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Biomedical subjects

C M Hardy

Publications and source records attributed to C M Hardy.

At least 19 recordsLinked to original sources
PubMed2006

A virus vector based on Canine Herpesvirus for vaccine applications in canids.

Canine Herpesvirus (CHV) is being developed as a virus vector for the vaccination of European red foxes. However, initial studies using recombinant CHV vaccines in foxes revealed viral attenuation and lack of antibody response to inserted foreign antigens. These findings were attributed both to inactivation of the thymidine kinase (TK) gene and excess foreign genetic material in the recombinant viral genome. In this study, we report an improved CHV-bacterial artificial chromosome (BAC) vector system designed to overcome attenuation in foxes. A non-essential region was identified in the CHV genome as an alternative insertion site for foreign genes. Replacement of a guanine/cytosine (GC)-rich intergenic region between UL21 and UL22 of CHV with a marker gene did not change growth behaviour in vitro, showing that this region is not essential for virus growth in cell culture. We subsequently produced a CHV-BAC vector with an intact TK gene in which the bacterial genes and the antigen expression cassette were inserted into this GC-rich locus. Unlike earlier constructs, the new CHV-BAC allowed self-excision of the bacterial genes via homologous recombination after transfection of BACs into cell culture. The BAC-CHV system was used to produce a recombinant virus that constitutively expressed porcine zona pellucida subunit C protein between the UL21 and UL22 genes of CHV. Complete self-excision of the bacterial genes from CHV was achieved within one round of replication whilst retaining antigen gene expression.

Animals↗
PubMed2006

Biological control of vertebrate pests using virally vectored immunocontraception.

Species-specific viruses are being genetically engineered to produce contraceptive biological controls for pest animals such as mice, rabbits and foxes. The virus vaccines are intended to trigger an autoimmune response in the target animals that interferes with their fertility in a process termed virally vectored immunocontraception. Laboratory experiments have shown that high levels of infertility can be induced in mice infected with recombinant murine cytomegalovirus and ectromelia virus expressing reproductive antigens as well as in rabbits using myxoma virus vectors. The strategies used to produce and deliver species-specific immunocontraceptive vaccines to free-living wildlife are presented in this review. Discussion includes coverage of the likely safety of the proposed vaccines as well as the implications of the approach for fertility control in other species.

Animals↗
PubMed2005

Dispersal and phylogeography of the agamid lizard Amphibolurus nobbi in fragmented and continuous habitat.

Approximately 90% of native vegetation has been cleared for agriculture in central New South Wales, Australia. Habitat loss has reduced and fragmented populations of the agamid lizard Amphibolurus nobbi. We compared genetic structure of populations of this species in an unmodified landscape with those from small nature reserves and linear remnants in farming areas. We ask: Is there evidence for reduced dispersal and population fragmentation among farm populations? Using 2008 bp mtDNA sequences and allozyme electrophoresis, we found that small populations in farming areas had as much genetic variation as populations in nature reserves. Application of nested clade analysis (NCA) indicated isolation-by-distance effects among populations from uncleared areas, but not among populations within farming locations. The genetic evidence therefore implied a high level of migration in the cleared landscapes. High dispersal after fragmentation may have resulted from either a burst of movement at the time of land clearing with dragons from many sources finding refuge in a few remnants, or from ongoing rapid dispersal through unsuitable habitat. A phylogeny based on mtDNA revealed that A. nobbi populations in the study area are deeply divided into two reciprocally monophyletic groups. Although we did not sample the entire species range, one of these evolutionarily significant units was only detected in remnant vegetation in the agricultural landscape. Therefore, a substantial subclade of this species may be vulnerable to extinction. Our findings emphasize that local populations of widespread species can harbour important intraspecific genetic diversity, supporting the case for maintaining widespread species throughout production landscapes.

Animals↗
PubMed2003

Contraceptive responses of mice immunized with purified recombinant mouse zona pellucida subunit 3 (mZP3) proteins.

