ruminants caused by bluetongue virus serotypes (BTV), which have caused serious outbreaks worldwide. Commercially available vaccines are live-attenuated or inactivated virus strains: these are effective, but there is the risk of reversion to virulence or reassortment with circulating strains for live virus, and residual live virus for the inactivated vaccines. The live-attenuated virus vaccines are not able to distinguish naturally infected animals from vaccinated animals (DIVA compliant). Recombinant vaccines are preferable to minimize the risks associated with these vaccines, and would also enable the development of candidate vaccines that are DIVA-compliant. RESULTS: In this study, two novel protein body (PB) plant-produced vaccines were developed, Zera®-VP2ep and Zera®-VP2. Zera®-VP2ep contained B-cell epitope sequences of multiple BTV serotypes and Zera®-VP2 contained the full-length BTV-8 VP2 codon-optimised sequence. In addition to fulfilling the DIVA requirement, Zera®-VP2ep was aimed at being multivalent with the ability to stimulate an immune response to several BTV serotypes. Both these candidate vaccines were successfully made in N. benthamiana via transient Agrobacterium-mediated expression, and in situ TEM analysis showed that the expressed proteins accumulated within the cytoplasm of plant cells in dense membrane-defined PBs. The peptide sequences included in Zera®-VP2ep contained epitopes that bound antibodies produced against native VP2. Preliminary murine immunogenicity studies showed that the PB vaccine candidates elicited anti-VP2 immune responses in mice without the use of adjuvant. CONCLUSIONS: These proof of concept results demonstrate that Zera®-VP2ep and Zera®-VP2 have potential as BTV vaccines and their development should be further investigated.
Open Access
Diamphotoxin: the arrow poison of the Kung bushmen
(1983) DE LA HARPE, J; REICH, E; REICH, K; DOWDLE, E
Purification de la diamphotoxine et étude de ses caractéristiques pharmacologiques et biochimiques.
Open Access
Differential Internalization of Mammalian and Non-mammalian Gonadotropin-releasing Hormone Receptors: UNCOUPLING OF DYNAMIN-DEPENDENT INTERNALIZATION FROM MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING
(2001) Hislop, James N; Everest, Helen M; Flynn, Andrea; Harding, Tom; Uney, James B; Troskie, Brigitte E; Millar, Robert P; McArdle, Craig A
Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.
Open Access
Discovery of parvovirus-related sequences in an unexpected broad range of animals
(2016) François, S; Filloux, D; Roumagnac, P; Bigot, D; Gayral, P; Martin, D P; Froissart, R; Ogliastro, M
Our knowledge of the genetic diversity and host ranges of viruses is fragmentary. This is particularly true for the Parvoviridae family. Genetic diversity studies of single stranded DNA viruses within this family have been largely focused on arthropod- and vertebrate-infecting species that cause diseases of humans and our domesticated animals: a focus that has biased our perception of parvovirus diversity. While metagenomics approaches could help rectify this bias, so too could transcriptomics studies. Large amounts of transcriptomic data are available for a diverse array of animal species and whenever this data has inadvertently been gathered from virus-infected individuals, it could contain detectable viral transcripts. We therefore performed a systematic search for parvovirus-related sequences (PRSs) within publicly available transcript, genome and protein databases and eleven new transcriptome datasets. This revealed 463 PRSs in the transcript databases of 118 animals. At least 41 of these PRSs are likely integrated within animal genomes in that they were also found within genomic sequence databases. Besides illuminating the ubiquity of parvoviruses, the number of parvoviral sequences discovered within public databases revealed numerous previously unknown parvovirus-host combinations; particularly in invertebrates. Our findings suggest that the host-ranges of extant parvoviruses might span the entire animal kingdom.
