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J R Glover

Publications and source records attributed to J R Glover.

At least 19 recordsLinked to original sources
PubMed2001

Crowbars and ratchets: hsp100 chaperones as tools in reversing protein aggregation.

Molecular chaperones have the capacity to prevent inappropriate interactions between aggregation-prone folding or unfolding intermediates created in the cell during protein synthesis or in response to physical and chemical stress. What happens when surveillance by molecular chaperones is evaded or overwhelmed and aggregates accumulate? Recent progress in the elucidation of Hsp100/Clp function suggests that intracellular aggregates or stable complexes can be progressively dissolved by the action of chaperones that act as molecular crowbars or ratchets. These insights set the stage for new progress in the understanding and treatment of diseases of protein folding.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

An automated system for epileptogenic focus localization in the electroencephalogram.

This paper describes an automated system for the detection and localization of foci of epileptiform activity in the EEG. The system detects sharp EEG transients (STs) in the process, but the emphasis is on epileptic focus localization. A combination of techniques involving signal processing, pattern recognition, and the expert rules of an experienced electroencephalographer, involving considerable spatiotemporal context information, is applied to multichannel EEG data. An overall emphasis on minimizing the number of false-positive sharp transient detections drives the system design. Tested on data from 13 subjects with epileptiform activity and 5 controls, all areas of focal epileptiform activity were detected by the system, although not all of the contributing foci were reported separately. Two false-positive foci were detected as well due to nonfocal spike activity and normal spike-like activity not present in the training set. The system detected 95.7% of the epileptiform events constituting the correctly detected foci, with a false detection rate of 11.1%.

Artifacts↗
PubMed1998

Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins.

Hsp104 is a stress tolerance factor that promotes the reactivation of heat-damaged proteins in yeast by an unknown mechanism. Herein, we demonstrate that Hsp104 functions in this process directly. Unlike other chaperones, Hsp104 does not prevent the aggregation of denatured proteins. However, in concert with Hsp40 and Hsp70, Hsp104 can reactivate proteins that have been denatured and allowed to aggregate, substrates refractory to the action of other chaperones. Hsp104 cooperates with the chaperones present in reticulocyte lysates but not with DnaK of E. coli. We conclude that Hsp104 has a protein remodeling activity that acts on trapped, aggregated proteins and requires specific interactions with conventional chaperones to promote refolding of the intermediates it produces.

Adenosine Triphosphatases↗
PubMed1997

Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae.

The [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of [PSI+].

Fungal Proteins↗
PubMed1997

Radiotherapy enhances laser palliation of malignant dysphagia: a randomised study.

BACKGROUND/AIMS: A major drawback of laser endoscopy in the palliation of malignant dysphagia is the need for repeated treatments. This study was designed to test whether external beam radiotherapy would reduce the necessity for repeated laser therapy. PATIENTS/METHODS: Sixty seven patients with inoperable oesophageal or gastric cardia cancers and satisfactory swallowing after initial laser recanalisation were randomised to palliative external beam radiotherapy (30 Gy in 10 fractions) or no radiotherapy. All patients underwent a 'check' endoscopy five weeks after initial recanalisation and were subsequently reendoscoped only for recurrent dysphagia, which occurred in 59 patients. RESULTS: Dysphagia was relieved equally well in both groups and the improvement was maintained with further endoscopic treatment. The initial dysphagia controlled interval and the duration between procedures required to maintain lifelong palliation (treatment interval) increased from five to nine weeks (median) in the radiotherapy group (p < 0.01 both parameters). Radiotherapy was well tolerated in all but three patients. One perforation occurred and two fistulae opened after dilatation in patients who received radiotherapy. CONCLUSION: Additional radiotherapy reduces the necessity for therapeutic endoscopy for a patient's remaining life. It has an important role in relatively well patients who are likely to survive long enough to benefit.

Aged↗
PubMed1996

Support for the prion hypothesis for inheritance of a phenotypic trait in yeast.

A cytoplasmically inherited genetic element in yeast, [PSI+], was confirmed to be a prionlike aggregate of the cellular protein Sup35 by differential centrifugation analysis and microscopic localization of a Sup35-green fluorescent protein fusion. Aggregation depended on the intracellular concentration and functional state of the chaperone protein Hsp104 in the same manner as did [PSI+] inheritance. The amino-terminal and carboxy-terminal domains of Sup35 contributed to the unusual behavior of [PSI+]. [PSI+] altered the conformational state of newly synthesized prion proteins, inducing them to aggregate as well, thus fulfilling a major tenet of the prion hypothesis.

