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C B Shoemaker

Publications and source records attributed to C B Shoemaker.

At least 37 records · Page 2Linked to original sources
PubMed1999

Variation in the salivary peptide, maxadilan, from species in the Lutzomyia longipalpis complex.

Maxadilan is an approximately 7kDa peptide that occurs in the saliva of the sand fly Lutzomyia longipalpis. This peptide is a potent vasodilator and may also have immunomodulatory effects related to the pathogenesis of leishmanial infections. Variation in the primary DNA and inferred amino acid sequence of maxadilan is reported. Differences were found within and among natural field populations as well as among sibling species. Extensive amino acid sequence differentiation, up to 23%, was observed among maxadilan from different populations. This is a remarkable degree of polymorphism considering the small size of this peptide. The vasodilatory activity of maxadilan was equivalent among recombinant maxadilan variants. All maxadilan variants induce interleukin-6. Predicted secondary structure and hydrophobicity plots suggest that these characteristics are conserved among variant peptides. However, profiles based on the antigenic index do differ among peptides.

Animals↗
PubMed1998

Functional heterodimeric amino acid transporters lacking cysteine residues involved in disulfide bond.

The protein mediating system L amino acid transport, AmAT-L, is a disulfide-linked heterodimer of a permease-related light chain (AmAT-L-lc) and the type II glycoprotein 4F2hc/ CD98. The Schistosoma mansoni protein SPRM1 also heterodimerizes with h4F2hc, inducing amino acid transport with different specificity. In this study, we show that the disulfide bond is formed by heavy chain C109 with a Cys residue located in the second putative extracellular loop of the multi-transmembrane domain light chain (C164 and C137 for XAmAT-L-lc and SPRM1, respectively). The non-covalent interaction of Cys-mutant subunits is not sufficient to allow coimmunoprecipitation, but cell surface expression of the light chains is maintained to a large extent. The non-covalently linked transporters display the same transport characteristics as disulfide bound heterodimers, but the maximal transport rates are reduced by 30-80%.

Amino Acid Transport Systems↗
PubMed1998

Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family.

Amino-acid transport across cellular plasma membranes depends on several parallel-functioning (co-)transporters and exchangers. The widespread transport system L accounts for a sodium-independent exchange of large, neutral amino acids, whereas the system y(+)L exchanges positively charged amino acids and/or neutral amino acids together with sodium. The molecular nature of these transporters remains unknown, although expression of the human cell-surface glycoprotein 4F2 heavy chain (h4F2hc; CD98 in the mouse) is known to induce low levels of L- and/or y(+)L-type transport. This glycoprotein is found in activated lymphocytes, together with an uncharacterized, disulphide-linked lipophilic light chain with an apparent relative molecular mass of 40,000 (M(r) 40K). Here we identify the permease-related protein E16 as the first light chain of h4F2hc and show that the resulting heterodimeric complex mediates L-type amino-acid transport. The homologous protein from Schistosoma mansoni, SPRM1, also associates covalently with coexpressed h4F2hc glycoprotein, although it induces amino-acid transport of different substrate specificity. The coexpression of h4F2hc is required for surface expression of these permease-related light chains, which belong to a new family of amino-acid transporters that form heterodimers with cell-surface glycoproteins.

Amino Acid Transport Systems↗
PubMed1998

A divergent member of the transforming growth factor beta receptor family from Schistosoma mansoni is expressed on the parasite surface membrane.

To optimize reproductive success under the limitations determined by conditions within an individual host, parasitic helminths have evolved mechanisms that allow them to detect and respond to host factors such as species, age, sex, reproductive condition, and immune status. Using the model helminth Schistosoma mansoni, we have explored the possibility that parasitic helminths express signal-transducing receptor molecules on their surfaces. Here, we present the identification of a schistosome member of the transforming growth factor beta receptor family of cell-surface receptors, the first member of this family to be identified in a platyhelminth. The putative protein kinase domain of the schistosome receptor displays up to 58% amino acid identity to kinase domains of other type I receptor serine-threonine kinases, and contains a potential "GS domain," suggesting it is a divergent member of the type I receptor subfamily. This receptor is expressed on the surface of the parasite's syncytial tegument and expression of receptor messenger RNA and protein is up-regulated following infection of the mammalian host. The receptor protein can be isolated in a phosphorylated form from adult parasites, which together with its surface location, suggests that it functions in transducing signals across the parasite surface membrane.