Mouse zona pellucida subunit 3 (mZP3) was tested for efficacy as an immunocontraceptive antigen by comparing the fertility of mice immunized with recombinant mZP3 proteins. Recombinant protein was expressed using either the vaccinia virus T7 mammalian (vmZP3 protein) or baculovirus insect cell (bmZP3 protein)-expression systems. Female BALB/c or wild mice were immunized by i.p. injection using Freund's complete adjuvant and boosted three times with affinity purified recombinant proteins in Freund's incomplete adjuvant. Most mice developed antibodies that crossreacted to the respective mZP3 antigens by ELISA or western blot. In BALB/c mice immunized with vmZP3, fertility and mean litter size were reduced transiently to 25% and 10%, respectively, of those of control mice. However, immunization with bmZP3 did not affect either the fertility or mean litter sizes in BALB/c or wild mice immunized with bmZP3. The results demonstrate that reduction in fertility can be achieved in female BALB/c mice immunized using Freund's adjuvants and recombinant mZP3 protein produced in a mammalian, but not an insect, cell-expression system. Arguments are presented for the likely role of glycosylation of the mZP3 antigen in inducing contraceptive immune responses.

Animals↗
PubMed2002

Assessment of the immunocontraceptive effect of a zona pellucida 3 peptide antigen in wild mice.

Immunizing laboratory mice against a short peptide to mouse zona pellucida protein 3 (mZP3; amino acids 328-342) reduces fertility in some strains. This antigen was therefore tested to see if it is suitable for use in an immunocontraceptive vaccine to control wild mice. Mouse zona pellucida protein 3 peptide conjugated to a carrier protein (keyhole limpet hemocyanin) was considerably more immunogenic and effective in reducing fertility in wild mice when compared with inbred BALB/c mice. Fertility of the immunized wild mice was reduced by over 50% compared with controls, whereas BALB/c mice showed no reduction. Variation in the responses between individual animals to mZP3 peptide was observed and infertility correlated to the presence of cross-reacting antibodies to native zona pellucida in wild, but not BALB/c, mice.

Animals↗
PubMed2002

Mouse-specific immunocontraceptive polyepitope vaccines.

Two mouse-specific polyepitope protein antigens comprising different combinations of sequences chosen from the mouse fertility antigens zona pellucida proteins 1 and 3 (ZP1 and ZP3), prolactin, proliferin, granulocyte-macrophage colony-stimulating factor (GM-CSF), sperm protein SP56 and T-helper cell-stimulating epitopes were produced in bacterial protein expression systems. The recombinant proteins were fused to maltose binding protein (MBP) and used to immunize female mice; their effects on fertility were assessed. Controls were immunized with either MBP only or phosphate buffered saline (PBS). One antigen construct (MBP-polyepitope B), containing mouse-specific epitopes for ZP1, ZP3, SP56 and proliferin, significantly reduced the fertility of female BALB/c mice. Fertility in this group was decreased by > 40% compared with the MBP control and the number of viable embryos was decreased by > 60%. This construct will now be used to produce the antigen in a recombinant murine cytomegalovirus for assessment as a potential mouse-specific anti-fertility vaccine for use in the control of mice in the field.

Amino Acid Sequence↗
PubMed2000

The complete cDNA sequences of IL-2, IL-4, IL-6 AND IL-10 from the European rabbit (Oryctolagus cuniculus).

The cDNAs for four rabbit cytokine genes [interleukin 2 (IL-2), IL-4, IL-6 and IL-10] have been cloned from primary lymphocytes by polymerase chain reaction (PCR) methods. IL-2 and IL-10 are both highly conserved between rabbit and other species. IL-4 and IL-6 are less strongly conserved, at both nucleotide and amino acid levels, and exhibit structural differences. An extension of the coding region of rabbit IL-6 relative to all other reported IL-6 genes results from a mutation in the usual stop codon which allows translation to continue for a further 27 amino acids. Analysis of IL-6 from four other lagomorph species suggests that this mutation is specific to the European rabbit. Sequence and structural differences of IL-4 and IL-6, while presumably not altering function, may render them highly species-specific. Several alternatively spliced variants of IL-2 and IL-4 are also reported.

