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Morgan A.J.,University of Sydney | Riley L.G.,University of Sydney | Riley L.G.,Genetic Metabolic Disorders Research Unit | Sheehy P.A.,University of Sydney | And 2 more authors.
Journal of Dairy Research | Year: 2014

Colostrum consists of a number of biologically active proteins and peptides that influence physiological function and development of a neonate. The present study investigated the biological activity of peptides released from first day bovine colostrum through in vitro and in vivo enzymatic digestion. This was assessed for proliferative activity using a human intestinal epithelial cell line, T84. Digestion of the protein fraction of bovine colostrum in vitro was conducted with the enzymes pepsin, chymosin and trypsin. Pepsin and chymosin digests yielded protein fractions with proliferative activity similar to that observed with undigested colostrum and the positive control foetal calf serum (FCS). In contrast trypsin digestion significantly (P<0·05) decreased colostral proliferative activity when co-cultured with cells when compared with undigested colostrum. The proliferative activity of undigested colostrum protein and abomasal whey protein digesta significantly increased (P<0·05) epithelial cell proliferation in comparison to a synthetic peptide mix. Bovine colostrum protein digested in vivo was collected from different regions of the gastrointestinal tract (GIT) in newborn calves fed either once (n=3 calves) or three times at 12-h intervals (n=3 calves). Digesta collected from the distal duodenum, jejunum and colon of calves fed once, significantly (P<0·05) stimulated cell proliferation in comparison with comparable samples collected from calves fed multiple times. These peptide enriched fractions are likely to yield candidate peptides with potential application for gastrointestinal repair in mammalian species. © 2014 Proprietors of Journal of Dairy Research. Source


Tucker E.J.,Murdoch Childrens Research Institute | Tucker E.J.,University of Melbourne | Wanschers B.F.J.,Radboud University Nijmegen | Szklarczyk R.,Radboud University Nijmegen | And 23 more authors.
PLoS Genetics | Year: 2013

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis. © 2013 Tucker et al. Source


Lim S.C.,Murdoch Childrens Research Institute | Lim S.C.,University of Melbourne | Lim S.C.,Center for Cancer Biology | Smith K.R.,Walter and Eliza Hall Institute of Medical Research | And 27 more authors.
American Journal of Human Genetics | Year: 2014

Leigh syndrome (LS) is a severe neurodegenerative disorder with characteristic bilateral lesions, typically in the brainstem and basal ganglia. It usually presents in infancy and is genetically heterogeneous, but most individuals with mitochondrial complex IV (or cytochrome c oxidase) deficiency have mutations in the biogenesis factor SURF1. We studied eight complex IV-deficient LS individuals from six families of Lebanese origin. They differed from individuals with SURF1 mutations in having seizures as a prominent feature. Complementation analysis suggested they had mutation(s) in the same gene but targeted massively parallel sequencing (MPS) of 1,034 genes encoding known mitochondrial proteins failed to identify a likely candidate. Linkage and haplotype analyses mapped the location of the gene to chromosome 19 and targeted MPS of the linkage region identified a homozygous c.3G>C (p.Met1?) mutation in C19orf79. Abolishing the initiation codon could potentially still allow initiation at a downstream methionine residue but we showed that this would not result in a functional protein. We confirmed that mutation of this gene was causative by lentiviral-mediated phenotypic correction. C19orf79 was recently renamed PET100 and predicted to encode a complex IV biogenesis factor. We showed that it is located in the mitochondrial inner membrane and forms a ∼300 kDa subcomplex with complex IV subunits. Previous proteomic analyses of mitochondria had overlooked PET100 because its small size was below the cutoff for annotating bona fide proteins. The mutation was estimated to have arisen at least 520 years ago, explaining how the families could have different religions and different geographic origins within Lebanon. © 2014 The American Society of Human Genetics. Source


Menezes M.J.,Genetic Metabolic Disorders Research Unit | Menezes M.J.,University of Sydney | Guo Y.,Childrens Hospital of Philadelphia | Zhang J.,Fudan University | And 23 more authors.
Human Molecular Genetics | Year: 2015

Functional defects of the mitochondrial translation machinery, as a result of mutations in nuclear-encoded genes, have been associated with combined oxidative phosphorylation (OXPHOS) deficiencies. We report siblings with congenital sensorineural deafness and lactic acidemia in association with combined respiratory chain (RC) deficiencies of complexes I, III and IV observed in fibroblasts and liver. One of the siblings had a more severe phenotype showing progressive hepatic and renal failure. Wholeexome sequencing revealed a homozygous mutation in the gene encoding mitochondrial ribosomal protein S7 (MRPS7), a c.550A>G transition that encodes a substitution of valine for a highly conserved methionine (p.Met184Val) in both affected siblings. MRPS7 is a 12S ribosomal RNA-binding subunit of the small mitochondrial ribosomal subunit, and is required for the assembly of the small ribosomal subunit. Pulse labeling of mitochondrial protein synthesis products revealed impaired mitochondrial protein synthesis in patient fibroblasts. Exogenous expression of wild-type MRPS7 in patient fibroblasts rescued complexes I and IV activities, demonstrating the deleterious effect of the mutation on RC function. Moreover, reduced 12S rRNA transcript levels observed in the patient's fibroblasts were also restored to normal levels by exogenous expression of wild-type MRPS7. Our data demonstrate the pathogenicity of the identified MRPS7 mutation as a novel cause of mitochondrial RC dysfunction, congenital sensorineural deafness and progressive hepatic and renal failure. © The Author 2015. Source


Ho G.,Genetic Metabolic Disorders Research Unit | Ho G.,University of Sydney | Yonezawa A.,Kyoto University | Masuda S.,Kyoto University | And 11 more authors.
Human Mutation | Year: 2011

Riboflavin, or vitamin B2, is a precursor to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) molecules, required in biological oxidation-reduction reactions. We previously reported a case of a newborn female who had clinical and biochemical features of multiple acyl-CoA dehydrogenation deficiency (MADD), which was corrected by riboflavin supplementation. The mother was then found to be persistently riboflavin deficient, suggesting that a possible genetic defect in riboflavin transport in the mother was the cause of the transient MADD seen in the infant. Two recently-identified riboflavin transporters G protein-coupled receptor 172B (GPR172B or RFT1) and riboflavin transporter 2 (C20orf54 or RFT2) were screened for mutations. Two missense sequence variations, c.209A>G [p.Q70R] and c.886G>A [p.V296M] were found in GPR172B. In vitro functional studies of both missense variations showed that riboflavin transport was unaffected by these variations. Quantitative real-time PCR revealed a de novo deletion in GPR172B spanning exons 2 and 3 in one allele from the mother. We postulate that haploinsufficiency of this riboflavin transporter causes mild riboflavin deficiency, and when coupled with nutritional riboflavin deficiency in pregnancy, resulted in the transient riboflavin-responsive disease seen in her newborn infant. This is the first report of a genetic defect in riboflavin transport in humans. © 2010 Wiley-Liss, Inc. Source

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