Cloning and characterization of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus.

In order to characterise the integral membrane glycoprotein H11 from the intestinal microvilli of the nematode Haemonchus contortus, cDNA libraries prepared using mRNA from adult worms from the UK and Australia were immunoscreened with anti-H11 sera. Antibodies affinity purified on the protein expressed by insert DNA (295 bp) of a positive clone from a UK library bound specifically to H11. A longer clone (948 bp) was obtained from the Australian library by hybridisation. Using a primer based on sequence common to these, a polymerase chain reaction product of 3.3 kb was generated from cDNA from UK H. contortus. The sequences from the UK and Australian nematodes were essentially identical over the 929 bp region in which both were represented. All three cloned DNAs hybridised to mRNA of about 3.5 kb. Analysis of the deduced amino acid sequence, which showed 32% identity with those of mammalian microsomal aminopeptidases, indicated that H11 has a short N-terminal cytoplasmic tail, a single transmembrane region and a long extracellular region with putative N-linked glycosylation sites and the HEXXHXW motif characteristic of microsomal aminopeptidases. Microsomal aminopeptidase activity co-purifies with H11. It is inhibited by bestatin, phenanthroline and amastatin. The recombinant protein has been expressed in active form in insect cells.

The sequence of foot-and-mouth disease virus RNA to the 5' side of the poly(C) tract.

The nucleotide sequence of foot-and-mouth disease virus (FMDV) RNA to the 5' side of the poly(C) tract (S fragment) has been determined for representatives of the A and O serotypes of the virus. The two S fragments differ in length by five nucleotides (nt), with 367 nt for O1 compared with 362 nt for A10, due to a number of insertions/deletions. However, the two sequences show 86% homology. There are no conserved open reading frames (ORFs). Secondary structure predictions reveal a high degree of potential base-pairing, such that the entire S fragment sequence can be folded into a hairpin structure.

Molecular aspects of sexual development and reproduction in nematodes and schistosomes.

In contrast to the free-living nematode Caenorhabditis elegans, surprisingly little is known about the molecular aspects of reproduction in parasitic helminths. Investigations into such aspects would provide an improved understanding of the fundamentals of sexual differentiation, development, maturation and behaviour, as well as sex-specific genes and their expression. Such knowledge could lead to new means of parasite control by interfering with or disrupting one or more of these processes, which is particularly important given the emerging problems with genetic resistance in parasitic nematodes against anthelmintic drugs. This chapter brings together some relevant information on the sexual biology of C. elegans, summarizes studies of gender-specific expression in selected parasitic helminths of socio-economic significance, describes advanced molecular techniques for the analysis of gender-specific genes, and indicates the prospects for genomic research on reproductive processes and the implications thereof for controlling parasitic helminths.

The organization of a galectin gene from Teladorsagia circumcincta.

Galectins are a family of soluble beta-galactoside-binding lectins that are conserved amongst a broad range of organisms. We have previously isolated cDNA clones coding for galectins from the sheep gastrointestinal nematode parasites Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis, revealing a high level of identity between these molecules. This subsequent study reports the organization of the T. circumcincta Tci-gal-1 galectin gene. The coding region is broken into eight exons covering 6.6 kbp, with introns ranging in size from 55 base pairs (bp) to 2.8 kbp. Comparisons with recently reported galectin structures from Caenorhabditis elegans reveal strong architectural similarity between galectins from the parasitic and free-living nematodes, but this structure is not conserved in mammalian galectins.

cDNA cloning of galectins from third stage larvae of the parasitic nematode Teladorsagia circumcincta.

A monoclonal antibody raised to a Teladorsagia circumcincta 31-33 kDa doublet antigen was used to immunoscreen a T. circumcincta cDNA expression library. Sheep antibodies eluted from the proteins expressed by two clones immunopositive with the monoclonal antibody specifically recognised the doublet antigen on Western blots of third stage larval extract, confirming that these clones coded for the antigen. Database searches revealed high levels of similarity with beta-galactoside-binding lectin-like proteins (Ga1BPs or galectins) from Caenorhabditis elegans and Onchocerca volvulus. By analogy with these sequences, both T. circumcincta cDNA clones contain the full-length protein coding region. The native doublet proteins could be preferentially extracted from homogenates of third stage larvae with lactose and could be affinity purified on an asialofetuin column, confirming the identity of these bands as galectins. Reverse transcriptase-polymerase chain reaction amplification using a primer based on the C. elegans Spliced Leader SL1 sequence showed that the corresponding T. circumcincta mRNAs are also trans-spliced at their 5' ends. While there are considerable nucleotide differences between the two clones, the majority are located in the non-coding regions. Within the coding region there are 87 nucleotide differences but only three of these result in amino acid substitutions.

