Hannah Research Institute

Ayr, United Kingdom

Hannah Research Institute

Ayr, United Kingdom
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Lenton S.,Keele University | Lenton S.,Laue Langevin Institute | Lenton S.,University of Leeds | Grimaldo M.,Laue Langevin Institute | And 12 more authors.
Biophysical Journal | Year: 2017

The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals. © 2017 Biophysical Society


Clegg R.A.,Hannah Research Institute | Bowen L.C.,University of Liverpool | Bicknell A.V.,University of Liverpool | Tabish M.,University of Liverpool | And 4 more authors.
Archives of Biochemistry and Biophysics | Year: 2012

Multiple isoforms of the cyclic AMP-dependent protein kinase (PK-A) catalytic (C) subunit, arise as a consequence of the use of alternative splicing strategies during transcription of the kin-1 gene in the nematode, Caenorhabditis elegans. N-myristoylation is a common co-translational modification of mammalian PK-A C-subunits; however, the major isoform (N′3), originally characterised in C. elegans, is not N-myristoylated. Here, we show that N′1 isoforms are targets for N-myristoylation in C. elegans. We have demonstrated the in vivo incorporation of radioactivity into N′1 C-subunit isoforms, following incubation of nematodes with [ 3H]-myristic acid. HPLC and MALDI-TOF MS analysis of proteolytic digests of immunoprecipitates confirmed the presence of myristoyl-glycine in the C-subunit. In order to better understand the impact of the N′1 N-terminal sequence, and its myristoylation, on C-subunit activity, a chimerical C-subunit, consisting of the N′1 N-terminus from C. elegans and a murine core and C-terminal sequence was expressed. Myristoylation had no appreciable effect on the catalytic properties of the chimeric protein. However, the myristoylated chimeric protein did exhibit enhanced apolar targeting compared to the myristoylated wild-type murine polypeptide. This behaviour may reflect the inability of the N′1-encoded N-terminus sequence to correctly dock with a hydrophobic domain on the surface of the C-subunit. © 2012 Elsevier Inc. All rights reserved.


Huber R.C.,University of Porto | Kolb A.F.,Hannah Research Institute | Kolb A.F.,University of Aberdeen | Lillico S.,Roslin Institute | And 6 more authors.
Nutritional Neuroscience | Year: 2013

Objectives: Early malnutrition is a highly prevalent condition in developing countries. Different rodent models of postnatal early malnutrition have been used to approach the subject experimentally, inducing early malnutrition by maternal malnutrition, temporal maternal separation, manipulation of litter size or the surgical nipple ligation to impair lactation. Studies on the behaviour of (previously) malnourished animals using animal models have produced sometimes contradictory results regarding the effects of early postnatal malnutrition and have been criticized for introducing potential confounding factors. The present paper is a first report on the behavioural effects of early malnutrition induced by an alternative approach: mice nursed by α-casein-deficient knockout dams showed a severe growth delay during early development and substantial catch-up growth after weaning when compared with animals nursed by wild-type females. Methods: Established behavioural tests were used to study the consequences of early postnatal malnutrition on mouse pups at weaning and after partial weight recovery. Results: Despite the impaired growth, the only behavioural difference between malnourished and normally growing animals was found in exploratory behaviour during acute malnutrition at the time of weaning. After partial catch-up in weight early protein malnourished animals showed no indication of lasting effects on general activity, emotionality and exploration, memory, and pain reactivity. Discussion: These results suggest that the role of early nutrition on behavioural development after recovery in animal models may have been overestimated. Further careful examination of this animal model in terms of maternal care and offspring behaviour will be necessary to confirm if mice nursed by α-casein-deficient dams offer an alternative to existing models while eliminating potential confounding factors.


Sorrell D.A.,Hannah Research Institute | Robinson C.J.,Hannah Research Institute | Smith J.-A.,Hannah Research Institute | Kolb A.F.,Hannah Research Institute | Kolb A.F.,University of Aberdeen
Nucleic Acids Research | Year: 2010

Recombinase mediated cassette exchange (RMCE) is a process in which site-specific recombinases exchange one gene cassette flanked by a pair of incompatible target sites for another cassette flanked by an identical pair of sites. Typically one cassette is present in the host genome, whereas the other gene cassette is introduced into the host cell by chemical or biological means. We show here that the frequency of cassette exchange is dependent on the relative and absolute quantities of the transgene cassette and the recombinase. We were able to successfully modify genomic targets not only by electroporation or chemically mediated gene transfer but also by using an adenovirus vector carrying both the transgene cassette to be inserted and the recombinase coding region. RMCE proceeds efficiently in cells in which the adenovirus vector is able to replicate. In contrast, insufficient quantities of the transgene cassette are produced in cells in which the virus cannot replicate. Additional transfection of the transgene cassette significantly enhances the RMCE frequency. This demonstrates that an RMCE system in the context of a viral vector allows the site directed insertion of a transgene into a defined genomic site. © The Author(s) 2010. Published by Oxford University Press.


Kolb A.F.,Hannah Research Institute | Kolb A.F.,University of Aberdeen | Huber R.C.,University of Porto | Huber R.C.,Roslin Institute | And 10 more authors.
PLoS ONE | Year: 2011

The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate. We have analysed the role of the milk protein α-casein by inactivating the corresponding gene in mice. Absence of α-casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for α-casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of α-casein also significantly inhibits transcription of the other casein genes. α-Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for α-casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight. © 2011 Kolb et al.