Open Access
Diversification into novel habitats in the Africa clade of Dioscorea (Dioscoreaceae): erect habit and elephant’s foot tubers
(2016) Maurin, Olivier; Muasya, A Muthama; Catalan, Pilar; Shongwe, Eugene Z; Viruel, Juan; Wilkin, Paul; van der Bank, Michelle
Abstract Background Dioscorea is a widely distributed and highly diversified genus in tropical regions where it is represented by ten main clades, one of which diversified exclusively in Africa. In southern Africa it is characterised by a distinct group of species with a pachycaul or “elephant’s foot” structure that is partially to fully exposed above the substrate. In contrast to African representatives of the genus from other clades, occurring mainly in forest or woodland, the pachycaul taxa and their southern African relatives occur in diverse habitats ranging from woodland to open vegetation. Here we investigate patterns of diversification in the African clade, time of transition from forest to more open habitat, and morphological traits associated with each habitat and evaluate if such transitions have led to modification of reproductive organs and mode of dispersal. Results The Africa clade originated in the Oligocene and comprises four subclades. The Dioscorea buchananii subclade (southeastern tropical Africa and South Africa) is sister to the East African subclade, which is respectively sister to the recently evolved sister South African (e. g., Cape and Pachycaul) subclades. The Cape and Pachycaul subclades diversified in the east of the Cape Peninsula in the mid Miocene, in an area with complex geomorphology and climate, where the fynbos, thicket, succulent karoo and forest biomes meet. Conclusions Diversification out of forest is associated with major shifts in morphology of the perennial tuber (specifically an increase in size and orientation which presumably led them to become pachycaul) and rotation of stem (from twining to non-twining). The iconic elephant's foot morphology, observed in grasslands and thicket biomes, where its corky bark may offer protection against fire and herbivory, evolved since mid Miocene. A shift in pollination trait is observed within the forest, but entry into open habitat does not show association with reproductive morphology, except in the seed wing, which has switched from winged all round the seed margin to just at the base or at the apex of it, or has been even replaced by an elaiosome.
Open Access
Female begging calls reflect nutritional need of nestlings in the hen harrier Circus cyaneus
(2017) Redpath, Steve; Thompson, Alex; AMAR, ARJUN
BACKGROUND: Most birds exhibit bi-parental care with both sexes providing food for their young. Nestling signal food needs through begging. However, for some species, males rarely visit the nest, so have limited opportunity for gaining information directly from the chicks. Instead, females beg when males deliver food. We tested whether this calling signalled nutritional need and specifically the needs of the female (Breeder Need hypothesis) or that of their chicks (Offspring Need hypothesis). RESULTS: We observed begging and provisioning rates at 42 nests of hen harrier (Circus cyaneus) in Scotland, explored the factors associated with variation in begging rate and the relationship between begging and provisioning. We also tested the impact of food on begging and provisioning through a feeding experiment. Female begging rate increased up to a chick age of 3 weeks and then tailed off. In addition, begging increased when broods were large. CONCLUSIONS: Our data provided support for the Offspring Need hypothesis. At nests where adlib food was provided females reduced their begging rate. These patterns suggested that female begging was an honest signal of need. However, begging continued even with adlib food and was only weakly associated with greater provisioning by males, suggesting that these calls may also play an additional role, possibly reflecting sexual or parent-offspring conflict.
Open Access
Functional microdomains in G-protein-coupled receptors: the conserved arginine-cage motif in the gonadotropin-releasing hormone receptor
(1998) Ballesteros, Juan; Kitanovic, Smiljka; Guarnieri, Frank; Davies, Peter; Fromme, Bernard J; Konvicka, Karel; Chi, Ling; Millar, Robert P; Davidson, James S; Weinstein, Harel; Sealfon, Stuart C
An Arg present in the third transmembrane domain of all rhodopsin-like G-protein-coupled receptors is required for efficient signal transduction. Mutation of this Arg in the gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished or severely impaired agonist-stimulated inositol phosphate generation, consistent with Arg having a role in receptor activation. To investigate the contribution of the surrounding structural domain in the actions of the conserved Arg, an integrated microdomain modeling and mutagenesis approach has been utilized. Two conserved residues that constrain the Arg side chain to a limited number of conformations have been identified. In the inactive wild-type receptor, the Arg side chain is proposed to form an ionic interaction with Asp3.49(138). Experimental results for the Asp3. 49(138) --> Asn mutant receptor show a modestly enhanced receptor efficiency, consistent with the hypothesis that weakening the Asp3. 49(138)-Arg3.50(139) interaction by protonation of the Asp or by the mutation to Asn favors activation. With activation, the Asp3. 49(138)-Arg3.50(139) ionic bond would break, and the unrestrained Arg would be prevented from orienting itself toward the water phase by a steric clash with Ile3.54(143). The mutation Ile3.54(143) --> Ala, which eliminates this clash in simulations, causes a marked reduction in measured receptor signaling efficiency, implying that solvation of Arg3.50(139) prevents it from functioning in the activation of the receptor. These data are consistent with residues Asp3.49(138) and Ile3.54(143) forming a structural motif, which helps position Arg in its appropriate inactive and active receptor conformations.
