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J G Hickford

Publications and source records attributed to J G Hickford.

At least 19 recordsLinked to original sources
PubMed2001

Rapid and accurate typing of Dichelobacter nodosus using PCR amplification and reverse dot-blot hybridisation.

Here we describe an approach to genotyping D. nodosus, based on variation in the fimbrial subunit gene (fimA), which uses polymerase chain reaction (PCR) amplification and hybridisation to immobilised oligonucleotides (PCR/oligotyping). The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments hybridising were detected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (SCIP). The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing [Vet. Microbiol. 71 (2000) 113], was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types of the bacterium were present in the footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus strains present in a footrot sample can be accurately identified in less than 2 days.

Amino Acid Sequence↗
PubMed2001

Dichelobacter nodosus serotype M fimbrial subunit gene: implications for serological classification.

Dichelobacter nodosus fimbrial subunit gene (fimA) from a serotype M strain (M-SPAHL) was investigated in this study. A primer set targeting the relatively conserved fimA regions and based on the published sequence from Nepalese serogroup M isolates (Nepalese M), failed to amplify the fimA of M-SPAHL. However, when the downstream primer was substituted with a primer that is specific for other serogroups of D. nodosus, the fimA was successfully amplified. Cloning followed by DNA sequencing, revealed that the M-SPAHL fimA was different to the Nepalese M fimA. The predicted amino acid sequence of the M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not Nepalese M. The consequences of serological relatedness and sequence dissimilarity are discussed.

Amino Acid Sequence↗
PubMed2001

A polymorphic marker for the human cathepsin B gene.

Human cathepsin B (CTSB) is a proteolytic enzyme implicated in tumor invasion and metastasis. We describe a PCR-based polymorphic marker for this gene comprising two amplimers differing in length by 19 consecutive nucleotides in intron 7, near the exon 8 splice acceptor site, identifying two gene alleles (A and B). Allele frequencies were 0.614 for A and 0.386 for the B allele, with an observed heterozygosity of 0.457 in a cohort of 70 non-related Australian blood donors. One additional nucleotide difference was also revealed through sequencing. The human CTSB gene is located on chromosome 8 and the alleles described here can potentially be used as markers in linkage and association studies of cancers and other diseases.

Alleles↗
PubMed2001

Differential expression of a gene homologous to a G-alpha protein gene in neonatal mouse skin during development of hair follicles.

The development of mouse hair follicles depends on the proliferation, differentiation and migration of epithelial matrix cells in the follicle bulb. In particular, induction of the proliferation of epithelial cells is thought to be signalled by the dermal papilla at the base of the bulb. Neonatal mouse skin is useful for studying changes in gene expression during development of the follicles, as the mitotic activity of skin cells changes shortly after birth. Using RNA differential display, a 248-bp message has been identified, which is expressed in the skin, specifically on day 2 and day 3 but not on day 4 after birth. Confirmation of expression of this gene by ribonuclease protection assay showed that strong expression is seen on day 2 and day 3, but weak expression is also shown on day 1, day 4 and day 5. In situ hybridization data revealed that it is mainly localized in the dermal papilla. Analysis of its nucleotide sequence showed 99% identity between nucleotide 2 and 232 of the mouse uncoupled S49 cell mRNA for stimulatory GTP-binding protein (G(S)) alpha subunit, suggesting it is a segment of G(S)alpha. As the G(S)alpha subunit is involved in transducing extracellular signals across the cell, the finding of its expression in the papilla suggests it may be a molecular signal to the induction of epithelial proliferation in the follicle bulb. Evidence of strong expression on day 2, at the time when the mitotic activity of epithelial matrix cells starts to increase, also suggests that the G(S)alpha is a potential candidate for involvement in the initiation of follicle growth.

Aging↗
PubMed2000

Novel fimbrial subunit genes of Dichelobacter nodosus: recombination in vivo or in vitro?

