Conraths FJ et al. (1997) Exp Parasitol "Expression of the microfilarial sheath protein 2 (shp2) of the filarial parasites Litomosoides sigmodontis and Brugia malayi."

Hobom G, Zahner H, Conraths FJ, Hirzmann J

[Exp Parasitol, 1997]

The microfilarial sheaths of the filarial parasites Brugia malayi, Brugia pahangi, and Litomosoides sigmodontis consist of several parasite proteins, probably ranging between 7 and 10. The gene encoding sheath protein 2 (shp2), which is the object of this study, is transcribed in embryos and in the uterine epithelium; at least in B. malayi, it is translated in both tissues. Apparently, shp2 is synthesized as a monomer, exported by the respective cells, and integrated into the microfilarial sheath. In the sheath, it exists as a highly polymerized molecule cross-linked by cysteine formation and other covalent bonds, presumably epsilon-(gamma-glutamyl)-lysine links.

Garate T et al. (1990) Trop Med Parasitol "Cloning of specific diagnostic antigens of Onchocerca volvulus."

Parkhouse RM, Harnett W, Conraths FJ, Buttner DW, Garate T

[Trop Med Parasitol, 1990]

Specific, serological diagnosis is one of the main goals in onchocerciasis research. To date this objective has been hampered by (a) scarcity of parasite material, and (b) antigenic cross-reaction between Onchocerca volvulus and other nematode species. In order to obtain specific antigens, and in amounts suitable for study, molecular biological techniques have been adopted. A lambda gt11 cDNA expression library prepared from O. volvulus adult female worms was screened using infected human sera from onchocerciasis patients and rabbit hyperimmune sera raised against Onchocerca and genus-specific Onchocerca antigen extracts. Five clones were selected and their inserts expressed as beta-galactosidase fusion proteins. The fusion proteins were examined using individual sera from patients with O. volvulus or Wuchereria bancrofti infections. Three of the fusion proteins were recognised by more than 80% of O. volvulus sera and exhibited weak reactivity with a few W. bancrofti sera. One of these three clones was recognised to a significantly greater degree by sera from sowda than from generalised onchocerciasis patients.

Hirzmann J et al. (2002) J Biol Chem "Cloning and expression analysis of two mucin-like genes encoding microfilarial sheath surface proteins of the parasitic nematodes Brugia and Litomosoides."

Geyer R, Kasper M, Hirzmann J, Hobom G, Taubert A, Zahner H, Shresta TR, Conraths FJ, Hintz M, Stirm S

[J Biol Chem, 2002]

In several filarial genera the first stage larvae (microfilariae) are enclosed by an eggshell-derived sheath that provides a major interface between the parasite and the host immune system. Analysis of the polypeptide constituents of the microfilarial sheath from the cotton rat filaria Litomosoides sigmodontis identified two abundant surface glycoproteins: Shp3a and Shp3. The corresponding genes and the orthologues of the human parasite Brugia malayi and the rodent filaria Brugia pahangi were cloned and sequenced. They encode secreted, mucin-like proteins with N-terminal Ser/Thr-rich repeats and a C-terminal anchor domain rich in aromatic amino acids. About 75% of the protein molecular masses result from post-translational modifications. The Ser/Thr-rich motifs are supposed to serve as targets for dimethylaminoethanol-phosphate substitutions. These modifications were detected only on the sheaths of the late developmental stage of stretched microfilariae, corresponding with the expression of the proteins in the epithelium of the distal part of the uterus and the specific transcription of shp3 and shp3a in the anterior female worm segment. Genomic analysis of all three species demonstrated a conserved linkage of the two genes. Their transcripts undergo cis- and trans-splicing. The transcription start sites of the primary transcripts were determined for the L. sigmodontis genes. The core promoter regions are remarkably conserved between the paralogue genes Ls-shp3a and Ls-shp3 and their orthologues in Brugia, implicating conserved regulatory elements.