Open Access
Glutamate 301 of the mouse gonadotropin-releasing hormone receptor confers specificity for arginine 8 of mammalian gonadotropin-releasing hormone
(1994) Flanagan, C A; Becker, I I; Davidson, J S; Wakefield, I K; Zhou, W; Sealfon, S C; Millar, R P
The Arg residue at position 8 of mammalian GnRH is necessary for high affinity binding to mammalian GnRH receptors. This requirement has been postulated to derive from an electrostatic interaction of Arg8 with a negatively charged receptor residue. In order to identify such a residue, 8 conserved acidic residues of the mouse GnRH receptor were mutated to isosteric Asn or Gln. Mutant receptors were tested for decreased preference for Arg8-containing ligands by ligand binding and inositol phosphate production. One of the mutants, in which the Glu301 residue was mutated to Gln, exhibited a 56-fold decrease in apparent affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys8]GnRH, but its affinity for [Gln8]GnRH was unchanged compared with the wild type receptor. The apparent affinity of the mutant receptor for the acidic analogue, [Glu8]GnRH, was increased more than 10-fold. The mutant receptor did not, therefore, distinguish mammalian GnRH from analogues with amino acid substitutions at position 8 as effectively as the wild type receptor. This loss of discrimination was specific for the residue at position 8, because the mutant receptor did distinguish mammalian GnRH from analogues with favorable substitutions at positions 5, 6, and 7. These findings show that Glu301 of the GnRH receptor plays a role in receptor recognition of Arg8 in the ligand and are consistent with an electrostatic interaction between these 2 residues.
Open Access
Identification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form
(1997) Yu, X Christopher; Sturrock, Edward D; Wu, Zhuchun; Biemann, Klaus; EHLERS, Mario R W; Riordan, James F
The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.
Open Access
Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE
(2003) Clausen, Johannes D; McIntosh, David B; Vilsen, Bente; Woolley, David G; Andersen, Jens Peter
Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.
Open Access
Influence of Tumour Necrosis Factor Alpha on the Outcome of Ischaemic Postconditioning in the Presence of Obesity and Diabetes
(2012) Lacerda, Lydia; Opie, Lionel H; Lecour, Sandrine
Obesity and diabetes contribute to cardiovascular disease and alter cytokine profile. The cytokine, tumour necrosis factor alpha (TNFα), activates a protective signalling cascade during ischaemic postconditioning (IPostC). However, most successful clinical studies with IPostC have not included obese and/or diabetic patients. We aimed to investigate the influence of TNFα on the outcome of IPostC in obese or diabetic mice. TNF knockout or wildtype mice were fed for 11 weeks with a high carbohydrate diet (HCD) to induce modest obesity. Diabetes was induced in a separate group by administration of a single intraperitoneal injection of streptozotocin. Hearts were then isolated and subjected to ischaemia (35 min of global ischaemia) followed by 45 min of reperfusion. HCD increased body weight, plasma insulin and leptin levels while the glucose level was unchanged. In streptozotocin-treated mice, blood glucose, plasma leptin and insulin were altered. Control, obese or diabetic mice were protected with IPostC in wiltype animals. In TNF knockout mice, IPostC failed to protect control and diabetic hearts while a slight protection was observed in obese hearts. Our data confirm a bidirectional role for TNFα associated with the severity of concomitant comorbidities and suggest that diabetic and/or modestly obese patients may still benefit from IPostC.