Kolb A.F.,University of Aberdeen | Kolb A.F.,Hannah Research Institute | Sorrell D.,Hannah Research Institute | Sorrell D.,Horizon Discovery | And 7 more authors.
Transgenic Research | Year: 2013

Development of the mammary gland requires the coordinated action of proteolytic enzymes during two phases of remodelling. Firstly, new ducts and side-branches thereof need to be established during pregnancy to generate an extensive ductal tree allowing the secretion and transport of milk. A second wave of remodelling occurs during mammary involution after weaning. We have analysed the role of the cell surface protease aminopeptidase N (Anpep, APN, CD13) during these processes using Anpep deficient and Anpep over-expressing mice. We find that APN deficiency significantly delays mammary gland morphogenesis during gestation. The defect is characterised by a reduction in alveolar buds and duct branching at mid-pregnancy. Conversely over-expression of Anpep leads to accelerated ductal development. This indicates that Anpep plays a critical role in the proteolytic remodelling of mammary tissue during adult mammary development. © 2012 Springer Science+Business Media B.V.


Noble R.C.,Hannah Research Institute | Moore J.H.,Hannah Research Institute | Harfoot C.G.,Hannah Research Institute
British Journal of Nutrition | Year: 2011

1. Studies have been made of the effects of different concentrations of either free or esterified linoleic acid on the biohydrogenation of linoleic acid by rumen micro-organisms in vitro. A comparison has been made with the changes which occurred in the fatty acid compositions of rumen free fatty acids and plasma triglycerides of sheep given intraruminal infusions of linoleic acid or maize oil. 2. In the in vitro experiments, with increasing concentrations of 18:2 added as the free fatty acid, a decreasing proportion of this 18:2 was hydrogenated to 18:0 and trans-11-octadecenoic acid accumulated. The accumulation of large amounts of trans-11-octadecenoic acid was accompanied in all instances by the accumulation of a conjugated diene identified as cis-9, trans-11-octadecadienoic acid. There appeared to be a product–precursor relationship between the conjugated diene and the trans-11 monoene. 3. When linoleic acid was presented in vitro as the triglyceride, the extent to which hydrogenation occurred was, in all instances, greater than when equivalent amounts of 18:2 were presented as the free acid. Only small amounts of the cis-9, trans-11 diene were detected, and there was no apparent product–precursor relationship between this conjugated diene and the C18 monoenoic acids. The C18 monoenoic acids that accumulated consisted of both cis and trans isomers; the cis isomers consisted largely of cis-9- and cis-11-octadecenoic acids, which together comprised about 30% of the C18 monoenoic acids present. 4. The infusion of free linoleic acid into the rumen of sheep resulted in an increase in the proportion of total 18:1 and a decrease in the proportions of 16:0 and 18:0 in the total rumen free fatty acids. This increase which occurred in the concentration of 18:1 consisted predominantly of the trans-11 isomer. A concomitant increase in the concentration of the C18 trans-11 acid was observed to occur in the fatty acids of the plasma triglycerides. Infusion of maize oil into the rumen of sheep resulted in little change in the fatty acid compositions of either the free fatty acids in the rumen or the triglycerides of the plasma. 5. The findings in vitro and in vivo are discussed with reference to each other and with reference to the possibility that biohydrogenation of 18:2 derived from the triglyceride proceeds by a different pathway from that of 18:2 presented as the free acid. © 1974, The Nutrition Society. All rights reserved.


PubMed | Hannah Research Institute
Type: Journal Article | Journal: Nucleic acids research | Year: 2010

Recombinase mediated cassette exchange (RMCE) is a process in which site-specific recombinases exchange one gene cassette flanked by a pair of incompatible target sites for another cassette flanked by an identical pair of sites. Typically one cassette is present in the host genome, whereas the other gene cassette is introduced into the host cell by chemical or biological means. We show here that the frequency of cassette exchange is dependent on the relative and absolute quantities of the transgene cassette and the recombinase. We were able to successfully modify genomic targets not only by electroporation or chemically mediated gene transfer but also by using an adenovirus vector carrying both the transgene cassette to be inserted and the recombinase coding region. RMCE proceeds efficiently in cells in which the adenovirus vector is able to replicate. In contrast, insufficient quantities of the transgene cassette are produced in cells in which the virus cannot replicate. Additional transfection of the transgene cassette significantly enhances the RMCE frequency. This demonstrates that an RMCE system in the context of a viral vector allows the site directed insertion of a transgene into a defined genomic site.


PubMed | Hannah Research Institute
Type: Journal Article | Journal: PloS one | Year: 2011

The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate.We have analysed the role of the milk protein -casein by inactivating the corresponding gene in mice. Absence of -casein protein significantly curtails secretion of other milk proteins and calcium-phosphate, suggesting a role for -casein in the establishment of casein micelles. In contrast, secretion of albumin, which is not synthesized in the mammary epithelium, into milk is not reduced. The absence of -casein also significantly inhibits transcription of the other casein genes. -Casein deficiency severely delays pup growth during lactation and results in a life-long body size reduction compared to control animals, but has only transient effects on physical and behavioural development of the pups. The data support a critical role for -casein in casein micelle assembly. The results also confirm lactation as a critical window of metabolic programming and suggest milk protein concentration as a decisive factor in determining adult body weight.


PubMed | Hannah Research Institute
Type: Journal Article | Journal: In vitro cellular & developmental biology. Animal | Year: 2016

Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveoluslike structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominately in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.

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