Amino Acid Sequence↗
PubMed1996

Modulation of the cleavage of glycosylphosphatidylinositol-anchored proteins by specific bacterial phospholipases.

Many enzymes are tethered to the extracellular face of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. These proteins can be released in soluble form by the action of GPI-specific phospholipase. Little is currently known about the factors modulating this release. We investigated the effects of several experimental variables on the cleavage of the GPI-anchored proteins 5'nucleotidase, acetylcholinesterase, and alkaline phosphatase by phospholipases from Bacillus thuringiensis and Staphylococcus aureus. Phospholipase activity was not inhibited by isotonic salt and was relatively unaffected by buffer type and concentration. In both cases, the optimum pH for cleavage was approximately 6.5. Over 80% of 5'-nucleotidase activity present in the lymphocyte plasma membrane was cleaved by the B. thuringiensis enzyme, and the initial rate of release was linear with phospholipase concentration. All three GPI-anchored proteins were released from lymphocyte plasma membrane at comparable phospholipase concentrations, suggesting that they have similar anchor structures. The catalytic activity of 5'-nucleotidase appeared to increase following conversion to the soluble form. The relative surface charge of the host plasma membrane modulated catalytic activity towards GPI-anchored proteins, depending on the net charge of the phospholipase. Studies on purified lymphocyte 5'-nucleotidase reconstituted into bilayers of dimyristoylphosphatidylcholine indicated that the efficiency of phospholipase cleavage was 12- to 50-fold lower when compared with the native plasma membrane. The ability of the phospholipase to cleave the GPI anchor was further reduced when the bilayer was in the gel phase.

5'-Nucleotidase↗
PubMed1996

HSP100/Clp proteins: a common mechanism explains diverse functions.

The HSP100/Clp proteins are a newly discovered family with a great diversity of functions, such as increased tolerance to high temperatures, promotion of proteolysis of specific cellular substrates and regulation of transcription. HSP100/Clp proteins are also synthesized in a variety of specific patterns and, in eukaryotes, are localized to different subcellular compartments. Recent data suggest that a common ability to disassemble higher-order protein structures and aggregates unifies the molecular functions of this diverse family.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1994

Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer.

The active conformation of native peroxisomal 3-ketoacyl-CoA thiolases (EC 2.3.1.16) is homodimeric. We have previously shown that a truncated Saccharomyces cerevisiae thiolase lacking its first 16 N-terminal amino acids fails to be translocated into peroxisomes but assembles into an enzymatically active form in the cytoplasm of a strain with a disrupted nuclear thiolase gene. We now report that when truncated thiolase is cosynthesized with full-length thiolase, approximately 50% of truncated thiolase cofractionates with the full-length thiolase to fractions enriched for peroxisomes and is translocated into peroxisomes as shown by its protection from the action of external proteases. We constructed an immunologically distinct cytosolic variant of thiolase by adding an influenza hemagglutinin epitope tag to the N terminus of the truncated thiolase. In a strain simultaneously expressing the full-length, truncated, and epitope-tagged truncated thiolases, we demonstrated that normally untargeted thiolase subunits are efficiently translocated into peroxisomes by dimerization with full-length thiolase subunits. Even though truncated and epitope-tagged truncated thiolase subunits are translocated into peroxisomes in this strain, only the full-length thiolase subunit can be coimmunoprecipitated with the epitope-tagged truncated thiolase subunit from the peroxisomal matrix. This observation suggests that interactions between thiolase subunits are not disrupted during translocation.

Acetyl-CoA C-Acetyltransferase↗
PubMed1994

Targeting of passenger protein domains to multiple intracellular membranes.

The role of passenger domains in protein targeting was examined by fusing previously characterized targeting motifs to different protein sequences. To compare the targeting requirements for a variety of subcellular compartments, targeting of the fusion proteins was examined for endoplasmic reticulum, mitochondria and peroxisomes in vitro and in yeast. Although most passenger domains were only partially passive to translocation, motif-dependent targeting via motifs positioned at either end of one passenger domain (gPA) was demonstrated for all of the subcellular compartments tested. The data presented extend earlier suggestions that translocation competence is an intrinsic property of the passenger protein. However, the properties that determine protein targeting are not mutually exclusive for the compartments tested. Therefore, although the primary determinant of specificity is the targeting motif, our results suggest that translocation competence of the targeted protein augments the fidelity of transport.

