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Biomedical subjects

B Burke

Publications and source records attributed to B Burke.

At least 19 recordsLinked to original sources
PubMed1999

Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain.

We employed a phage display system to search for proteins that interact with transportin 1 (TRN1), the import receptor for shuttling hnRNP proteins with an M9 nuclear localization sequence (NLS), and identified a short region within the N-terminus of the nucleoporin Nup153 which binds TRN1. Nup153 is located at the nucleoplasmic face of the nuclear pore complex (NPC), in the distal basket structure, and functions in mRNA export. We show that this Nup153 TRN1-interacting region is an M9 NLS. We found that both import and export receptors interact with several regions of Nup153, in a RanGTP-regulated fashion. RanGTP dissociates Nup153-import receptor complexes, but is required for Nup153-export receptor interactions. We also show that Nup153 is a RanGDP-binding protein, and that the interaction is mediated by the zinc finger region of Nup153. This represents a novel Ran-binding domain, which we term the zinc finger Ran-binding motif. We provide evidence that Nup153 shuttles between the nuclear and cytoplasmic faces of the NPC. The presence of an M9 shuttling domain in Nup153, together with its ability to move within the NPC and to interact with export receptors, suggests that this nucleoporin is a mobile component of the pore which carries export cargos towards the cytoplasm.

Amino Acid Sequence↗
PubMed1999

Novel method for rapid determination of clarithromycin sensitivity in Helicobacter pylori.

A novel PCR-hybridization assay, performed in single closed capillaries, was developed to detect clarithromycin resistance-associated gene mutations in Helicobacter pylori. Mutations were detected by thermal analysis in 33 of 34 (97%) resistant isolates but not in 66 isolates determined to be sensitive by conventional antibiotic assays. The method was rapid and reproducible, and it reduced PCR product contamination risk.

Anti-Bacterial Agents↗
PubMed1999

Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis.

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. With a mass of about 125 MDa, NPCs are thought to be composed of 50 or more distinct protein subunits, each present in multiple copies. During mitosis in higher cells the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized. Using both conventional and digital confocal immunofluorescence microscopy we have been able to define a time course of post-mitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a component of the nuclear basket, associates with chromatin towards the end of anaphase, in parallel with the inner nuclear membrane protein, LAP2. However, immunogold labeling suggests that the initial Nup153 chromatin association is membrane-independent. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, 54, 45) during mitosis and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates.

Animals↗
PubMed1999

Function and assembly of nuclear pore complex proteins.

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. The current view of NPC organization features a massive symmetrical framework that is embedded in the double membranes of the nuclear envelope. It embraces a central channel of as yet ill-defined structure but which may accommodate particles with diameters up to 26 nm provided that they bear specific import/export signals. Attached to both faces of the central framework are peripheral structures, short cytoplasmic filaments, and a nuclear basket assembly, which interact with molecules transiting the NPC. The mechanisms of assembly and the nature of NPC structural intermediates are still poorly understood. However, mutagenesis and expression studies have revealed discrete sequences within certain NPC proteins that are necessary and sufficient for their appropriate targeting. In addition, some details are emerging from observations on cells undergoing mitosis where the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized to form nuclear envelopes in the two daughter cells. To date, it has been possible to define a time course of postmitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral inner nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a dynamic component of the nuclear basket, associates with chromatin towards the end of anaphase coincident with, although independent of, the inner nuclear membrane protein, LAP2. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, p54, p45) during mitosis, and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates and which may therefore represent an essential component of the central framework of the NPC.

Animals↗
PubMed1998

Functional analysis of Tpr: identification of nuclear pore complex association and nuclear localization domains and a role in mRNA export.

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

Animals↗
PubMed1998

Species-specific differences in the operational RNA code for aminoacylation of tRNAPro.

