PubMed Health⌕ Search

Biomedical subjects

D J Burke

Publications and source records attributed to D J Burke.

At least 19 recordsLinked to original sources
PubMed2001

The Heartmate II: design and development of a fully sealed axial flow left ventricular assist system.

Our group is developing the control and power transmission components required to implement a permanent and fully sealed left ventricular assist system (LVAS). Starting with the percutaneously powered HeartMate II blood pump, our development efforts are focused in the following areas: a complete redesign of the transcutaneous energy transmission system (TETS) to include a rectification network and autonomous voltage regulation within the secondary coil, a hermetically sealed electronics package containing a miniaturized implementation of the existing redundant drive and control electronics with several power-input options, an implanted rechargeable lithium ion battery pack capable of providing up to 1 h of untethered operation, implantable electrical connectors that allow components to be connected after placement in the body or to be replaced if needed, and a radio telemetry subsystem to transmit diagnostic information and to permit remote adjustment of selected parameters.

Electric Power Supplies↗
PubMed2001

The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain.

We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2000

The spindle checkpoint of Saccharomyces cerevisiae responds to separable microtubule-dependent events.

The spindle checkpoint regulates microtubule-based chromosome segregation and helps to maintain genomic stability [1,2]. Mutational inactivation of spindle checkpoint genes has been implicated in the progression of several types of human cancer. Recent evidence from budding yeast suggests that the spindle checkpoint is complex. Order-of-function experiments have defined two separable pathways within the checkpoint. One pathway, defined by MAD2, controls the metaphase-to-anaphase transition and the other, defined by BUB2, controls the exit from mitosis [3-6]. The relationships between the separate branches of the checkpoint, and especially the events that trigger the pathways, have not been defined. We localized a Bub2p-GFP fusion protein to the cytoplasmic side of the spindle pole body and used a kar9 mutant to show that cells with misoriented spindles are arrested in anaphase of mitosis. We used a kar9 bub2 double mutant to show that the arrest is BUB2 dependent. We conclude that the separate pathways of the spindle checkpoint respond to different classes of microtubules. The MAD2 branch of the pathway responds to kinetochore microtubule interactions and the BUB2 branch of the pathway operates within the cytoplasm, responding to spindle misorientation.

Calcium-Binding Proteins↗
PubMed2000

Complexity in the spindle checkpoint.

Cell viability requires accurate chromosome segregation at mitosis. The spindle checkpoint ensures that anaphase is not attempted until the sister chromatids of each chromosome are attached to spindle microtubules from opposite poles. The checkpoint mechanism involves a signal transduction cascade that is more complex than was originally envisioned.

Animals↗
PubMed2000

The spindle checkpoint: two transitions, two pathways.

The spindle checkpoint is an evolutionarily conserved mitotic regulatory mechanism that ensures that anaphase is not attempted until chromosomes are properly aligned on the spindle. Two different cell-cycle transitions must be inhibited by the spindle checkpoint to arrest cells at metaphase and prevent mitotic exit. The checkpoint proteins interact in ways that are more complex than was originally envisioned. This review summarizes the evidence for two pathways of spindle-checkpoint regulation in budding yeast. We describe how the proteins are involved in these pathways and discuss the ways in which the spindle checkpoint inhibits the cell-cycle machinery.

Animals↗
PubMed1998

Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events.

We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.

Anaphase↗
PubMed1998

Cell cycle arrest in cdc20 mutants of Saccharomyces cerevisiae is independent of Ndc10p and kinetochore function but requires a subset of spindle checkpoint genes.