Amino Acid Sequence↗
PubMed1999

Expression of recombinant mouse sperm protein sp56 and assessment of its potential for use as an antigen in an immunocontraceptive vaccine.

Recombinant mouse sp56 protein was produced for testing as an antigen in an immunocontraceptive vaccine. The coding sequence for the mature sp56 protein was cloned into the bacterial expression system pFLAG using a PCR-based method on mouse testis cDNA. Polyclonal antisera were raised in mice against affinity purified recombinant sp56 fusion protein (sp56FLAG) or an artificial sp56 peptide fused to a carrier protein (KLH) and shown to cross-react to a protein band of 75 kD in detergent extracts of mouse sperm by Western immunoblot analysis under reducing conditions. The antisera to sp56FLAG also immunolocalized over the entire acrosome of mouse sperm. Female BALB/c mice were immunized intraperitoneally with sp56FLAG in a fertility trial with 20 microg sp56FLAG in Freund's Complete Adjuvant and boosted three to five times with 20 microg sp56FLAG in Freund's Incomplete Adjuvant. Litter sizes of sp56FLAG-treated mice were significantly smaller than control-treated animals after five boosts.

Amino Acid Sequence↗
PubMed1998

Characterisation of two highly conserved but non-allelic cellular disintegrins from rabbit testis.

Two novel cellular disintegrin genes (termed ADAM 6d and ADAM 6e: Gen Bank accession numbers U82750 and U82751) were isolated and characterised from a rabbit testicular cDNA library. The cDNAs have open reading frames encoding for proteins of 731 and 730 amino acids, respectively. They share an amino-acid homology of greater than 89% and a nucleotide base matching of 94% in both the coding and non-coding regions. PCR of DNA extracted from both the parents and progeny of wild rabbits was used to demonstrate that the genes are non-allelic. Recombinant ADAM 6e fused to maltose binding protein was prepared and polyclonal antibodies produced in mice. These polyclonal antibodies recognised two bands with molecular masses of 42 kDa and 46 kDa on Western blots of rabbit sperm extracts run on SDS PAGE reducing gels. The implications of the presence of these two highly conserved proteins in rabbit testis and on sperm are discussed.

ADAM Proteins↗
PubMed1997

Examination of the immunocontraceptive potential of recombinant rabbit fertilin subunits in rabbit.

Recombinant fertilin subunits produced in a bacterial expression systems were used to test fertilin as an immunocontraceptive antigen in the European rabbit (Oryctolagus cuniculus). Wild female rabbits (n = 40) were immunized with either recombinant rabbit fertilin alpha or beta subunits by the s.c. or intra-Peyer's patch route. High titers of serum anti-fertilin polyclonal IgG antibodies were achieved in all rabbits after repeated boosts, with fertilin-specific IgG but not IgA antibodies detected in vaginal lavages of all animals. The serum IgG antibodies recognized polypeptides in detergent extracts of rabbit sperm with relative molecular masses of 48, 53, and 85 kDa on reducing SDS-PAGE gels and were shown to bind to the head region of methanol-fixed and live caudal rabbit sperm. Preincubation of rabbit sperm with these anti-fertilin IgG antibodies at concentrations of 400 micrograms/ml blocked sperm binding to zona-intact oocytes and inhibited fertilization in vitro by 60-80%. However, despite the levels of circulating and vaginal IgG antibodies achieved, only 4 immunized does failed to become pregnant out of 33 that ovulated. The remaining animals either showed no effect on fertility (n = 29) relative to control animals or failed to ovulate (n = 7). All control animals ovulated and were either fully fertile (n = 15) or were mated to infertile males (n = 4). In addition, proven-fertile male domestic rabbits (n = 3) were immunized s.c. and boosted three times with fertilin beta. Only one animal subsequently showed impaired fertility. These results show that in the rabbit, high levels of circulating sperm-reactive anti-fertilin antibodies and the presence of vaginal IgG does not ensure infertility.

ADAM Proteins↗
PubMed1996

Cloning and expression of recombinant rabbit fertilin.