An operational RNA code relates amino acids to specific structural features located in tRNA acceptor stems. In contrast to the universal nature of the genetic code, the operational RNA code can vary in evolution due to coadaptations of the contacts between aminoacyl-tRNA synthetases and the acceptor stems of their cognate tRNA substrates. Here we demonstrate that, for class II prolyl-tRNA synthetase (ProRS), functional coadaptations have occurred in going from the bacterial to the human enzyme. Analysis of 20 ProRS sequences that cover all three taxonomic domains (bacteria, eucarya, and archaea) revealed that the sequences are divided into two evolutionarily distant groups. Aminoacylation assays showed that, while anticodon recognition has been maintained through evolution, significant changes in acceptor stem recognition have occurred. Whereas all tRNAPro sequences from bacteria strictly conserve A73 and C1.G72, all available cytoplasmic eukaryotic tRNAPro sequences have a C73 and a G1.C72 base pair. In contrast to the Escherichia coli synthetase, the human enzyme does not use these elements as major recognition determinants, since mutations at these positions have only small effects on cognate synthetase charging. Additionally, E. coli tRNAPro is a poor substrate for human ProRS, and the presence of the human anticodon-D stem biloop domain was necessary and sufficient to confer efficient aminoacylation by human ProRS on a chimeric tRNAPro containing the E. coli acceptor-TpsiC stem-loop domain. Our data suggest that the two ProRS groups may reflect coadaptations needed to accommodate changes in the operational RNA code for proline.

Acylation↗
PubMed1998

Amino-terminal sequences that direct nucleoporin nup153 to the inner surface of the nuclear envelope.

Nup153 is a large O-linked glycoprotein that is a component of the basket-like structure that forms the nucleoplasmic face of nuclear pore complexes (NPCs). The Nup153 molecule has a tripartite structure consisting of N- and C-terminal domains flanking a central zinc finger domain. All of the targeting and assembly information contained within Nup153 is contributed by the N-domain. In fact this region of the molecule can target a cytosolic protein, pyruvate kinase, to the nucleoplasmic face of the NPC. The zinc finger and C-terminal domains appear to have no role in these targeting and assembly activities. Deletion analysis reveals that there are two distinct regions within the Nup153 N-domain that contain different targeting functions. One of these is directly involved in assembly into the NPC while a second overlapping region may target Nup153, as well as other reporter molecules, to the inner face of the nuclear envelope.

Amino Acid Substitution↗
PubMed1998

Three distinct steps in transport of vesicular stomatitis virus glycoprotein from the ER to the cell surface in vivo with differential sensitivities to GTP gamma S.

Microinjected GTP gamma S revealed three distinct steps in the exocytic transport of the temperature sensitive glycoprotein of vesicular stomatitis virus (ts-O45-G) from the ER to the cell surface in intact Vero cells. While COPII dependent export of ts-O45-G from the ER is blocked in cells injected with recombinant protein of a dominant mutant of SAR1a (SAR1a[H79G]) inhibited in GTP hydrolysis, neither injected GTP gamma S nor antibodies against beta-COP (anti-EAGE) interfere with this transport step significantly. In contrast, transport to the Golgi complex is blocked by 50 microM GTP gamma S, a dominant mutant of ARF1 (ARF1[Q71L]) inhibited in GTP hydrolysis, or microinjected anti-EAGE, but injected Sar1a[H79G]p has no effect. Microinjection of GTP gamma S or expression of ARF[Q71L] rapidly induces accumulation of COPI coated vesicular structures lacking ts-O45-G. Finally, transport of ts-O45-G from the trans-Golgi network (TGN) to the cell surface is inhibited only by high concentrations of GTP gamma S (500 microM). Interestingly, this step is only partially brefeldin A sensitive, and injected antibodies against beta-COP and p200/myosin II, a TGN membrane associated protein, have no effect. These data provide first strong in vivo evidence for at least three distinct steps in the exocytic pathway of mammalian cells regulated by different sets of GTPases and coat proteins. COPII, but not COPI, is required for ER export of ts-O45-G. COPI plays a role in subsequent transport to the Golgi complex, and a so far unidentified GTP gamma S sensitive coat appears to be involved in transport from the TGN to the cell surface.

Animals↗
PubMed1997

Nup84, a novel nucleoporin that is associated with CAN/Nup214 on the cytoplasmic face of the nuclear pore complex.