Magalska A et al. (2000) European Worm Meeting "The worm is not enough - the construction of cDNA and gDNA libraries from Ancylostoma ceylanicum"

Kapulkin V, Ciesielski A, Wedrychowicz HZ, Magalska A

[European Worm Meeting, 2000]

We are interested in usage of C. elegans as system for expression of hookworm genes. Hookworms (Ancylostomatideae ) are economically and medically important blood feeding nematodes which cause intestinal infections in humans and domestic animals. It has been reported recently that above 1 billion of human beings are currently infected with these worms. The application of C. elegans as a heterologous host for hookworm genes is limited mainly by the lack of hookworm genomic data. For this purpose we have constructed cDNA and gDNA libraries from the Ancylostoma ceylanicum -important human/animal parasite related to C. elegans. The cDNA library is based on PCR mediated amplification of spliced leader of parasite cDNA reported previously ( Martin SA, Thompson FJ, Devaney E. Mol. Biochem. Parasitol. 1995 Mar;70(1-2):241-5.). PCR amplified cDNA has been gel fractionated and cloned. The most abundant library is available now from adult mixed sex A. ceylanicum (above 6000 clones), smaller library was constructed from infective L3 larvae. The large inserts gDNA libraries were constructed by ligation of PFGE fractionated Ancylostoma genomic DNA into the pBACe3.6 (provided by Pieter deJong), both digested with EcoRI. Ligation mixture was precipitated with ethanol in the presence of yeast tRNA prior electroporation into DH10B host. Both cDNA and gDNA libraries were arrayed in 96 well format, and stored at -70C. Randomly picked clones were sequenced from both ends, blastn and blastx searches were performed. For cDNA clones our sequencing results show clear similarities to C. elegans genes : gene F38E11.2 hsp-12.6; similar to Human Alpha crystallin B chain, gene C15C7.6; similar to syntaxin-6, gene T20F5.2; similar to the S25B family of peptidases. For BAC clones, end sequencing was also performed ( in collaboration with M. obocka, Institute of Biochemistry and Biophysics ) , however no significant similarities were found (yet!).

Carolina Ibanez-Ventoso et al. (2007) International Worm Meeting "MicroRNAs in Aging and Healthspan."

Carolina Ibanez-Ventoso, Monica Driscoll, Mehul Vora

[International Worm Meeting, 2007]

Over 200 mutations that affect C. elegans longevity have been identified. To date, much of the research on C. elegans aging has focused on factors that extend lifespan with less emphasis on their effects on healthspan (the period of mid life vigor that precedes decline). Our lab has been interested in identifying genes that extend healthspan, conferring longer periods of youthful behavior. We have identified genetic and pharmacological manipulations that can extend healthspan with the use of new aging biomarkers and assays we''ve developed: decline of muscle integrity (physical markers and automated computer vision swimming analysis), age-associated accumulation of autofluorescent cellular material (lipofuscin and age pigments), and micoRNA (miRNA) abundance. miRNAs are small molecules prevalent in metazoans that regulate cellular expression by binding to partially homologus sites on gene transcripts to down-regulate translation. Our interest in these broad conserved regulators has been influenced by the suggestion that they might function in biology to limit stochastic biological processes—lost of robustness and increase in stochastic events is associated with aging. To address whether miRNAs could influence the aging process, we conducted the first genome wide-analysis of how miRNAs change with age in any organism. This work showed that about half of C. elegans miRNAs change significantly during adult lifespan and could thus contribute to healthspan [1]. Indeed the miRNA lin-4 influences lifespan and the expression of healthspan indicators like lipofuscin [2], work we have confirmed. Our preliminary work supports that several other miRNAs also impact lifespan and the quality of aging. We are exploiting the powerful experimental advantages of the nematode C. elegans to decipher roles of miRNAs in nematode healthspan and to test whether engineered expression of these small molecules can confer anti-aging therapies in C. elegans. Since many miRNAs are conserved in expression pattern and function, we anticipate that our exploratory work in this area may suggest specific working hypotheses for mammalian aging, perhaps influencing design of novel means of clinical intervention. 1.Ibanez-Ventoso et al. (2006) Modulated microRNA expression during adult lifespan in C. elegans. Aging Cell 5: 235-246. 2.Boehm M, Slack FJ (2005) A developmental timing microRNA and its target regulate life span in C. elegans. Science 310: 1954-1957.