Biomedical subjects

Patrick J Skelly

Publications and source records attributed to Patrick J Skelly.

8 recordsLinked to original sources

PubMed2006

Making sense of the schistosome surface.

The syncytial cytoplasmic layer, termed the tegument, which covers the entire surface of adult schistosomes, is a major interface between the parasite and its host. Since schistosomes can survive for decades within the host bloodstream, they are clearly able to evade host immune responses, and their ability is dependent on the properties of the tegument surface. We review here the molecular organization and biochemical functions of the tegument, combining the extensive literature over the last three decades with recent proteomic studies. We have interpreted the organization of the tegument surface as bounded by a conventional plasma membrane overlain by a membrane-like secretion, the membranocalyx, with which host molecules can associate. The range of parasite proteins, glycans and lipids found in the surface complex is evaluated, together with the host molecules detected. We consider the way in which the tegument surface is formed after cercarial penetration into the skin, and changes that occur as parasites develop to maturity. Lastly, we review the evidence on surface dynamics and turnover.

Animals↗

PubMed2004

Aromatic amino acid transporter AAT-9 of Caenorhabditis elegans localizes to neurons and muscle cells.

The Caenorhabditis elegans genome encodes nine homologues of mammalian glycoprotein-associated amino acid transporters. Two of these C. elegans proteins (AAT-1 and AAT-3) have been shown to function as catalytic subunits (light chains) of heteromeric amino acid transporters. These proteins need to associate with a glycoprotein heavy chain subunit (ATG-2) to reach the cell surface in a manner similar to that of their mammalian homologues. AAT-1 and AAT-3 contain a cysteine residue in the second putative extracellular loop through which a disulfide bridge can form with a heavy chain. In contrast, six C. elegans members of this family (AAT-4 to AAT-9) lack such a cysteine residue. We show here that one of these transporter proteins, AAT-9, reaches the cell surface in Xenopus oocytes without an exogenous heavy chain and that it functions as an exchanger of aromatic amino acids. Two-electrode voltage clamp experiments demonstrate that AAT-9 displays a substrate-activated conductance. Immunofluorescence shows that it is expressed close to the pharyngeal bulbs within C. elegans neurons. The selective expression of an aat-9 promoter-green fluorescent protein construct in several neurons of this region and in wall muscle cells around the mouth supports and extends these localization data. Taken together, the results show that AAT-9 is expressed in excitable cells of the nematode head and pharynx in which it may provide a pathway for aromatic amino acid transport.

Amino Acid Transport Systems↗

PubMed2004

Cloning and expression of a Na(+), K(+)-ATPase alpha-subunit from Taenia solium (TNaK1alpha).

The Na(+), K(+)-ATPase are membrane-associated enzymes that transport Na(+) and K(+) across the membrane generating chemical and electrical gradients, essential to maintain the resting potential for the excitation of myocytons and neurons and for transport of nutrients. The cDNA encoding a full-length isoform of Taenia solium Na(+), K(+)-ATPase alpha-subunit (TNaK1alpha) was isolated from a cysticercal cDNA library. TNaK1alpha has 1014 amino acids and a predicted molecular mass of 111,989Da. The protein displays strong sequence homology and conserved motifs typical of Na(+), K(+)-ATPase alpha-subunits. Northern and Southern hybridizations reveal a TNaK1alpha mRNA of about 3.7kb, which is encoded by a single gene. Polyclonal antibodies raised against a synthetic peptide corresponding to the NH(2)-terminal sequence of TNaK1alpha recognized a 100-kDa polypeptide in the membrane fraction of adult and larval stages of T. solium and other Taenia species. Immunolocalization studies using the same antibodies revealed that the TNaK1 is preferentially localized in muscle cells and protonephridial ducts, and in small quantities in the tegument of T. solium cysticerci.

Amino Acid Sequence↗

PubMed2004

Intravascular schistosomes and complement.

Schistosomes are exposed to a variety of immunological effectors, such as host complement, in the bloodstream of their definitive hosts. The parasites are reported to possess a plethora of regulatory proteins, including molecules acquired from the host, which impede the complement cascade. Evidence for the presence of a surface C2-binding protein, a C3-binding protein and a C8- and C9-binding protein has been reported. In addition, a surface Fc receptor might bind immunoglobulin and limit its ability to fix complement. However, the actual protective role of these proteins in vivo remains unresolved.

Animals↗

PubMed2003

Functional characterization of Caenorhabditis elegans heteromeric amino acid transporters.

Mammalian heteromeric amino acid transporters (HATs) are composed of a multi-transmembrane spanning catalytic protein covalently associated with a type II glycoprotein (e.g. 4F2hc, rBAT) through a disulfide bond. Caenorhabditis elegans has nine genes encoding close homologues of the HAT catalytic proteins. Three of these genes (designated AAT-1 to AAT-3) have a much higher degree of similarity to the mammalian homologues than the other six, including the presence of a cysteine residue at the position known to form a disulfide bridge to the glycoprotein partner in mammalian HATs. C. elegans also has two genes encoding homologues of the heteromeric amino acid transporter type II glycoprotein subunits (designated ATG-1 and ATG-2). Both ATG, and/or AAT-1, -2, -3 proteins were expressed in Xenopus oocytes and tested for amino acid transport function. This screen revealed that AAT-1 and AAT-3 facilitate amino acid transport when expressed together with ATG-2 but not with ATG-1 or the mammalian type II glycoproteins 4F2hc and rBAT. AAT-1 and AAT-3 covalently bind to both C. elegans ATG glycoproteins, but only the pairs with ATG-2 traffic to the oocyte surface. Both of these functional, surface-expressed C. elegans HATs transport most neutral amino acids and display the highest transport rate for l-Ala and l-Ser (apparent K(m) 100 microm range). Similar to their mammalian counterparts, the C. elegans HATs function as (near) obligatory amino acid exchangers. Taken together, this study demonstrates that the heteromeric structure and the amino acid exchange function of HATs have been conserved throughout the evolution of nematodes to mammals.