Amino Acid Sequence↗
PubMed1994

Mutagenesis of the amino targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals conserved amino acids required for import into peroxisomes in vivo.

Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA thiolase is a soluble matrix protein that does not end in a consensus peroxisomal targeting signal-1. The amino terminus of S. cerevisiae peroxisomal thiolase is conserved in 6 of 11 residues with the amino terminus of rat thiolase B, shown to act as a peroxisomal targeting signal-2 (Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S. (1991) EMBO J. 10, 3255-3262). Unlike mammalian peroxisomal thiolases, there is no extensive cleavage of S. cerevisiae thiolase upon import into peroxisomes. We demonstrate by in vivo expression that the amino-terminal 16 amino acids of S. cerevisiae thiolase are necessary and sufficient for targeting to peroxisomes. This result implies that yeast, like mammalian cells, can target proteins to the peroxisomal matrix by at least two different routes. We also demonstrate by targeted mutagenesis and in vivo expression of mutated thiolase genes that three amino acids conserved in the amino termini of all known thiolases are critical for efficient targeting of S. cerevisiae thiolase to peroxisomes.

Acetyl-CoA C-Acyltransferase↗
PubMed1994

PAY4, a gene required for peroxisome assembly in the yeast Yarrowia lipolytica, encodes a novel member of a family of putative ATPases.

PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica. Here we characterize one mutant, pay4, and describe the cloning and sequencing of the PAY4 gene. The pay4 mutant shows no identifiable peroxisomes by biochemical and morphological criteria. The complementing PAY4 gene encodes a polypeptide, Pay4p, 1025 amino acids in length and having a predicted molecular mass of 112,258 Da. The predicted Pay4p sequence contains two putative ATP-binding domains and shows structural relationships to other potential ATP-binding proteins involved in biological processes as diverse as peroxisome biogenesis, vesicle-mediated protein transport, cell cycle control, and transcriptional regulation. These proteins all share a highly conserved stretch of approximately 175 amino acids that contains a consensus sequence for ATP binding. Pay4p shows sequence conservation with Pas1p and Pas5p, putative ATPases required for peroxisomal assembly in the yeasts Saccharomyces cerevisiae and Pichia pastoris, respectively. Pay4p, Pas1p, and Pas5p are presumably related members of a family of putative ATPases involved in peroxisome biogenesis. Pay4p is synthesized in low amounts in Y. lipolytica cells grown in glucose, and there is a rapid and pronounced increase in the levels of Pay4p upon transfer of the cells to a medium containing oleic acid as the sole carbon source.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1992

Peroxisome biogenesis in yeast.

Eukaryotic cells have evolved a complex set of intracellular organelles, each of which possesses a specific complement of enzymes and performs unique metabolic functions. This compartmentalization of cellular functions provides a level of metabolic control not available to prokaryotes. However, it presents the eukaryotic cell with the problem of targeting proteins to their specific location(s). Proteins must be efficiently transported from their site of synthesis in the cytosol to their specific organelle(s). Such a process may require translocation across one or more hydrophobic membrane barriers and/or asymmetric integration into specific membranes. Proteins carry cis-acting amino acid sequences that serve to act as recognition motifs for protein sorting and for the cellular translocation machinery. Sequences that target proteins to the endoplasmic reticulum/secretory pathway, mitochondria, and chloroplasts are often present as cleavable amino-terminal extensions. In contrast, most peroxisomal proteins are synthesized at their mature size and are translocated to the organelle without any post-translational modification. This review will summarize what is known about how yeast solve the problem of specifically importing proteins into peroxisomes and will suggest future directions for investigations into peroxisome biogenesis in yeast.

Amino Acid Sequence↗
PubMed1992

Endoscopic ultrasound for localisation of islet cell tumours.

In a prospective study endoscopic ultrasound localisation of pancreatic endocrine tumours was attempted in 21 patients with clinically suspected islet cell tumours. Most patients were referred after the failure of conventional imaging methods. Endoscopic ultrasound correctly identified the site of 12 of 15 insulinomas, one glucagonoma, and a diffuse pancreatic abnormality in a patient with multiple endocrine adenopathy. There were two true negative examinations and one technical failure. The sensitivity of endoscopic ultrasound was much greater than that of computed tomography or conventional transabdominal ultrasonography.

Adenoma, Islet Cell↗