The short filaments extending from the cytoplasmic face of nuclear pore complexes are thought to contain docking sites for nuclear import substrates. One component of these filaments is the large O-linked glycoprotein CAN/Nup214. Immunoprecipitation studies carried out under nondenaturing conditions, and using a variety of antibodies, reveal a novel nonglycosylated nucleoporin, Nup84, that is tightly associated with CAN/Nup214. Consistent with such an association, Nup84 is found to be exposed on the cytoplasmic face of the nuclear pore complex. cDNA sequence analyses indicate that Nup84 contains neither the GLFG nor the XFXFG repeats that are a characteristic of a number of other nuclear pore complex proteins. Secondary structure predictions, however, suggest that Nup84 contains a coiled-coil COOH-terminal domain, a conclusion supported by the observation of significant sequence similarity between this region of the molecule and various members of the tropomyosin family. Mutagenesis and expression studies indicate that the putative coiled-coil domain is required for association with the cytoplasmic face of the nuclear pore complex, whereas it is the NH2-terminal region of Nup84 that contains the site of interaction with CAN/Nup214. These findings suggest a model in which Nup84 may function in the attachment of CAN/Nup214 to the central framework of the nuclear pore complex. In this way, Nup84 could play a central role in the organization of the interface between the pore complex and the cytoplasm.

Amino Acid Sequence↗
PubMed1997

Brain anomalies, retardation of mentality and growth, ectodermal dysplasia, skeletal malformations, Hirschsprung disease, ear deformity and deafness, eye hypoplasia, cleft palate, cryptorchidism, and kidney dysplasia/hypoplasia (BRESEK/BRESHECK): new X-linked syndrome?

Two half brothers (maternally related) had a similar syndrome of microhydrocephaly in both brothers and dilatation of the spinal canal with fusion of thalami in one brother. Primordial growth delay was noted in both brothers, with severe mental retardation in the surviving brother. Both had ectodermal dysplasia with scaling, hyperkeratosis, and generalized alopecia, but normal sweat and sebaceous glands. Skeletal anomalies included hemivertebrae with abnormal segmentation in one and scoliosis with polydactyly in the other. Ears were apparently low set, large, and protruding, with mixed hearing loss in the brother who survived. Eye anomalies included maldevelopment of one eye in Patient 1 and small optic nerves more noticeable on one side in Patient 2. Both had cryptorchidism and dysplastic/hypoplastic kidneys of varying severity that resulted in the early postnatal death of one sib. Manifestations present in only one or the other sib included submucous cleft palate, aganglionosis of the rectum and colon, agenesis of one testicle, and single umbilical artery. This syndrome has not been described previously and may be due to an X-linked mutation. The acronym BRESEK reflects the common findings, whereas BRESHECK denotes all manifestations of both patients: brain, retardation, ectodermal dysplasia, skeletal deformities, Hirschsprung disease, ear/eye anomalies, cleft palate/cryptorchidism, and kidney dysplasia/hypoplasia. In addition to an X-linked mutation, a contiguous gene deletion or maternal mosaicism of an autosomal dominant gene must be considered.

Abnormalities, Multiple↗
PubMed1997

Valproate-induced liver failure in one of two siblings with Alpers disease.

Alpers disease is a neurodegenerative disorder of childhood characterized by early developmental delay, intractable seizures, and death in childhood. Neuropathologic changes are most severe in the gray matter and consist of diffuse neuronal loss, spongiform changes, and astrocytosis. We report 2 siblings with Alpers disease who were discordant for exposure to valproate (VPA). Both had developmental delay, and a progressive seizure disorder beginning at 5 years of age. The proband died at age 8 years of complications of ongoing seizures, including epilepsia partialis continua, with only minimal liver abnormalities. Her younger brother was treated with VPA for new-onset seizures and developed fulminant liver failure 6 months later, which led to his death at 5 years of age. Neuropathologic abnormalities of both siblings were consistent with Alpers disease. These observations support classification of Alpers disease and Alpers disease with liver cirrhosis as a single disease. They also confirm previous reports indicating that VPA may accelerate fulminant liver failure in Alpers disease. We recommend that a diagnosis of Alpers disease be considered in children with unexplained early developmental delay, cerebellar signs, or partial seizures, especially epilepsia partialis continua. When Alpers disease is strongly suspected, use of VPA should be avoided.

Anticonvulsants↗
PubMed1997

Glomerular volume in congestive cardiac failure.