Fertilin is a sperm surface protein complex which is reported to play an essential role in sperm-egg fusion in mammals. It is comprised of two related subunits, alpha and beta, both of which are glycosylated and have cytoplasmic and extracellular domains. This protein has been reported to play an essential role in sperm-egg fusion in mammals. We report on the cloning and sequencing of the complete cDNA sequences of both subunits from rabbit testis, and the production of recombinant proteins for testing their potential as antigens for use in an immunocontraceptive vaccine to control wild rabbit populations. The cDNAs for rabbit fertilin alpha and beta (Genbank accession numbers, U46069 and U46070) are predicted to encode proteins of 919 and 751 amino acids, respectively, and to show significant levels of homology to fertilin subunits isolated from other species. Analysis of the predicted protein sequences of fertilin alpha but not beta reveals the presence of 21 direct repeats of the hexameric sequence A/PPPPEA at the extreme carboxy terminus, similar to what has been described for a fertilin alpha gene isoform in the monkey. DNA sequences corresponding to the predicted mature alpha and beta fertilin subunits were individually cloned into a bacterial expression system, and the recombinant proteins were used to raise polyclonal antibodies in mice. These antibodies detect components of the native fertilin complex from rabbit sperm.

ADAM Proteins↗
PubMed1991

Nucleotide sequence of the COX1 gene in Kluyveromyces lactis mitochondrial DNA: evidence for recent horizontal transfer of a group II intron.

The cytochrome oxidase subunit 1 gene (COX1) in K. lactis K8 mtDNA spans 8,826 bp and contains five exons (termed E1-E5) totalling 1,602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S. cerevisiae. The four introns (termed K1 cox1.1-1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes. The first intron belongs to group II whereas the remaining three are group I type B. Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5 a, and 1.5 b respectively from S. cerevisiae. Horizontal transfer of an intron between recent progenitors of K. lactis and S. cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching. Sequence comparisons between K1 cox1.3/Sc cox1.5 a and K1 cox1.4/Sc cox1.5 b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts. Intron K1 cox1.2 is not found in S. cerevisiae and appears at an unique location in K. lactis. A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions. Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S. cerevisiae mtDNA.

Amino Acid Sequence↗
PubMed1991

A mobile group II intron of a naturally occurring rearranged mitochondrial genome in Kluyveromyces lactis.

Mitochondrial intron content is variable in the yeast Kluyveromyces lactis. Strains can be divided into three classes depending on the structure of the cytochrome oxidase subunit 1 (COX1) gene: (1) those containing intron K1 cox1.1, (2) those containing K1 cox1.2, 3 and 4 and, (3) those that contain all four introns. In addition, strains belonging to the first class (designated Type B strains), have an altered mitochondrial gene order relative to strains from classes (2) and (3) (Type A, Hardy et al. 1989). Crossing experiments reveal that K1 cox1.1 (a group II intron) transfers at high frequency (89%) to mitochondrial genomes lacking this intron. By contrast, the mobility of the remaining introns (all group I) is of the order of 7%.

Chromosome Mapping↗
PubMed1989

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA.

Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble rho 0 mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit-) or nuclear lesions (pet-) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (rho-) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G + C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A + T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.

Base Sequence↗
PubMed1989

Cocoa feeding and human lactose intolerance.

On the basis of evidence of the suppressing effect of cocoa on human lactose intolerance, a feeding study was conducted on 35 subjects with a commercial chocolate-milk formula. Variables studied were breath hydrogen level (BHL), symptoms, and onset time. Data from repeated feeding were analyzed by paired t tests. The addition of cocoa significantly reduced BHL (p less than 0.005) as well as the symptom score of both bloating (p less than 0.05) and cramping (p less than 0.025). Individual lactose intolerance levels ranged from 5% (basal milk) to 12.5% with an average of 7.8% upon consumption of 250 mL milk. Having both plain and cocoa formulas contain sucrose and carrageenan led to a conclusion that the suppressive effect of cocoa observed was independent of the presence of sucrose and carrageenan. Ninety-five percent of the subjects responded positively (BHL greater than 17 ppm) to the plain and 51% to the cocoa formula.

Animals↗