Open Access
Interaction of nucleotides and cations with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum as determined by fluorescence changes of bound 1-anilino-8-naphthalenesulfonate
(1983) Arav, R; Aderem, A A; Berman, M C
The changes in fluorescence of 1-anilino-8-naphthalenesulfonate (ANS-) have been used to determine binding of ligands to the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles, isolated from rabbit skeletal muscle. ANS- binds to sarcoplasmic reticulum membranes with an apparent Kd of 3.8 X 10(-5) M. The binding of ANS- had no effect on Ca2+ transport or Ca2+-dependent ATPase activity. EGTA, by binding endogenous Ca2+, increased the fluorescence intensity of bound ANS- by 10-12%. Subsequent addition of ATP, ADP, or Ca2+, in the presence or absence of Mg2+, reversed this change of fluorescence. The binding parameters, as determined by these decreases in fluorescence intensity, were as follows: for ATP, Kd = 1.0 X 10(-5) M, nH = 0.80; for ADP, Kd = 1.2 X 10(-5) M, nH = 0.89; and for Ca2+, Kd = 3.4 X 10(-7) M, nH = 1.8. The binding parameters for ITP and for the nonhydrolyzable analogue, adenyl-5'-yl-beta, gamma-methylene)diphosphate, were similar to those of ATP, but GDP, IDP, CDP, AMP, and cAMP had lower apparent affinities. Millimolar concentrations of pyrophosphate also decreased the fluorescence of bound ANS-, whereas orthophosphate caused a small (2-3%) increase in fluorescence in Ca2+-free media. Vanadate, in the presence of EGTA, decreased the fluorescence of bound ANS-with half-maximal effect at 4 X 10(-5) M. The changes of fluorescence intensity of bound ANS- appear to reflect conformational changes of the (Ca2+, Mg2+)-ATPase, consequent to ligand binding, with the low and high fluorescence intensity species corresponding to the E1 and E2 conformations, respectively. These appear to reflect similar conformational states of the (Ca2+, Mg2+)-ATPase to those reported by changes in intrinsic tryptophan fluorescence (DuPont, Y. (1976) Biochem, Biophys. Res. Commun. 71, 544-550).
Open Access
Interference with Hemozoin Formation Represents an Important Mechanism of Schistosomicidal Action of Antimalarial Quinoline Methanols
(2009) Corrêa Soares, Juliana B R; Menezes, Diego; Vannier-Santos, Marcos A; Ferreira-Pereira, Antonio; Almeida, Giulliana T; Venancio, Thiago M; Verjovski-Almeida, Sergio; Zishiri, Vincent K; Kuter, David; Hunter, Roger; Egan, Timothy J; Oliveira, Marcus F
BackgroundThe parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel remains the main drug used for schistosomiasis treatment, and reliance on the single therapy has been prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within the S. mansoni gut is a major heme detoxification route with lipid droplets involved in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of three antimalarial compounds, quinine (QN), quinidine (QND) and quinacrine (QCR) in a murine schistosomiasis model by using a combination of biochemical, cell biology and molecular biology approaches.Methodology/Principal FindingsTreatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN, and QND (75 mg/kg/day) from the 11th to 17th day after infection caused significant decreases in worm burden (39%–61%) and egg production (42%–98%). Hz formation was significantly inhibited (40%–65%) in female worms recovered from QN- and QND-treated mice and correlated with reduction in the female worm burden. We also observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reaction to parasite eggs trapped in the liver. Microarray gene expression analysis indicated that QN treatment increased the expression of transcripts related to musculature, protein synthesis and repair mechanisms.ConclusionsThe overall significant reduction in several disease burden parameters by the antimalarial quinoline methanols indicates that interference with Hz formation in S. mansoni represents an important mechanism of schistosomicidal action of these compounds and points out the heme crystallization process as a valid chemotherapeutic target to treat schistosomiasis.Author SummaryHeme is an essential molecule to most living organisms, but once in a free state it exerts toxic effects. Blood-feeding organisms evolved efficient ways to detoxify free heme derived from hemoglobin digestion. A key mechanism present in some hematophagous organisms consists of the crystallization of heme into a pigment named hemozoin. Schistosoma mansoni is one of the etiologic agents of human schistosomiasis, a parasitic disease that affects over 200 million people in tropical and subtropical areas. Hemozoin formation represents the main heme detoxification pathway in S. mansoni. Here, we report that the antimalarial quinoline methanols quinine and quinidine exert schistosomicidal effects notably due to their capacity to interfere with hemozoin formation. When quinine or quinidine were administered intraperitoneally during seven days to S. mansoni-infected mice (75 mg/kg/day), both worm and eggs burden were significantly reduced. Interestingly, hemozoin content in female worms was drastically affected after treatment with either compound. We also found that quinine caused important changes in the cellular organization of worm gastrodermis and increased expression of genes related to musculature, protein synthesis and repair mechanisms. Together, our results indicate that interference with hemozoin formation is a valid chemotherapeutic target for development of new schistosomicidal agents.