Amino Acid Sequence↗
PubMed1998

The vasoactive peptide maxadilan from sand fly saliva inhibits TNF-alpha and induces IL-6 by mouse macrophages through interaction with the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor.

Maxadilan is a vasodilatory peptide encoded by a gene cloned from Lutzomyia longipalpis salivary glands. In this study we investigated the effects of maxadilan on macrophage functions. Maxadilan treatment of LPS-stimulated BALB/c macrophages inhibited TNF-alpha release but increased IL-6. Further, it also induced IL-6 release in a dose-dependent manner from unstimulated macrophages. Maxadilan increased production of PGE2, and the inhibition of TNF-alpha was completely abrogated by indomethacin. Others have recently shown that maxadilan is a selective agonist of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor. Treatment with the receptor antagonist PACAP 6-38 blocked maxadilan activities on macrophages. The natural endogenous ligand, PACAP 38, had the same effects as maxadilan on TNF-alpha and IL-6 production. Finally, in a dose- and time-dependent fashion, maxadilan induced the intracellular accumulation of cAMP in macrophages. Taken together, the results presented here indicate a modulatory effect of ligands of PACAP type I receptor on cytokine production by macrophages and suggest that activation of this receptor, with the subsequent elevation of intracellular cAMP in macrophages, could participate in a negative-feedback mechanism that controls certain inflammatory responses.

Adenylyl Cyclases↗
PubMed1998

The PACAP-type I receptor agonist maxadilan from sand fly saliva protects mice against lethal endotoxemia by a mechanism partially dependent on IL-10.

Sand fly saliva contains maxadilan, a peptide that causes vasodilation and modifies the secretion of pro-inflammatory cytokines by macrophages. We show that 1 to 10 microg maxadilan protected BALB/c mice against a lethal dose of LPS. Maxadilan reduced serum levels of TNF-alpha by approximately tenfold, while it caused a threefold increase in IL-6 and IL-10. The protective effect of maxadilan is partially dependent on its ability to induce IL-10 production since maxadilan did not prevent death from endotoxic shock in IL-10(-/-) mice. Finally, maxadilan is a selective agonist of the pituitary adenylate cyclase-activating peptide (PACAP) type I receptor, and we found that the natural ligand of this receptor (PACAP 38) also protected mice against lethal endotoxemia. These results indicate that activation of the PACAP type I receptor may contribute to the control of systemic inflammation by a mechanism that is partially dependent on IL-10.

Animals↗
PubMed1998

Glucose Transport and Metabolism in Mammalian-stage Schistosomes.

Adult schistosomes transport nutrients from the host bloodstream across their outer body covering or tegument. The tegument is a cytologically unusual structure; it is a syncytium bounded externally by two lipid bilayer membranes. In this review, Patrick Skelly, Louis Tielens and Chuck Shoemaker reconsider our understanding of how glucose enters schistosomes across this unusual outer covering in the light of recent papers characterizing glucose transport proteins and glucose metabolism pathways in these parasites.

Journal Article↗
PubMed1998

Molecular and functional characterization and tissue localization of 2 glucose transporter homologues (TGTP1 and TGTP2) from the tapeworm Taenia solium.

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers D-glucose and D-mannose but not by L-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

Amino Acid Sequence↗
PubMed1998

T and B cell reactivity to a 42-kDa protein is associated with human resistance to both schistosomiasis mansoni and haematobium.