Alanine↗

PubMed2003

Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference.

In this paper, we used the genetic manipulation technique known as RNA-interference to suppress the expression of a target, cathepsin B, gene in the platyhelminth parasite, Schistosoma mansoni. Parasites were cultured for 6 days in the presence of double stranded RNA derived from the cathepsin B cDNA sequence or from two control sequences. Relative to the controls, the cathepsin B double stranded RNA-treated group exhibited lower levels of cathepsin B as determined by immuno-staining and by enzyme activity measurements. Additionally, using the reverse transcriptase-PCR, suppression was seen in the inability to detect cathepsin B cDNA, using RNA obtained from those parasites. This ability to manipulate gene expression represents a powerful new tool for investigating gene function in these debilitating human parasites.

Animals↗

PubMed2002

Comparative efficacy of the Schistosoma mansoni nucleic acid vaccine, Sm23, following microseeding or gene gun delivery.

Sm23 is an integral membrane protein expressed widely in the human parasitic worm Schistosoma mansoni. Sm23 has already been shown to elicit protective immune responses following immunization with peptides or DNA constructs. In this study, we evaluated the immunogenicity and the protective efficacy of the Sm23 DNA vaccine using two different intradermal DNA delivery methods: microseeding and gene gun. Using both techniques, all mice immunized with the Sm23-pcDNA construct generated Sm23-specific immunoglobulin (Ig)G antibody, while mice immunized with the control plasmid, pcDNA, did not. Antibody isotypes analysis revealed that microseeding elicited mainly IgG2a and IgG2b antibodies, with relatively low levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio was 0.03, indicative of a Th1 type immune response. In contrast, gene gun immunization resulted in significantly higher levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio in this case was 11, indicative of a Th2 type immune response. No significant difference in the levels of IgG2b was observed. Coimmunization with plasmid DNA encoding either interleukin (IL)-12 or IL-4 by microseeding did not affect the levels of IgG1, while the levels of IgG2a and IgG2b were reduced. On the other hand, the levels of IgG3 were significantly increased by IL-4, but unchanged by IL-12. Importantly, in all experiments, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection. Microseeding immunizations resulted in higher levels of protection (31-34% protection) than gene gun immunization (18% protection). This suggests that the Th1 type immune response elicited by microseeding immunization was responsible for the higher protection levels. However, the protective effect of the vaccine was not affected by coadministering plasmids encoding either IL-12 or IL-4 using the microseeding technique.

Animals↗

PubMedDate not supplied

A DNA-prime/protein-boost vaccination regimen enhances Th2 immune responses but not protection following Schistosoma mansoni infection.

DNA immunization represents a promising vaccine strategy that has been reasonably successful, and will likely play an even greater role in vaccine development as these vaccines continue to be improved. We have developed a partially protective DNA vaccine against schistosome infection based on a 23-kDa integral membrane protein, Sm23. The focus of this study was to compare immunogenicity and efficacy of vaccination regimens utilizing Sm23 DNA vaccine alone vs. regimens that utilized both Sm23 DNA and Sm23 in recombinant protein form. We found that priming and boosting with the Sm23 DNA construct (Sm23-pcDNA) resulted in a significant level of protection against challenge infection (36-44%). In contrast, altering this protocol by changing the boost from Sm23 DNA to boosting with recombinant Sm23 protein (rSm23) formulated in aluminium hydroxide (alum) failed to induce a significant reduction in worm burdens. Similarly, mice primed and boosted with the rSm23 in alum also did not develop significant levels of protection against challenge infection. We hypothesize that the differences in the ability to drive protective immunity using the DNA prime-DNA boost strategy and the inability to do so when recombinant Sm23 in alum was substituted for Sm23 DNA is due to driving of different immune responses. In support of this, we found that mice primed and boosted with Sm23-pcDNA had Th1-type immune responses characterized by low anti-Sm23 IgG1 : IgG2a antibody isotype ratios, whereas mice boosted with rSm23 had higher IgG1 : IgG2a ratios. In addition, priming and boosting with rSm23 elicited mainly IgG1 antibodies with no detectable IgG2a, indicative of a polarized Th2-type immune response. Thus, similar to our earlier work, the results of this study show that protective vaccination using Sm23 is associated with a Th1 immune response, and efficacy is diminished using protocols that diminish this Th1 bias. In our study, this was likely due to the use of the Th2-driving adjuvant alum, and future studies are planned where we will compare the protective efficacy of rSm23 administered with Th1-type adjuvants.

Animals↗