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Biomedical subjects

C Mullins

Publications and source records attributed to C Mullins.

At least 19 recordsLinked to original sources
PubMed2005

Tryptophan hydroxylase 2 (TPH2) gene variants associated with ADHD.

Genetic and pharmacological studies have emphasised the role of serotonin 5-hydroxytryptamine (5-HT) as a possible etiologic factor in the development of attention-deficit hyperactivity disorder (ADHD). Tryptophan hydroxylase (TPH) is a rate-limiting enzyme in the biosynthesis of serotonin from tryptophan. Originally, the TPH gene was thought to be widely expressed, but a second form of TPH, TPH2, was recently identified and the TPH2 gene was found to be solely expressed in the brain. We examined eight single nucleotide polymorphisms (SNP) in the TPH2 gene for association with ADHD in 179 Irish nuclear families. Transmission disequilibrium test analysis revealed significant association between the T allele of marker rs1843809 with the disorder (chi2=12.2, P=0.0006, OR=2.36). Stratifying data by the sex of the transmitting parent showed that this association was enhanced when paternal transmission was considered (OR=3.7). In addition, several haplotypes (all including the associated marker) were associated with ADHD. These preliminary findings suggest that TPH2 is a susceptibility locus for ADHD. Further confirmation, preferably from different ethnic groups, is required to firmly implicate TPH2 in the pathophysiology of ADHD.

Attention Deficit Disorder with Hyperactivity↗
PubMed2001

The molecular machinery for lysosome biogenesis.

The lysosome serves as a site for delivery of materials targeted for removal from the eukaryotic cell. The mechanisms underlying the biogenesis of this organelle are currently the subject of renewed interest due to advances in our understanding of the protein sorting machinery. Genetic model systems such as yeast and Drosophila have been instrumental in identifying both protein and lipid components of this machinery. Importantly, many of these components, as well as the processes in which they are involved, are proving conserved in mammals. Other recently identified components, however, appear to be unique to higher eukaryotes. BioEssays 23:333-343, 2001. Published 2001 John Wiley & Sons, Inc.

ADP-Ribosylation Factors↗
PubMed2001

Structural requirements for function of yeast GGAs in vacuolar protein sorting, alpha-factor maturation, and interactions with clathrin.

The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells. Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p. We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of alpha-factor. A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important. We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro. While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements. Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and alpha-factor maturation and identify structural determinants that are critical for these functions.

ADP-Ribosylation Factors↗
PubMed2000

GGAs: a family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi complex.

Formation of intracellular transport intermediates and selection of cargo molecules are mediated by protein coats associated with the cytosolic face of membranes. Here, we describe a novel family of ubiquitous coat proteins termed GGAs, which includes three members in humans and two in yeast. GGAs have a modular structure consisting of a VHS domain, a region of homology termed GAT, a linker segment, and a region with homology to the ear domain of gamma-adaptins. Immunofluorescence microscopy showed colocalization of GGAs with Golgi markers, whereas immunoelectron microscopy of GGA3 revealed its presence on coated vesicles and buds in the area of the TGN. Treatment with brefeldin A or overexpression of dominant-negative ADP ribosylation factor 1 (ARF1) caused dissociation of GGAs from membranes. The GAT region of GGA3 was found to: target a reporter protein to the Golgi complex; induce dissociation from membranes of ARF-regulated coats such as AP-1, AP-3, AP-4, and COPI upon overexpression; and interact with activated ARF1. Disruption of both GGA genes in yeast resulted in impaired trafficking of carboxypeptidase Y to the vacuole. These observations suggest that GGAs are components of ARF-regulated coats that mediate protein trafficking at the TGN.

ADP-Ribosylation Factor 1↗
PubMed2000

Distinct requirements for the AP-3 adaptor complex in pigment granule and synaptic vesicle biogenesis in Drosophila melanogaster.

