PubMed Health⌕ Search

Biomedical subjects

A Rowley

Publications and source records attributed to A Rowley.

At least 19 recordsLinked to original sources
PubMed2000

Proteomics characterization of abundant Golgi membrane proteins.

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Amino Acid Sequence↗
PubMed2000

The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae.

To investigate the factors involved in the sorting of cargo proteins into COPII endoplasmic reticulum (ER) to Golgi apparatus transport vesicles, we have created a strain of S. cerevisiae (p24Delta8) that lacks all eight members of the p24 family of transmembrane proteins (Emp24p, Erv25p, and Erp1p to Erp6p). The p24 proteins have been implicated in COPI and COPII vesicle formation, cargo protein sorting, and regulation of vesicular transport in eukaryotic cells. We find that p24Delta8 cells grow identically to wild type and show delays of invertase and Gas1p ER-to-Golgi transport identical to those seen in a single Deltaemp24 deletion strain. Thus, p24 proteins do not have an essential function in the secretory pathway. Instead, they may serve as quality control factors to restrict the entry of proteins into COPII vesicles.

Base Sequence↗
PubMed2000

Applications of protein mass spectrometry in cell biology.

Advances in mass spectrometry combined with accelerated progress in genome sequencing projects have facilitated the rapid identification of proteins by enzymatic digestion, mass analysis, and sequence database searching. Applications for this technology range from the surveillance of protein expression in cells, tissues, and whole organisms, to the identification of proteins and posttranslational modifications. Here we consider practical aspects of the application of mass spectrometry in cell biology and illustrate these with examples from our own laboratories.

Amino Acid Sequence↗
PubMed1999

Erp1p and Erp2p, partners for Emp24p and Erv25p in a yeast p24 complex.

Six new members of the yeast p24 family have been identified and characterized. These six genes, named ERP1-ERP6 (for Emp24p- and Erv25p-related proteins) are not essential, but deletion of ERP1 or ERP2 causes defects in the transport of Gas1p, in the retention of BiP, and deletion of ERP1 results in the suppression of a temperature-sensitive mutation in SEC13 encoding a COPII vesicle coat protein. These phenotypes are similar to those caused by deletion of EMP24 or ERV25, two previously identified genes that encode related p24 proteins. Genetic and biochemical studies demonstrate that Erp1p and Erp2p function in a heteromeric complex with Emp24p and Erv25p.

Amino Acid Sequence↗
PubMed1999

Report of the National Institutes of Health Workshop on Kawasaki Disease.

The National Institute of Allergy and Infectious Disease, National Institutes of Health convened a workshop on Kawasaki disease, May 1997, co-chaired by Drs. Karyl Barron and Stanford Shulman. The goal of the workshop was to review the latest scientific advances relating to the epidemiology, etiology, pathogenesis, treatment, and complications of Kawasaki disease, along with future therapeutic options and proposed future research directions.

Coronary Disease↗
PubMed1998

The yeast protein complex containing cdc68 and pob3 mediates core-promoter repression through the cdc68 N-terminal domain.

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.

Adenosine Triphosphatases↗
PubMed1997

Patterns of Kawasaki syndrome presentation.

Kawasaki syndrome (KS) is a systemic disorder of unknown etiology that can lead to coronary artery aneurysm and thrombosis in a significant number of children. It is defined by a number of clinical guidelines set by the Centers for Disease Control (Rauch, A.M., Hurwitz, E.S. (1985) Centers for Disease Control (CDC). Case Definition for Kawasaki syndrome. Pediatr. Infect. Dis. 4, 702-703). Many of the symptoms of this illness may lead the patient to the otolaryngologist. These criteria include injected or fissured lips, injected pharynx, strawberry tongue and cervical lymphadenopathy. When administered in the first 10 days of the illness, gamma globulin has been demonstrated to reduce the prevalence of coronary artery abnormalities (Newburger, J.W., Takahashi, M., Burns, J.C. et al. (1986) Treatment of Kawasaki syndrome with intravenous gamma globulin. N. Engl. J. Med. 315, 341-347). Unfortunately, when a diagnosis of KS is not considered or if a patient presents with unusual symptoms that are not consistent with the CDC guidelines, the diagnosis and treatment of KS can be delayed or even missed. We present a series of patients with KS to illustrate its patterns of presentation.

