Publications by authors named "Hamish Ward"

5 Publications

  • Page 1 of 1

Phenotypic population screen identifies a new mutation in bovine DGAT1 responsible for unsaturated milk fat.

Sci Rep 2015 Feb 26;5:8484. Epub 2015 Feb 26.

1] ViaLactia Biosciences (NZ) Ltd., Auckland, New Zealand [2] School of Biological Sciences, University of Auckland, Auckland, New Zealand.

Selective breeding has strongly reduced the genetic diversity in livestock species, and contemporary breeding practices exclude potentially beneficial rare genetic variation from the future gene pool. Here we test whether important traits arising by new mutations can be identified and rescued in highly selected populations. We screened milks from 2.5 million cows to identify an exceptional individual which produced milk with reduced saturated fat content, and improved unsaturated and omega-3 fatty acid concentrations. The milk traits were transmitted dominantly to her offspring, and genetic mapping and genome sequencing revealed a new mutation in a previously unknown splice enhancer of the DGAT1 gene. Homozygous carriers show features of human diarrheal disorders, and may be useful for the development of therapeutic strategies. Our study demonstrates that high-throughput phenotypic screening can uncover rich genetic diversity even in inbred populations, and introduces a novel strategy to develop novel milks with improved nutritional properties.
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http://dx.doi.org/10.1038/srep08484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4341421PMC
February 2015

Metabolic proteomics of the liver and mammary gland during lactation.

J Proteomics 2012 Jul 24;75(14):4429-35. Epub 2012 Apr 24.

Centre for Biodiscovery and School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by β-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and β-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than β-oxidation.
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http://dx.doi.org/10.1016/j.jprot.2012.04.019DOI Listing
July 2012

Profiling the metabolic proteome of bovine mammary tissue.

Proteomics 2008 Apr;8(7):1502-15

Centre for Biodiscovery and School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

2-DE and MALDI mass fingerprinting were used to analyse mammary tissue from lactating Friesian cows. The goal was detection of enzymes in metabolic pathways for synthesis of milk molecules including fatty acids and lactose. Of 418 protein spots analysed by PMF, 328 were matched to database sequences, resulting in 215 unique proteins. We detected 11 out of the 15 enzymes in the direct pathways for conversion of glucose to fatty acids, two of the pentose phosphate pathway enzymes and two of the enzymes for lactose synthesis from glucose. We did not detect enzymes that catalyse the first three reactions of glycolysis. Our results are typical of enzyme detection using 2-DE of mammalian tissues. We therefore advocate caution when relating enzyme abundances measured by 2-DE to metabolic output as not all relevant proteins are detected. 2-D DIGE was used to measure interindividual variation in enzyme abundance from eight animals. We extracted relative protein abundances from 2-D DIGE data and used a logratio transformation that is appropriate for compositional data of the kind represented in many proteomics experiments. Coefficients of variation for abundances of detected enzymes were 3-8%. We recommend use of this transformation for DIGE and other compositional data.
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http://dx.doi.org/10.1002/pmic.200700864DOI Listing
April 2008

The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering.

J Neurosci 2004 Jun;24(25):5816-26

Department of Pharmacology, The School of Pharmacy, London WC1N 1AX, United Kingdom.

Glycine receptors (GlyRs) and specific subtypes of GABA(A) receptors are clustered at synapses by the multidomain protein gephyrin, which in turn is translocated to the cell membrane by the GDP-GTP exchange factor collybistin. We report the characterization of several new variants of collybistin, which are created by alternative splicing of exons encoding an N-terminal src homology 3 (SH3) domain and three alternate C termini (CB1, CB2, and CB3). The presence of the SH3 domain negatively regulates the ability of collybistin to translocate gephyrin to submembrane microaggregates in transfected mammalian cells. Because the majority of native collybistin isoforms appear to harbor the SH3 domain, this suggests that collybistin activity may be regulated by protein-protein interactions at the SH3 domain. We localized the binding sites for collybistin and the GlyR beta subunit to the C-terminal MoeA homology domain of gephyrin and show that multimerization of this domain is required for collybistin-gephyrin and GlyR-gephyrin interactions. We also demonstrate that gephyrin clustering in recombinant systems and cultured neurons requires both collybistin-gephyrin interactions and an intact collybistin pleckstrin homology domain. The vital importance of collybistin for inhibitory synaptogenesis is underlined by the discovery of a mutation (G55A) in exon 2 of the human collybistin gene (ARHGEF9) in a patient with clinical symptoms of both hyperekplexia and epilepsy. The clinical manifestation of this collybistin missense mutation may result, at least in part, from mislocalization of gephyrin and a major GABA(A) receptor subtype.
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http://dx.doi.org/10.1523/JNEUROSCI.1184-04.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6729214PMC
June 2004

Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor, and mutation analysis in hyperekplexia.

J Biol Chem 2003 Jul 8;278(27):24688-96. Epub 2003 Apr 8.

Department of Molecular Medicine, Faculty of Medical and Health Sciences, University of Auckland, Private bag 92019, New Zealand.

Gephyrin (GPHN) is an organizational protein that clusters and localizes the inhibitory glycine (GlyR) and GABAA receptors to the microtubular matrix of the neuronal postsynaptic membrane. Mice deficient in gephyrin develop a hereditary molybdenum cofactor deficiency and a neurological phenotype that mimics startle disease (hyperekplexia). This neuromotor disorder is associated with mutations in the GlyR alpha1 and beta subunit genes (GLRA1 and GLRB). Further genetic heterogeneity is suspected, and we hypothesized that patients lacking mutations in GLRA1 and GLRB might have mutations in the gephyrin gene (GPHN). In addition, we adopted a yeast two-hybrid screen, using the GlyR beta subunit intracellular loop as bait, in an attempt to identify further GlyR-interacting proteins implicated in hyperekplexia. Gephyrin cDNAs were isolated, and subsequent RT-PCR analysis from human tissues demonstrated the presence of five alternatively spliced GPHN exons concentrated in the central linker region of the gene. This region generated 11 distinct GPHN transcript isoforms, with 10 being specific to neuronal tissue. Mutation analysis of GPHN exons in hyperekplexia patients revealed a missense mutation (A28T) in one patient causing an amino acid substitution (N10Y). Functional testing demonstrated that GPHNN10Y does not disrupt GlyR-gephyrin interactions or collybistininduced cell-surface clustering. We provide evidence that GlyR-gephyrin binding is dependent on the presence of an intact C-terminal MoeA homology domain. Therefore, the N10Y mutation and alternative splicing of GPHN transcripts do not affect interactions with GlyRs but may affect other interactions with the cytoskeleton or gephyrin accessory proteins.
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http://dx.doi.org/10.1074/jbc.M301070200DOI Listing
July 2003