Polymerase chain reaction (PCR) was used to amplify the variable region of the fimbrial subunit encoding gene (fimA) of Dichelobacter nodosus from sheep and goats infected with footrot. Two amplimers (designated X and Y) generated single-strand conformation polymorphism (SSCP) patterns different to those of previously identified serogroups and serotypes. DNA sequencing revealed that these two fragments were novel. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1, whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype F1 and the upstream primer specific for serotype M or E1, generated PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artifacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It, therefore, seems more likely that fragments X and Y reflect genuine fimA genes of D. nodosus which have resulted from in vivo DNA recombination rather than from a PCR recombination artifact.

Animals↗
PubMed2000

Extensive diversity in New Zealand Dichelobacter nodosus strains from infected sheep and goats.

Footrot is a contagious bacterial disease of ruminants spread by the Gram-negative, anaerobic organism, Dichelobacter nodosus. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. Using the polymerase chain reaction (PCR) to amplify fragments of the fimbrial gene (fimA), D. nodosus was detected in 14 hoof scrapings, sampled from six farming regions within New Zealand. DNA sequencing revealed 15 strains covering eight serogroups on the New Zealand farms. The predominant serogroup was B which contained six strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Eleven out of the 15 D. nodosus strains had fimbriae sequences different to those previously reported and the presence of multiple strains on a single hoof was common (86% samples). Individual sheep from the same farm, or the same paddock, were often infected by a different range of strains, which suggests a host role in mediating footrot infection.

Amino Acid Sequence↗
PubMed2000

Evolution of the ovine MHC DQA region.

Southern hybridisation was used to define an apparent gene duplication event at the ovine DQA2 locus. Approximately 500 sheep from five different breeds were genotyped at their DQA1 and DQA2 loci. A subset of these were selected for further characterisation. Southern hybridisation of TaqI digested DNA revealed no DQA1 region in some sheep. It was also noted in these DQA1 null animals the DQA2 specific probe hybridised to two bands. An EcoRV-RFLP designed to distinguish copy number confirmed this duplication of the DQA2 region. The results showed that the duplication was exclusively associated with the DQA1 null haplotype and occurred only in alleles DQA2-F, -G, -I and -J. Comparison with bovine MHC genes revealed that they also contained a DQA1 null haplotype and that this haplotype was associated with a putative DQA3 gene. The potential for an ovine DQA3 locus is discussed.

Alleles↗
PubMed2000

Single nucleotide polymorphisms in an intron of the ovine calpastatin gene.

Calpastatin is the specific inhibitor of the ubiquitous calcium-dependent proteases mu-calpain and m-calpain. Enzyme assay data from sheep and cattle inversely correlates post-mortem muscle calpastatin levels with ultimate meat tenderness. Genetic markers of meat quality may therefore be found linked to the calpastatin gene (CAST). A three-allele system detected by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) has been observed in the ovine CAST. The three allele amplimers have been fully nucleotide sequenced and their differences in terms of single nucleotide polymorphism (SNPs) in the intron region of the amplimer are reported and compared to a consensus sequence of the orthologous region of the cattle CAST. A PCR-RFLP for more rapid CAST genotyping of all three ovine alleles was also developed.

Alleles↗
PubMed1999

Increased vibrissa growth in transgenic mice expressing insulin-like growth factor 1.

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 has been reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in hair follicles, transgenic mice were produced by pronuclear microinjection of a construct containing a mouse ultra-high sulfur keratin (UHS-KER) promoter linked to an ovine IGF-1 cDNA. This UHS-KER promoter has previously been shown to direct expression of a reporter gene to the hair follicles of transgenic mice. Four transgenic mouse lines were established as a result of microinjection of 435 embryos. Transgene expression was found in skin at day 8 and day 15 of age in three of the lines. Progeny tests were carried out by mating two of the transgenic expressing males to nontransgenic females. Mice from one line were all nonexpressors while four of the 12 mice from the other showed integration of the transgene and three expressed transgene IGF-1 mRNA in the skin. Vibrissa growth at 11-21 d of age was significantly greater in transgenic expressors than in their nontransgenic littermates. Specifically, the increase in vibrissa length for transgenics at days 11-16 (20.5%) is approximately 2-fold compared with days 16-21 (11.9%). These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable of stimulating vibrissa growth during the first neonatal hair cycle.