Isolation and characterisation of sex-specific transcripts from Oesophagostomum dentatum by RNA arbitrarily-primed PCR.

In light of the lack of molecular data on the sexual differentiation, maturation and interaction of parasitic nematodes of livestock, the present study investigated sex-specific gene expression in the nodule worm, Oesophagostomum dentatum (Strongylida). Using the technique of RNA arbitrarily-primed polymerase chain reaction (RAP-PCR), 31 expressed sequence tags (ESTs) differentially-displayed between the sexes were cloned. Northern blot analysis proved ten ESTs to be expressed exclusively in males (adults and fourth-stage larvae), while two were expressed solely in female stages. None of the ESTs were expressed in infective third-stage larvae. Sequence analysis and subsequent database searches revealed two male-specific ESTs to have significant similarity to Caenorhabditis elegans (predicted) proteins, a protein containing an EGF-like cysteine motif and a serine/threonine phosphatase. Another two male-specific ESTs had similarity to non-nematode sequences. The two female-specific ESTs had similarity to vitellogenin-5 and endonuclease III (predicted) from C. elegans. The remaining ESTs had no similarity to any nucleic acid or protein sequences contained in the databases. The isolation and characterisation of sex-specific ESTs from O. dentatum provides a unique opportunity for studying the reproductive biology of parasitic nematodes at the molecular level, with a view toward novel approaches for parasite control.

Progress on vaccination against Haemonchus contortus.

Control of Haemonchus contortus at present is largely by the use of anthelmintics, assisted in some regions by management programs. Widespread development of resistance, particularly in South Africa and Australia, and concerns associated with the manufacture and use of chemicals have led to increasing interest in vaccination as an alternative means of control. Vaccination strategies basically fall into 2 categories, 'hidden' antigens (usually derived from the gastrointestinal tract of the adult parasite), or 'natural' antigens (those exposed to the immune system of the host during the course of infection, usually derived from the infective larval stage). Particularly promising results have been obtained using the hidden gut antigen H11, or H110D, and more recently with another hidden antigen, H-gal-GP. The use of a natural antigen vaccine, however, would provide advantages such as boosting of the immune response by field challenge. This article will review recent developments in both types of vaccines against H. contortus and consider the advantages and disadvantages of the 2 approaches.

Genomic and genetic research on bursate nematodes: significance, implications and prospects.

Molecular genetic research on parasitic nematodes (order Strongylida) is of major significance for many fundamental and applied areas of medical and veterinary parasitology. The advent of gene technology has led to some progress for this group of nematodes, particularly in studying parasite systematics, drug resistance and population genetics, and in the development of diagnostic assays and the characterisation of potential vaccine and drug targets. This paper gives an account of the molecular biology and genetics of strongylid nematodes, mainly of veterinary socio-economic importance, indicates the implications of such research and gives a perspective on genome research for this important parasite group, in light of recent technological advances and knowledge of the genomes of other metazoan organisms.

Protection against multiply drug-resistant and geographically distant strains of Haemonchus contortus by vaccination with H11, a gut membrane-derived protective antigen.

H11 is a "hidden antigen" derived from adult Haemonchus contortus which is capable of conferring a high degree of protection against subsequent challenge. To be commercially useful, any vaccine based on this antigen must protect against field strains and drug-resistant worms. The results show that vaccination with H11 affords high levels of protection against the multiply drug-resistant (MDR) Lawes strain of Haemonchus contortus. Moreover, there was no difference in protection whether the H11 was extracted from drug-resistant or susceptible worms, indicating that multiple drug-resistance does not significantly alter the immunogenicity of H11. Ideally, a commercial vaccine based on this antigen should also be capable of protecting against strains from geographically separate areas, so that it is not necessary to manufacture "region-specific" vaccines. Vaccination with H11 purified from either Australian or U.K. Haemonchus contortus was effective in protecting against subsequent challenge with Australian larvae, indicating that one type of H11 should have world-wide application.