The AP-3 adaptor protein complex has been implicated in the biogenesis of lysosome-related organelles, such as pigment granules/melanosomes, and synaptic vesicles. Here we compare the relative importance of AP-3 in the biogenesis of these organelles in Drosophila melanogaster. We report that the Drosophila pigmentation mutants orange and ruby carry genetic lesions in the sigma3 and beta3-adaptin subunits of the AP-3 complex, respectively. Electron microscopy reveals dramatic reductions in the numbers of electron-dense pigment granules in the eyes of these AP-3 mutants. Mutant flies also display greatly reduced levels of pigments housed in these granules. In contrast, electron microscopy of retinula cells reveals numerous synaptic vesicles in both AP-3 mutant and wild-type flies, while behavioral assays show apparently normal locomotor ability of AP-3 mutant larvae. Together, these results demonstrate that Drosophila AP-3 is critical for the biogenesis of pigment granules, but is apparently not essential for formation of a major population of synaptic vesicles in vivo.

Adaptor Protein Complex alpha Subunits↗
PubMed2000

Lysosome-related organelles.

Lysosomes are membrane-bound cytoplasmic organelles involved in intracellular protein degradation. They contain an assortment of soluble acid-dependent hydrolases and a set of highly glycosylated integral membrane proteins. Most of the properties of lysosomes are shared with a group of cell type-specific compartments referred to as 'lysosome-related organelles', which include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and Drosophila pigment granules. In addition to lysosomal proteins, these organelles contain cell type-specific components that are responsible for their specialized functions. Abnormalities in both lysosomes and lysosome-related organelles have been observed in human genetic diseases such as the Chediak-Higashi and Hermansky-Pudlak syndromes, further demonstrating the close relationship between these organelles. Identification of genes mutated in these human diseases, as well as in mouse and Drosophila: pigmentation mutants, is beginning to shed light on the molecular machinery involved in the biogenesis of lysosomes and lysosome-related organelles.

Albinism, Oculocutaneous↗
PubMed1999

The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases.

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.

Amino Acid Sequence↗
PubMed1999

AP-4, a novel protein complex related to clathrin adaptors.

Here we report the identification and characterization of AP-4, a novel protein complex related to the heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein sorting in the endocytic and late secretory pathways. The key to the identification of this complex was the cloning and sequencing of two widely expressed, mammalian cDNAs encoding new homologs of the adaptor beta and sigma subunits named beta4 and sigma4, respectively. An antibody to beta4 recognized in human cells an approximately 83-kDa polypeptide that exists in both soluble and membrane-associated forms. Gel filtration, sedimentation velocity, and immunoprecipitation experiments revealed that beta4 is a component of a multisubunit complex (AP-4) that also contains the sigma4 polypeptide and two additional adaptor subunit homologs named mu4 (mu-ARP2) and epsilon. Immunofluorescence analyses showed that AP-4 is associated with the trans-Golgi network or an adjacent structure and that this association is sensitive to the drug brefeldin A. We propose that, like the related AP-1, AP-2, and AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of integral membrane proteins in mammalian cells.

Adaptor Protein Complex alpha Subunits↗
PubMed1999

Defective expression of the mu3 subunit of the AP-3 adaptor complex in the Drosophila pigmentation mutant carmine.

The adaptor protein (AP) complexes AP-1, AP-2, and AP-3 mediate coated vesicle formation and sorting of integral membrane proteins in the endocytic and late exocytic pathways in mammalian cells. A search of the Drosophila melanogaster expressed sequence tag (EST) database identified orthologs of family members mammalian medium (mu) chain families mu1, mu2, and mu3, of the corresponding AP complexes, and delta-COP, the analogous component of the coatomer (COPI) complex. The Drosophila orthologs exhibit a high degree of sequence identity to mammalian medium chain and delta-COP proteins. Northern analysis demonstrated that medium chain and delta-COP mRNAs are expressed uniformly throughout fly development. Medium chain and delta-COP genes were cytologically mapped and the mu3 gene was found to localize to a region containing the pigmentation locus carmine (cm). Analysis of genomic DNA of the cm1 mutant allele indicated the presence of a large insertion in the coding region of the mu3 gene and Northern analysis revealed no detectable mu3 mRNA. Light microscopy of the cm1 mutant showed a reduction in primary, secondary, and tertiary pigment granules in the adult eye. These findings provide evidence of a role for mu3 in the sorting processes required for pigment granule biogenesis in Drosophila.