Egyptian subjects living in areas endemic for Schistosoma mansoni or Schistosoma haematobium were selected on the basis of their apparent extremes of resistance or susceptibility to schistosomiasis and examined for T and B cell responses against the major electrophoretically resolved protein species from soluble adult worm extracts. A 42-kDa band was specifically recognized by a significant majority of subjects resistant to schistosomiasis. The 42-kDa species (p-42) from S. mansoni and S. haematobium were immunologically cross-reactive and induced significant protection in mice and hamsters against infection with cercariae. Amino acid sequence analysis of S. mansoni p-42 showed that it consists predominantly of glyceraldehyde 3-P dehydrogenase (G3PDH), which has been shown to be preferentially recognized by the sera of Brazilian subjects resistant to schistosomiasis mansoni. The present data extend the previous findings and imply that S. mansoni-derived G3PDH represents a target of protective T and B cell-mediated antischistosomiasis immunity in humans.

Adolescent↗
PubMed1996

The Schistosoma mansoni epidermal growth factor receptor homologue, SER, has tyrosine kinase activity and is localized in adult muscle.

DNA encoding the full-length Schistosoma mansoni epidermal growth factor receptor, SER, was obtained by combining partial cDNA clones. Three different anti-SER antibody preparations, specific either to the SER amino-terminus or the predicted ligand binding domain were generated with recombinant or synthetic peptides and purified by antigen affinity. Recombinant SER was expressed within insect cells using the baculovirus expression system and detected by each of the anti-SER antibodies as well as anti-phosphotyrosine antibodies. By in vitro phosphorylation of immunoprecipitated recombinant SER, the protein has been shown to be capable of tyrosine autophosphorylation but this activity is not affected by human epidermal growth factor. Native SER is detected as a 170 kDa protein in Western blots of S. mansoni adult worm membrane preparations. Adult worm sections, labeled with anti-SER antibodies, localize SER predominantly to the muscle of adult male and female worms. These results confirm a place for SER in the EGFR family of tyrosine kinases and strongly suggest that it participates in schistosome signal transduction, perhaps related to muscle development or function.

Amino Acid Sequence↗
PubMed1996

Rapid appearance and asymmetric distribution of glucose transporter SGTP4 at the apical surface of intramammalian-stage Schistosoma mansoni.

Adult Schistosoma mansoni blood flukes reside in the mesenteric veins of their vertebrate hosts, where they absorb immense quantities of glucose through their tegument by facilitated diffusion. Previously, we obtained S. mansoni cDNAs encoding facilitated-diffusion schistosome glucose transporter proteins 1 and 4 (SGTP1 and SGTP4) and localized SGTP1 to the basal membranes of the tegument and the underlying muscle. In this study, we characterize the expression and localization of SGTP4 during the schistosome life cycle. Antibodies specific to SGTP4 appear to stain only the double-bilayer, apical membranes of the adult parasite tegument, revealing an asymmetric distribution relative to the basal transporter SGTP1. On living worms, SGTP4 is available to surface biotinylation, suggesting that it is exposed at the hose-parasite interface. SGTP4 is detected shortly after the transformation of free-living, infectious cercariae into schistosomula and coincides with the appearance of the double membrane. Within 15 min after transformation, anti-SGTP4 staining produces a bright, patchy distribution at the surface of schistosomula, which becomes contiguous over the entire surface of the schistosomula by 24 hr after transformation. SGTP4 is not detected in earlier developmental stages (eggs, sporocysts, and cercariae) that do not possess the specialized double membrane. Thus, SGTP4 appears to be expressed only in the mammalian stages of the parasite's life cycle and specifically localized within the host-interactive, apical membranes of the tegument.

Amino Acid Sequence↗
PubMed1996

Schistosoma mansoni: the glucose transport protein SGTP4 is present in tegumental multilamellar bodies, discoid bodies, and the surface lipid bilayers.