Analysis of developmentally regulated genes of the parasite Haemonchus contortus.

Differential regulation of gene expression in the development of Haemonchus contortus was analysed using RNA arbitrarily-primed PCR. A study of third-stage larval and adult H. contortus revealed large differences between the two stages; 32 and 30% unique third-stage larval and adult RNA arbitrarily-primed PCR products, respectively. This finding is consistent with a high degree of differential gene expression between these developmental stages. A number of adult products were sequenced, revealing 11 molecules to be similar to deposits within sequence databases. Four other molecules that did not have significant similarity to sequences in the databases may represent developmentally regulated genes specific to H. contortus. Northern analysis of the putative adult-expressed molecules with homologues in the databases confirmed that four were expressed only in adults, while four were expressed in both stages, but had different sized transcripts. This may reflect differential splicing, or expression of closely related but different molecules at different life cycle stages. Two molecules were present in mRNA populations from both stages, suggesting these were false stage-associated molecules. No transcript was detected for one molecule by Northern analysis, probably due to low level of expression. In situ hybridisation analysis was used to localise expression of transcripts in the adult parasite, in particular, to gain some insight into the nature of those molecules with no known predicted function.

Identification and cDNA cloning of two novel low molecular weight host-protective antigens from Taenia ovis oncospheres.

Oncosphere antigens of Taenia ovis were solubilised in sodium dodecyl sulphate and separated by electrophoresis in polyacrylamide gels (SDS-PAGE). Antigen-containing gel fractions cut from the region covering 18-12 kDa were shown to be highly immunogenic in sheep challenge experiments. Specific antisera against 2 candidate antigens at 18 and 16 kDa were used to screen a cDNA library prepared from T. ovis oncosphere mRNA. Recombinant proteins selected with antibody to the 16 and 18 kDa native antigens were expressed as GST fusion proteins. Vaccination trials using either of the 2 fusion proteins To16.17-GST and To18-GST, revealed that each was capable of inducing high levels of immunity in sheep against challenge infection with T. ovis eggs. Antibodies induced by vaccination with the recombinant antigens reacted specifically with their respective 18 or 16 kDa native oncosphere antigens. There was no apparent homology between the T. ovis cDNA coding for To18 and To16.17, or with another host-protective antigen, To45W, described previously. These additional host-protective antigens should prove a valuable adjunct to To45W and permit the development of effective vaccination strategies.

Total ankle arthroplasty. Clinical study of fifty cases.

A prosthesis for the ankle joint that I designed in 1973 was implanted in fifty patients who otherwise would have required fusion of the ankle to relieve severe pain. The design of the device allows about as much motion as in the normal ankle. During implantation of the prosthesis the dome of the talus is not resected and only one centimeter of the distal end of the tibia is removed. If the prosthetic replacement is unsuccessful, fusion of the ankle can more easily be achieved secondarily if it should become necessary. Review of the cases of these fifty patients showed that the best results were obtained in the patients with degenerative arthritis whose ankles were stable and not unduly deformed. A few patients with rheumatoid arthritis (four of ten) who were not on long-term steroid therapy also did well. Contraindications revealed by my experience include avascular necrosis of the talus, pseudarthrosis at the site of a previously attempted ankle fusion, and conditions causing talar tilt of 20 degrees or more.

A single-chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gastrointestinal nematode parasite Haemonchus contortus.

Sheep immunoglobulin (Ig) heavy-chain (V(H)DJ(H)) and lambda light-chain variable region (V(lambda)J(lambda)) nucleotide coding sequence was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from abomasal lymph node (ALN) B cells of immune sheep challenged with the gastrointestinal nematode parasite Haemonchus contortus. Single-chain antibodies (scFv) were then constructed with the purified V(H)DJ(H) and V(lambda)J(lambda) Ig gene region DNA using oligonucleotides to PCR and join the variable regions to a central [Gly(4)Ser](3)-linker. In a similar fashion 5'-SfiI and 3'-NotI restriction endonuclease sites were added for cloning into a phagemid expression vector. Expression of sheep scFv from pHFA phagemid in an amber-suppresser strain of Escherichia coli, after infection with filamentous phage, resulted in 10(9) sheep scFv antibodies displayed as a library on phagemid particles. Western blot analysis demonstrated sheep scFv gene expression in E. coli cell lysate and on purified library phage. In addition, four rounds of scFv-library selection against H. contortus surface antigen resulted in a 300-fold increase in the elution titre of phage recovered from parasite surface antigen. Nearly 1000 of the selected and eluted scFvs were expressed in an attempt to identify monoclonal sheep scFv against parasite antigen. Only low affinity clones were isolated during screening of this sheep scFv-library, suggesting different strategies will be needed for isolation of specific high affinity recombinant antibody in future studies.