Adaptor Protein Complex 1↗
PubMed1998

Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene.

Prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane protein with folate hydrolase and NAALAdase activity. PSMA is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter NAAG (N-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia. To facilitate analysis of the role of PSMA in carcinoma we have determined the structural organization of the gene. The gene consists of 19 exons spanning approximately 60 kb of genomic DNA. A 1244 nt portion of the 5' region of the PSMA gene was able to drive the firefly luciferase reporter gene in prostate but not breast-derived cell lines. We have mapped the gene encoding PSMA to 11p11-p12, however a gene homologous, but not identical, to PSMA exists on chromosome 11q14. Analysis of sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.

Antigens, Surface↗
PubMed1998

Effect of altering the daily herbage allowance to cows in mid lactation on the composition, ripening and functionality of low-moisture, part-skim Mozzarella cheese.

Milk was collected from three spring-calving herds, on different daily herbage allowances (DHA) of perennial rye-grass (16, 20 or 24 kg dry matter (DM)/cow for a 17 week period. On five occasions, at weekly intervals in the middle of the period, the three different milks were converted into low-moisture part-skim Mozzarella cheese. Increasing the DHA resulted in significant increases in the concentrations of protein in the cheesemilk (P < 0.05) and cheese whey (P < 0.02). The moisture-adjusted cheese yield increased significantly (P < 0.01) on raising the DHA from 16 to 24 kg grass DM/cow. DHA had no significant effects on any of the gross compositional values of the cheese (although moisture and fat-in-DM levels tended to decrease and increase respectively with increasing DHA). The hardness of the uncooked cheese and functionality of cooked cheese (i.e. melt time, flowability, stretch and viscosity) were not significantly influenced by DHA over the 115 d ripening period at 4 degrees C.

Animals↗
PubMed1997

In addition to SEC11, a newly identified gene, SPC3, is essential for signal peptidase activity in the yeast endoplasmic reticulum.

Among the three characterized subunits comprising the signal peptidase complex of the yeast Saccharomyces cerevisiae (Sec11p, Spc1p, and Spc2p), only Sec11p is essential for cell growth, signal peptide cleavage, and signal peptidase-dependent protein degradation. Here we report the cloning of the SPC3 gene encoding the homolog to mammalian signal peptidase subunit SPC22/23. We find that Spc3p is also required for cell growth and signal peptidase activity within the yeast endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1997

Mutations in the arginine-rich protein gene (ARP) in pancreatic cancer.

The ARP gene encodes a highly conserved arginine-rich protein from chromosomal band 3p21.1. At the cytogenetic level this region is frequently deleted in a variety of different solid tumors, although not in pancreatic cancer. We have reported the presence of a specific mutation (ATG50-->AGG) or deletion of codon 50 of the ARP gene in different tumor types (Shridhar et al., 1996, 1996a). In the present study, we have observed mutations involving codon 50 in 11 of 37 pancreatic tumors. The frequency of codon 50 mutation is roughly the same in pancreatic tumors as in the other types of tumors previously examined. In addition, we have detected mutations at codon 51 in multiple PCR subclones in two other pancreatic tumors. Mutations in the ARP gene are thus commonly observed in pancreatic cancer, as well as many other cancers.

Adenocarcinoma↗
PubMed1997

Aphidicolin-induced FRA3B breakpoints cluster in two distinct regions.

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE and Alu repetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.