Schistosomes metabolize large quantities of glucose obtained from the host serum by facilitated diffusion through the tegument. Here we have used rabbit antibodies affinity purified against the carboxyl terminus of a facilitated glucose transporter, SGTP4, to localize the antigen in both schistosomula and adults. By ultrastructural immunocytochemical analysis, SGTP4 was localized to both lipid bilayers that cover the tegumental surface of adults and schistosomula. In the inner bilayer of adults, SGTP4 was apparently oriented with the carboxyl terminus on the internal side of the bilayer. SGTP4 was also present in the discoid and multilamellar bodies in adults and the membranous bodies in schistosomula. Finally, the affinity purified antibodies against SGTP4 bound nonspecifically to the head glands and postacetabular glands in schistosomula. The localization of the antigen in the two surface lipid bilayers suggests that SGTP4 may be responsible for transporting glucose from mammalian host serum into the tegument.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of the hexokinase from Schistosoma mansoni, expressed in Escherichia coli.

The hexokinase (ATP;D-hexose 6-phosphotransferase, EC 2.7.1.1) of Schistosoma mansoni has been expressed in Escherichia coli as a fusion protein including an N-terminal polyhistidine tag and enterokinase cleavage site. The enzyme was purified by metal chelate chromatography to > 95% homogeneity, based on analysis by SDS-gel electrophoresis and isoelectric focusing. The absorbance at 280 nm was 0.54 for a 1 mg/ml solution (molar extinction coefficient 2.7 x 10(4) cm2 mol). The pI of the S. mansoni hexokinase was 6.0-6.2, slightly more acidic than the rat Type I isozyme (pI 6.35). The S. mansoni enzyme migrated as a single band of activity during nondenaturing cellulose acetate electrophoresis; the mobility was slightly greater than the rat Type I isozyme, consistent with the estimated pI. The Km values for substrates glucose and ATP were 128 +/- 10 and 927 +/- 41 microM, respectively. In accord with a previous report, the S. mansoni hexokinase exhibited moderate sensitivity to inhibition (competitive vs ATP) by the product, glucose 6-phosphate, with a Ki approximately 150 microM; the product analog, 1,5-anhydroglucitol 6-phosphate, was somewhat less effective as an inhibitor, with Ki approximately 500 microM. These kinetic properties were not altered by removal of the N-terminal fusion partner by enterokinase treatment. Immunological crossreactivity between the rat Type I isozyme and the S. mansoni hexokinase was demonstrated by immunoblotting, but this was markedly dependent on the preparation of antiserum used. The activity of the enzyme is apparently highly dependent on maintenance of free sulfhydryl groups. Activity was maintained during storage in the presence of monothioglycerol; activity lost during storage in the absence of monothioglycerol could be partially restored by treatment with this reagent.

Adenosine Triphosphate↗
PubMed1995

Schistosoma mansoni hexokinase: cDNA cloning and immunogenicity studies.

DNA encoding a Schistosoma mansoni hexokinase (SHEX) was amplified from cDNA by the polymerase chain reaction using opposing oligonucleotide primers designed to hybridize with two short segments of hexokinase coding sequences that are well-conserved through evolution. The resulting DNA fragment was then used as a probe to identify a full-length hexokinase cDNA clone. SHEX cDNA encodes a 50-kDa protein that is approximately 46% homologous to rat hexokinase, 40% to rat glucokinase, and 34% to yeast hexokinase A. SHEX coding DNA was expressed within Escherichia coli cells and the 50-kDa recombinant product (rSHEX) was partially purified. Mice repeatedly immunized with rSHEX produced antibodies which recognize rSHEX but this offered no significant protection against subsequent cercarial challenge. On Western blots, rSHEX is weakly recognized by antisera against rat brain hexokinase but not by sera from three strains of mice experimentally infected with S. mansoni parasites or from numerous human schistosomiasis patients. Thus, unlike other reported S. mansoni glycolytic enzymes, hexokinase appears to be poorly immunogenic during schistosome infection and of limited potential as a vaccine candidate.

Amino Acid Sequence↗
PubMed1995

Immunolocalization of a Schistosoma mansoni facilitated diffusion glucose transporter to the basal, but not the apical, membranes of the surface syncytium.