Open Access
Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths
(2017) Pelly, Victoria S; Gialitakis, Manolis; Okoye, Isobel S; RYckerl, Dominik; Allen, Judith E; Brombacher, Frank; Wilson, Mark S
Immunity to intestinal helminth infections requires the rapid activation of T helper 2 cells (Th2 cells). However, simultaneous expansion of CD4 + Foxp3 + regulatory T cells (T reg cells) impedes protective responses, resulting in chronic infections. The ratio between T reg and effector T cells can therefore determine the outcome of infection. The redifferentiation of T reg cells into Th cells has been identified in hyperinflammatory diseases. In this study, we asked whether ex–T reg Th2 cells develop and contribute to type-2 immunity. Using multigene reporter and fate-reporter systems, we demonstrate that a significant proportion of Th2 cells derive from Foxp3 + cells after Heligmosomoides polygyrus infection and airway allergy. Ex-Foxp3 Th2 cells exhibit characteristic Th2 effector functions and provide immunity to H. polygyrus . Through selective deletion of Il4ra on Foxp3 + cells, we further demonstrate IL-4 is required for the development of ex-Foxp3 Th2 cells. Collectively, our findings indicate that converting T reg cells into Th2 cells could concomitantly enhance Th2 cells and limit T reg cell–mediated suppression.
Open Access
Is multiple nest building an adequate strategy to cope with inter-species nest usurpation?
(2016) Sumasgutner, Petra; Millán, Juan; CURTIS, ODETTE; Koelsag, Ann; AMAR, ARJUN
Abstract Background Black sparrowhawks (Accipiter melanoleucus) recently colonised the Cape Peninsula, South Africa, where the species faces competition for their nest sites from Egyptian geese (Alopochen aegyptiaca) which frequently usurp black sparrowhawk nests. In this paper, we test the hypothesis that multiple nest building by black sparrowhawks is a strategy to cope with this competitor, based on a 14-year long term data set. Results Two main results support the hypothesis: first, the numbers of intact nests per breeding season in black sparrowhawk territories increased as levels of geese interactions increased, specifically when usurpation occurred. Usurpation occurred significantly more often at nests later in the season, and may provide a further explanation for the advancement of the black sparrowhawk breeding season towards earlier breeding attempts which results in an overall extension of the breeding period (over 9 months) that has been found in our study population. Second, nest usurpation had a negative impact on black sparrowhawks’ reproductive performance at the ‘nest’ level, but not at the ‘territory’ level when multiple nests were available within the same breeding season, suggesting that this strategy was effective for dealing with this competitor. However, our results do not rule out long term negative consequences of these interactions, for example, reduced adult survival rates or reduced lifetime reproductive success, due to the higher energy demand required to build several nests each breeding season. Conclusions Our results suggest that black sparrowhawks avoid direct conflict with this large and aggressive competitor and instead choose the passive strategy in allocating more resources to multiple nest building. Our research further highlights the importance of behavioural plasticity, which might be especially important for city-dwelling species in the face of global urbanisation.
Open Access
Long-Term Left Ventricular Remodelling in Rat Model of Nonreperfused Myocardial Infarction: Sequential MR Imaging Using a 3T Clinical Scanner
(2012) Saleh, Muhammad G; Sharp, Sarah-Kate; Alhamud, Alkathafi; Spottiswoode, Bruce S; van der Kouwe, André J W; Davies, Neil H; Franz, Thomas; Meintjes, Ernesta M
Purpose. To evaluate whether 3T clinical MRI with a small-animal coil and gradient-echo (GE) sequence could be used to characterize long-term left ventricular remodelling (LVR) following nonreperfused myocardial infarction (MI) using semi-automatic segmentation software (SASS) in a rat model. Materials and Methods. 5 healthy rats were used to validate left ventricular mass (LVM) measured by MRI with postmortem values. 5 sham and 7 infarcted rats were scanned at 2 and 4 weeks after surgery to allow for functional and structural analysis of the heart. Measurements included ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV), and LVM. Changes in different regions of the heart were quantified using wall thickness analyses. Results. LVM validation in healthy rats demonstrated high correlation between MR and postmortem values. Functional assessment at 4 weeks after MI revealed considerable reduction in EF, increases in ESV, EDV, and LVM, and contractile dysfunction in infarcted and noninfarcted regions. Conclusion. Clinical 3T MRI with a small animal coil and GE sequence generated images in a rat heart with adequate signal-to-noise ratio (SNR) for successful semiautomatic segmentation to accurately and rapidly evaluate long-term LVR after MI.