Recidivism and return to work posttransplant. Recipients with substance abuse histories.

The purpose of this retrospective survey was to describe posttransplant recidivism and return to work rates for adult liver transplant recipients with histories of alcohol-related liver disease (ARLD) or ARLD and polysubstance use. Recidivism from alcohol was the primary substance analyzed. One hundred and twenty-two adult liver transplant recipients comprised the convenience sample; 98 had histories of ARLD and 24 had both ARLD and polysubstance use. Using a mailed survey, the recipients were asked about their drug and alcohol use (both prior to and following the transplant); family and social issues; and whether they were working posttransplant. The two subgroups of recipients differed with respect to the variables gender, age, and years posttransplant with the ARLD/polysubstance using recipients more likely to be male, younger, and more recent transplant recipients than those with ARLD, alone. Recidivism and return to work rates differed for the two subgroups.

Nursing theory across curricula: a status report from Midwest nursing schools.

This article reports on an informal survey conducted for the Theory Development Section of the Midwest Nursing Research Society. Its purposes were to describe current practices in teaching nursing theory at three levels of curricula and to compare these practices between nursing programs in liberal arts colleges and research universities, between schools with and without doctoral programs, and between faculty with nursing and nonnursing doctorates who teach nursing theory. Findings indicate greatest consistency of practices at baccalaureate and doctoral levels, though few baccalaureate programs have required courses in nursing theory. Few differences were found between types of schools, but faculty with nursing doctorates tended to emphasize theory-practice linkages at the baccalaureate level. Further study that uses this work as a platform is suggested.

Sexual dysfunction in men on chronic hemodialysis: a rehabilitation nursing concern.

The rehabilitation potential of hemodialysis patients is hampered by the many complications associated with a uremic state and hemodialysis therapy. One complication that is particularly disturbing to male hemodialysis patients is sexual dysfunction, which affects about 50% of them and whose exact cause is unknown. This article reviews the literature regarding factors associated with sexual dysfunction in male patients on dialysis and the implications for rehabilitation nursing practice.

Jordanian married Muslim women's intentions to use oral contraceptives.

BACKGROUND: High fertility rates and moderate to low contraceptive use in the Arab Islamic countries, and Jordan in particular, are issues of international nursing importance. AIM: By using the Theory of Planned Behaviour (TPB) as the conceptual framework, the relationships between married Jordanian Muslim women's previous contraceptive use, type of experience, and intention to use oral contraceptives (OCs) were examined. The relationship between selected demographic variables and intention to use OCs was also examined. METHODS: The study was part of a large cross-sectional survey. The Intention to Use Oral Contraceptive Tool was used to elicit participants' intentions and beliefs regarding oral contraceptive use. SAMPLE: The study sample was composed of 245 Jordanian married Muslim women. RESULTS: Results revealed a significant relationship between previous oral contraceptive use and intention to use OCs. The relationships between type of experience, whether successful or unsuccessful, with previous OC use, and intention to use OCs were not significant. CONCLUSION: Nursing implications regarding the provision of family planning services that are sensitive to Jordanian Muslim women's religious preferences are discussed.

Renal transplant recipients' and their physicians' expectations regarding return to work posttransplant.

The purpose of the study was to explore the return to work (RTW) expectations of adult renal transplant recipients and their transplant physicians. The design was a prospective survey. Data was collected from 25 adult end stage renal disease (ESRD) patients who were admitted to a large Midwestern transplant center for renal transplantation and the surgeons who performed their transplant operations. Self-administered questionnaires were completed pretransplant by the recipients and surgeons, and at 6 weeks posttransplant by the recipients. Recipients who were employed pretransplant and were more likely to expect to RTW posttransplant, did so at higher rates than recipients who were not employed pretransplant and had physicians who did not expect them to RTW. The results illustrate that posttransplant RTW expectations of both renal transplant recipients and their physicians, when assessed pretransplant, are often congruent and are indicative of recipient posttransplant vocational rehabilitation.