Child↗
PubMed1996

Disseminated herpes simplex virus infection presenting as fever in the newborn--a lethal outcome.

The clinical course of a neonate who presented with fever and tachypnea on day 6 of life is described. He developed disseminated intravascular coagulopathy, hepatic failure, coma and expired at 14 days of age. The post mortem viral cultures from liver, adrenal and lungs were positive for HSV type 2. The fatal outcome of this case of fever, due to HSV infection, emphasizes the need for early treatment of suspicious cases of HSV infection. HSV should be considered in the differential diagnosis of the newborn with persistent fever unresponsive to antibiotics.

Fatal Outcome↗
PubMed1995

Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC.

Characterization of the proteins that interact with replication origins, as well as characterization of the mechanisms by which the levels and activities of these proteins are regulated during the cell cycle, is required to understand the initiation of chromosomal DNA replication in eukaryotic cells. We have previously shown that the first detectable step in the assembly of initiation complexes in vivo involves the binding of the multisubunit origin recognition complex (ORC) and the general transcription/replication factor ABF1 protein to origins. In this paper we show that ORC is present in cells at low levels, corresponding to little more than one complete complex per replication origin, indicating that in vivo origin recognition by ORC is extremely efficient. We show that this efficient recognition requires two sequence elements, the essential A element containing the ARS consensus sequence and the functionally important B1 element, both in vitro and in vivo. Moreover, we show that origin binding by ORC in vivo does not require any other functional sequence element, indicating that it occurs independently of the binding of other factors, such as ABF1. Our results suggest a model for the roles of the individual elements of yeast replication origins.

Base Sequence↗
PubMed1995

Mutations sensitizing yeast cells to the start inhibitor nalidixic acid.

The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wild-type cells naturally become refractory to Nal. Three complementation groups of Nal-sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild-type chorismate mutase was unaffected, These aro7 alleles demonstrate 'recruitment', by mutation, of an innately indifferent protein to an inhibitor-sensitive form. The Nal-sensitive aro7 mutant cells were used to show that the resumption of Nal-inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal-supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal-mediated inhibition of Start.

Cell Cycle↗
PubMed1995

Thyrotoxic periodic paralysis: two case studies.

Thyrotoxic periodic paralysis (TPP) is a disorder that affects a small percentage of patients with hyperthyroidism; the patients are predominantly Asian men. Two typical cases are presented here; both patients came to the emergency room with complaints of sudden onset of weakness. Their symptoms were initially resolved through treatment for acute hypokalemia. Additional tests identified the patients as being thyrotoxic for which condition they underwent thyroid ablation with radioiodine. No further episodes were reported for either patient. Often the underlying cause of the weakness or paralysis is undetected because of the absence of clinical symptoms of hyperthyroidism and because clinicians are unfamiliar with the disorder. It is imperative that TPP be recognized in the differential diagnosis of weakness and that the underlying thyrotoxicity is treated to permanently alleviate the condition. The authors discuss the epidemiology, etiology, clinical manifestations, and treatments of TPP.

Adult↗
PubMed1995

Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle.

DNA replication is a pivotal event in the cell cycle and, as a consequence, is tightly controlled in eukaryotic cells. The initiation of DNA replication is dependent upon the completion of mitosis and upon the commitment to complete the cell cycle made during G(1). Characterisation of the protein factors required for initiating DNA replication is essential to understand how the cell cycle is regulated. Recent results indicate that initiation complexes assemble in multiple stages during the cell cycle. First, origins are bound by the multisubunit origin recognition complex (ORC) which is essential for DNA replication in vivo. ORC, present at little more than one complete complex per replication origin, binds to origins immediately after initiation in the previous cell cycle. ORC binding occurs by the recognition of a bipartite sequence that includes the essential ARS consensus sequence (ACS) and the functionally important B1 element adjacent to the ACS. A novel pre-replicative complex (pre-RC) assembles at origins at the end of mitosis in actively cycling cells and remains at origins until DNA replication initiates. Finally, Dbf4, which is periodically synthesised at the end of G(1), interacts with replication origins. Dbf4-origin interaction requires an intact ACS strongly suggesting that interaction occurs through ORC. Dbf4 interacts with and is required for the activation of the Cdc7 protein kinase and together, Dbf4 and Cdc7 are required for the G(1)-S transition. Separate regions of Dbf4 are required for Cdc7- and origin-interaction suggesting that Dbf4 may act to recruit Cdc7 to replication origins where phosphorylation of some key component may cause origin firing.