Glomerular volume has been reported to be increased in patients with congenital cyanotic heart disease and cor pulmonale; however it has not been systematically studied in patients with congestive cardiac failure (CCF). Glomerular volume was therefore measured by point-counting serially sectioned glomerular profiles of 25 randomly selected glomeruli using the Cavalieri principle in autopsy specimens from 8 patients dying from CCF and 6 age-matched controls with no renal or cardiac pathology. Mean glomerular volume was not different between patients dying from CCF and controls, 2.49 (0.21) vs. 2.25 (0.26) x 10(6) microm3, and the distribution of individual glomerular volumes was similar in the two groups. We conclude that severe CCF is not associated with significant glomerular enlargement and that the previously reported glomerular enlargement in cyanotic heart disease is likely to be mediated through hypoxemia.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Evaluation of pathologic criteria for acute renal allograft rejection: reproducibility, sensitivity, and clinical correlation.

This study was designed to evaluate the pathologic criteria used for acute renal allograft rejection that were developed by a panel of renal pathologists participating in the Cooperative Clinical Trials in Transplantation, a National Institutes of Health-supported, multicenter research group. The panel defined three categories of acute rejection. (1) Type I: mononuclear infiltrate in > or =5% of cortex, a total of at least three tubules with tubulitis in 10 consecutive high-power fields from the most severely affected areas, and at least two of the three following features: edema, activated lymphocytes, or tubular injury. (2) Type II: arterial, or arteriolar, endothelialitis with or without the preceding features. (3) Type III: arterial fibrinoid necrosis or transmural inflammation with or without thrombosis, parenchymal necrosis, or hemorrhage. Using these criteria, and without any knowledge of the clinical course or original diagnosis, a rotating panel of three pathologists agreed with the original study pathologist's diagnosis of the presence or absence of rejection in 259 of the 286 biopsies (91%) used for this analysis (kappa = 0.80). The sensitivity to establish the diagnosis of rejection was 91% for a single core and 99% for two cores. To validate the diagnostic criteria, the thresholds for number of tubules with tubulitis and the percent infiltrate were varied, and the pathologic diagnosis was compared with the clinical course. The greatest agreement occurred with a threshold of > or =1 tubule with tubulitis and > or =5% cortex with interstitial infiltrate (91%). Clinically severe rejection episodes were correlated with the type of rejection (type I, odds ratio [OR] 6.2; type II, OR 37.9). Type II rejection was more likely to be clinically severe than type I (OR 6.1). Analysis of other individual pathologic features revealed a correlation with clinical severity for endothelialitis (OR 13.2), interstitial hemorrhage (OR 13.2), and the presence of glomerulitis (OR 3.7) (all P < 0.05). The extent of tubulitis or of the interstitial infiltrate did not correlate with severity (P > 0.05). It is concluded that these criteria are simple, reproducible, and clinically relevant. These data should lead to further refinement of the diagnostic systems for renal allograft rejection.

Acute Disease↗
PubMed1997

Understanding species-specific differences in substrate recognition by Escherichia coli and human prolyl-tRNA synthetases.

Class II human prolyl-tRNA synthetase (ProRS) aminoacylates in vitro transcribed human tRNA(Pro) with kinetic parameters that are similar to those previously determined for aminoacylation of Escherichia coli tRNA(Pro) by its cognate synthetase. As in the bacterial system, large decreases in aminoacylation by human ProRS occur upon mutating anticodon positions G35 and G36 of human tRNA(Pro). The N73 'discriminator' base and the first and third base pairs of the acceptor stem vary between the E.coli and human isoacceptor groups. In contrast to the E. coli synthetase, the human enzyme does not appear to recognize these elements, since mutations at these positions do not significantly affect cognate synthetase charging. E. coli ProRS does not cross-aminoacylate human tRNA(Pro), and the bacterial tRNA(Pro) is a poor substrate for the human enzyme. Mutations in both the tRNAs and the synthetases have been made in an effort to identify elements in each system responsible for blocking cross-species aminoacylation. Alignment of all known ProRS primary sequences from different species reveals particularly low overall sequence homology, as well as two distinct groups of enzymes. The sequence divergence between E. coli and human ProRSs helps to explain the species-specific differences in the RNA code for aminoacylation of tRNA(Pro).

Amino Acid Sequence↗
PubMed1996

Rotavirus pathogenicity.

The pathogenicity of rotaviruses depends on multiple viral and host factors. In this review the evidence for the involvement of a number of viral genes in the ability to cause disease is presented. Different genes are of importance in different rotavirus-host systems: there is no single pathogenicity factor.

Animals↗