Animals↗
PubMed1999

Insulin-like growth factor 1 and hair growth.

Insulin-like growth factor 1 (IGF-1) has been identified as an important growth factor in many biological systems.[1] It shares considerable structural homology with insulin and exerts insulin-like effects on food intake and glucose metabolism. Recently it has been suggested to play a role in regulating cellular proliferation and migration during the development of hair follicles. [2,3] To exert its biological effects, the IGF-1 is required to activate cells by binding to specific cell-surface receptors. The type I IGF receptor (IGF-1R) is the only IGF receptor to have IGF-mediated signaling functions.[1] In circulation, this growth factor mediates endocrine action of growth hormone (GH) on somatic growth and is bound to specific binding proteins (BPs). The latter control IGF transport, efflux from vascular compartments and association with cell surface receptors.[4] In tissues, IGF-1 is produced by mesenchymal type cells and acts in a paracrine and autocrine fashion by binding to the IGF-1R. This binding activates the receptor tyrosine kinase (RTK) that triggers the downstream responses and finally stimulates cell division.[5] IGF-1 may therefore be able to stimulate the proliferation of hair follicle cells through cellular signaling pathways of its receptors. Local infusion of IGF-1 into sheep has been reported to be capable of stimulating protein synthesis in the skin.[6] It may also increase the production of wool keratin. Recently, transgenic mice overexpressing IGF-1 in the skin have been shown to have earlier hair follicle development than controls.[7] In addition, this growth factor plays an important role in many cell types as a survival factor to prevent cell death.[8] This anti-apoptotic function of IGF-1 may be important to the development of follicle cells as follicles undergo a growth cycle where the regressive, catagen phase is apoptosis driven. In this review, the effects of IGF-1 on follicle cell proliferation and differentiation are discussed. In particular, the paracrine versus endocrine action of IGF-1 on hair growth and the targeting of expression of the growth factor to the follicles of transgenic animals will be emphasized. The anti-apoptotic role of IGF-1 in hair follicles is also reviewed. Prospects for future studies on hair and fiber growth by IGF-1 are discussed.

Androgens↗
PubMed1996

Polymorphism at the ovine major histocompatibility complex class II loci.

Southern hybridization analysis of the ovine major histocompatibility complex (MHC) (MhcOvar) class II region, using sheep-specific probes for the DQA1, DQA2, DQB and DRA loci, has revealed extensive polymorphism. DQA1 and DQA2 had eight and 16 alleles respectively, DQB had six and DRA had three alleles. Little information was derived from the DRB locus owing to extensive cross-hybridization between the DRB probe and the DQB locus. Differences in allele frequency between breeds were revealed. At the DQA1 locus a null allele (DQA1-N) was observed with a frequency of between 27% and 45%, making this the most common DQA1 allele in all breeds examined. The frequency of DQA1-N homozygotes was between 11% and 18%, raising questions as to the functional significance of the DQA1 gene. Linkage analysis between the DQA1, DQA2, DQB and DRA loci did not reveal any recombination.

Alleles↗
PubMed1994

Polymorphism in two genes for B2 high sulfur proteins of wool.

Variation in the nucleotide sequence of the B2 high-sulfur protein genes has not been reported previously. This paper reports 15 nucleotide substitutions in each of the genes for the B2A and B2C proteins and a length of polymorphism in the B2A gene which translates to the insertion/deletion of one 30-nucleotide repeat sequence. Evidence is presented for gene conversion occurring within the B2 high-sulfur multigene family. These DNA polymorphisms may account for some of the microheterogeneity observed in the B2 high-sulfur proteins and may also be useful genetic markers of the B2 high-sulfur protein gene loci for future use in analysing wool fibre characteristics.

Alleles↗