Base Sequence↗
PubMed1995

A prospective trial comparing the efficacy and complications of the modified Dornier HM3 and MFL 5000 lithotriptors for solitary renal calculi.

A prospective randomized study of 198 patients was conducted to compare the efficacy of the modified Dornier HM3 lithotriptor to the MFL 5000 lithotriptor. Entrance criteria included solitary stones at any location within the upper collecting system that had not previously been treated with lithotripsy. Following lithotripsy the patients were evaluated by a blinded radiologist with a plain abdominal film, tomograms and renal ultrasound at 1, 4 and 12 weeks. Patients were classified at 12 weeks after lithotripsy as failing treatment if any stone fragments were imaged. Of the patients 170 were available for complete 3-month followup. No statistical or clinical difference in stone-free rates was apparent for calculi in the ureter or renal pelvis in either group. Of patients with lower caliceal stones 80% had no residual fragments visualized at 12 weeks when treated with the modified HM3 device versus 56% with the MFL 5000 lithotriptor (p = 0.05). Treatment time on the MFL 5000 unit was significantly prolonged compared with the modified HM3 device (0.7 hours versus 0.4 hours, respectively) resulting in fewer patients being treated in a given day (p < 0.001). No statistical difference in complication rates could be found between the 2 machines. Steinstrasse were noted in 10% of the patients treated with the modified HM3 device and 6% of the MFL 5000 group. Subcapsular hematomas were noted in 4% of the MFL 5000 treatment arm compared to 1% in the modified HM3 group. Overall, the MFL 5000 lithotriptor was believed to offer no significant clinical advantage over the modified HM3 device in terms of lithotripsy efficacy, although the multifunctional table did offer more versatility for stone treatment. For a busy lithotripsy center, the modified HM3 lithotriptor is still the most efficacious.

Adult↗
PubMed1994

Two steps in the assembly of complexes at yeast replication origins in vivo.

The integration of chromosomal DNA replication into the eukaryotic cell cycle might involve temporal regulation of interactions between cellular factors and replication origins. We show here that yeast replication origins exist in two chromatin states during the cell cycle. In the postreplicative state, genomic footprints closely resemble those produced in vitro by the purified ORC and ABF1 proteins, indicating that the binding of these proteins to replication origins is not sufficient to drive the initiation of DNA replication. The prereplicative state is characterized by an additional region of protection overlapping the ORC footprint. This prereplicative complex appears near the end of mitosis and persists through G1. After entry into S phase, origins return to the postreplicative state. Similarities in temporal regulation of the prereplicative state and the Xenopus licensing factor suggest that mechanisms limiting DNA replication to once per cell cycle may be conserved among eukaryotes.

Base Sequence↗
PubMed1993

Yeast origin recognition complex is involved in DNA replication and transcriptional silencing.

The HMR E silencer represses transcription of silent mating-type genes in the budding yeast Saccharomyces cerevisiae and contains three redundant regulatory elements A, E and B (ref. 1). The A element contains the 11 base pair consensus sequence that is essential for the firing of DNA replication origins. A multisubunit protein called the origin recognition complex (ORC) binds specifically to this consensus sequence within yeast origins in vitro and in vivo. We isolated mutants in A element-mediated silencing and report here that one of the genes we identified, RRR1, encodes ORC2, the 72K subunit of ORC. RRR1/ORC2 is an essential gene, but the rrr1-316 allele, which is viable, is defective in the replication of nuclear DNA and the maintenance of the 2-microns episomal DNA. This is, to our knowledge, the first genetic evidence that ORC is involved in DNA replication and silencing.

Amino Acid Sequence↗