Aphidicolin↗
PubMed1997

Frequent breakpoints in the region surrounding FRA3B in sporadic renal cell carcinomas.

The constitutive fragile site at chromosomal band 3p14.2, FRA3B, is the most active common fragile site in the human genome. We have localized aphidicolin-induced breakpoints to two distinct clusters, separated by 200 Kb, in FRA3B (Paradee et al., 1996). Sequence analysis of these regions identified two polymorphic microsatellite markers immediately adjacent to each of these breakpoint clusters. In this report we have used these two new microsatellites and 14 additional 3p microsatellites to analyse chromosome 3p breakage and loss in 94 sporadic RCC samples, including nonpapillary, papillary and oncocytomas. We have found heterozygous loss of 3p14 sequences in >60% of the RCC samples, including both clear cell and papillary renal cell carcinomas. We have found frequent breakage in the region immediately surrounding FRA3B, demonstrating that FRA3B does play a role in chromosome breakage and loss in RCC. In contrast to other reports, >50% of the papillary tumors also showed LOH of 3p markers. We also observed microsatellite instability (MIN) with most of the tested markers in seven of eight oncocytomas and one of 69 clear cell carcinomas. The MIN in some oncocytomas was of the RER+ (replication error) type I phenotype. None of the five 3p14.2 markers detected any homozygous deletions in tumor samples, but 69/94 (73%) of the tumors had LOH for the region, which includes the recently identified FHIT gene.

Adult↗
PubMed1996

Structurally related Spc1p and Spc2p of yeast signal peptidase complex are functionally distinct.

Two subunits of the mammalian signal peptidase complex, SPC12 and SPC25, share similar membrane topologies with the majority of each protein oriented toward the cytoplasm. Such similarities may suggest that these proteins perform redundant functions in signal peptidase activity. In the present study, we addressed this issue through analysis of the yeast homologs to SPC12 and SPC25, Spc1p and Spc2p. We show that both Spc1p and Spc2p are nonessential for signal peptidase activity and growth of yeast cells and that null mutations in the genes encoding Spc1p and Spc2p are synthetically lethal with a conditional mutation affecting Sec11p, an essential subunit of yeast signal peptidase. However, a high copy plasmid encoding Spc1p suppresses the conditional sec11 mutation, whereas the corresponding plasmid encoding Spc2p does not suppress sec11. Moreover, Spc2p, but not Spc1p, is important for signal peptidase activity and cell viability at high temperatures. These results indicate that although both Spc1p and Spc2p are noncatalytic, they are functionally distinct. Evidence is also presented that a double mutant lacking Spc1p and Spc2p grows well relative to wild type yeast cells, indicating that the signal peptidase complex missing at least two of its subunits is sufficient for signal peptidase activity in vivo.

Amino Acid Sequence↗
PubMed1996

The homologue of mammalian SPC12 is important for efficient signal peptidase activity in Saccharomyces cerevisiae.

The multisubunit signal peptidase catalyzes the cleavage of signal peptides and the degradation of some membrane proteins within the endoplasmic reticulum (ER). The only subunit of this enzyme functionally examined to date, yeast Sec11p, is related to signal peptidase I from bacteria. Since bacterial signal peptidase is capable of processing both prokaryotic and eukaryotic signal sequences as a monomer, it is unclear why the analogous enzyme in the ER contains proteins unrelated to signal peptidase I. To address this issue, the gene encoding Spc1p, the yeast homologue to mammalian SPC12, is isolated from the yeast Saccharomyces cerevisiae. Spc1p co-purifies and genetically interacts with Sec11p, but unlike Sec11p, Spc1p is not required for cell growth or the proteolytic processing of tested proteins in yeast. This indicates that only a subset of the ER signal peptidase subunits is required for signal peptidase and protein degradation activities in vivo. Through both genetic and biochemical criteria, Spc1p appears, however, to be important for efficient signal peptidase activity.

Amino Acid Sequence↗