Adult parasites of Schistosoma mansoni reside within vertebrate mesenteric veins where they consume immense quantities of host glucose after transporting the sugar through their surface syncytium or tegument. Previously we obtained cDNA clones encoding two functional facilitated diffusion glucose transporter proteins expressed by S. mansoni adult worms (Skelly et al. 1994). Antibodies specific for one transporter (SGTP1) have been generated against an extrafacial and an internal domain of the protein and used to localize the protein by light and electron microscopy. By light microscopy both antibodies stain a linear structure approximately 1-5 microns from the surface of the tegument of adult male and female schistosomes. Electron microscopic examination of frozen thin sections show binding of the antibodies to membranes in the base of the tegument and not to the membranes covering the outer surface or their invaginations. Analysis of the gold distribution suggests that the extrafacial domain is disposed toward the interstitial space beneath the tegument and the internal domain faces the syncytial plasm. The localization suggests that SGTP1 may function to transport free glucose from within the tegument and into the interstitial fluids that bathe the internal organs of these parasites.

Amino Acid Sequence↗
PubMed1994

The 50-kDa glucose 6-phosphate-sensitive hexokinase of Schistosoma mansoni.

Hexokinase has been purified from adult Schistosoma mansoni worms and the activity shown to be associated with a single protein species having an M(r) about 50,000. This protein is recognized on Western blots probed with antisera against rat Type I hexokinase or against a recombinant S. mansoni hexokinase that had been expressed in Escherichia coli using a previously cloned cDNA. An 18-residue N-terminal sequence determined for the purified S. mansoni hexokinase is identical to that deduced from the nucleotide sequence of the cDNA, consistent with the view that the cloned cDNA encodes the hexokinase characterized in the present study. The S. mansoni enzyme has a relatively low Km (approximately 60 microM) for glucose and is sensitive to inhibition (competitive versus ATP, Ki approximately 50 microM) by its product, glucose 6-phosphate (Glc-6-P). With these kinetic properties and 50 kDa molecular mass, S. mansoni hexokinase resembles the ancestral hexokinase predicted to have given rise, by gene duplication and fusion, to the present day 100-kDa Glc-6-P-sensitive mammalian hexokinases. The schistosomal hexokinase represents the first 50-kDa Glc-6-P-sensitive hexokinase whose sequence has been obtained. The schistosomal hexokinase does not bind to mitochondria, consistent with its lack of a hydrophobic segment at the N terminus which is required for binding of the mammalian Type I and II isoenzymes to mitochondria. The marked Crabtree effect exhibited by S. mansoni cercariae may be at least partly attributed to the expression of rather high levels of a hexokinase having a high affinity for glucose but only a moderate sensitivity to product inhibition by Glc-6-P.

Amino Acid Sequence↗
PubMed1994

Cloning, characterization, and functional expression of cDNAs encoding glucose transporter proteins from the human parasite Schistosoma mansoni.

The blood-dwelling adult form of the parasitic worm, Schistosoma mansoni, consumes prodigious quantities of host glucose following its transport across the tegument. Immunologic or pharmacologic targeting of the relevant transporter proteins might form the basis of a schistosome control strategy. Here we report the isolation and characterization of three different full-length cDNAs whose predicted protein sequences show a high degree of sequence and structural similarity to the facilitated diffusion transporter proteins of other animals, plants, and bacteria. Functional expression of two cDNAs has been achieved by injection of Xenopus oocytes with in vitro derived sense strand RNA. Injected oocytes have a significantly increased ability to take up radiolabeled glucose analogues over controls. S. mansoni glucose transporters expressed in oocytes exhibit stereospecificity for D-glucose, have relaxed specificity for different hexoses, exhibit sodium independence, and are markedly inhibited by phloretin and cytochalasin B. These two transporters, expressed in oocytes, have a Km for 3-O-methylglucose of 1.3 and 2 mM. A third glucose transporter homologue cDNA appears to derive from a recent pseudogene. Both of the functional S. mansoni glucose transporter genes are expressed in larval and adult male and female schistosomes.

Animals↗