The spindle checkpoint ensures accurate chromosome segregation by inhibiting anaphase onset in response to altered microtubule function and impaired kinetochore function. In this study, we report that the ability of the anti-microtubule drug nocodazole to inhibit cell cycle progression in Saccharomyces cerevisiae depends on the function of the kinetochore protein encoded by NDC10. We examined the role of the spindle checkpoint in the arrest in cdc20 mutants that arrest prior to anaphase with an aberrant spindle. The arrest in cdc20 defective cells is dependent on the BUB2 checkpoint and independent of the BUB1, BUB3, and MAD spindle checkpoint genes. We show that the lesion recognized by Bub2p is not excess microtubules, and the cdc20 arrest is independent of kinetochore function. We show that Cdc20p is not required for cyclin proteolysis at two points in the cell cycle, suggesting that CDC20 is distinct from genes encoding integral proteins of the anaphase promoting complex.

Animals↗
PubMed1997

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae.

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation.

Anaphase↗
PubMed1997

Three-dimensional analysis and ultrastructural design of mitotic spindles from the cdc20 mutant of Saccharomyces cerevisiae.

The three-dimensional organization of mitotic microtubules in a mutant strain of Saccharomyces cerevisiae has been studied by computer-assisted serial reconstruction. At the nonpermissive temperature, cdc20 cells arrested with a spindle length of approximately 2.5 microns. These spindles contained a mean of 81 microtubules (range, 56-100) compared with 23 in wild-type spindles of comparable length. This increase in spindle microtubule number resulted in a total polymer length up to four times that of wild-type spindles. The spindle pole bodies in the cdc20 cells were approximately 2.3 times the size of wild-type, thereby accommodating the abnormally large number of spindle microtubules. The cdc20 spindles contained a large number of interpolar microtubules organized in a "core bundle." A neighbor density analysis of this bundle at the spindle midzone showed a preferred spacing of approximately 35 nm center-to-center between microtubules of opposite polarity. Although this is evidence of specific interaction between antiparallel microtubules, mutant spindles were less ordered than the spindle of wild-type cells. The number of noncore microtubules was significantly higher than that reported for wild-type, and these microtubules did not display a characteristic metaphase configuration. cdc20 spindles showed significantly more cross-bridges between spindle microtubules than were seen in the wild type. The cross-bridge density was highest between antiparallel microtubules. These data suggest that spindle microtubules are stabilized in cdc20 cells and that the CDC20 gene product may be involved in cell cycle processes that promote spindle microtubule disassembly.

Cdc20 Proteins↗
PubMed1997

Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae.

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.

Cell Cycle Proteins↗
PubMed1997

Differential requirements for DNA replication in the activation of mitotic checkpoints in Saccharomyces cerevisiae.

Checkpoints prevent inaccurate chromosome segregation by inhibiting cell division when errors in mitotic processes are encountered. We used a temperature-sensitive mutation, dbf4, to examine the requirement for DNA replication in establishing mitotic checkpoint arrest. We used gamma-irradiation to induce DNA damage and hydroxyurea to limit deoxyribonucleotides in cells deprived of DBF4 function to investigate the requirement for DNA replication in DNA-responsive checkpoints. In the absence of DNA replication, mitosis was not inhibited by these treatments, which normally activate the DNA damage and DNA replication checkpoints. Our results support a model that indicates that the assembly of replication structures is critical for cells to respond to defects in DNA metabolism. We show that activating the spindle checkpoint with nocodazole does not require prior progression through S phase but does require a stable kinetochore.

Antineoplastic Agents↗
PubMed1996

Cotrel-dubousset instrumentation in children using simultaneous motor and somatosensory evoked potential monitoring.

STUDY DESIGN: To record prospectively combined motor- and somatosensory-evoked potentials in children during scoliosis surgery using Cotrel-Dubousset instrumentation, without using special anesthetic or muscle relaxant regimens. OBJECTIVE: To determine the outcome of scoliosis surgery guided by a new technique of monitoring motor- and somatosensory-evoked potentials simultaneously. SUMMARY OF BACKGROUND DATA: Other techniques used to assess cord function generally are limited by special anesthetic requirements or assess only a limited part of the cord or monitor motor function separately from somatosensory function. METHODS: Spinal cord function was monitored using epidural leads to record simultaneously the descending motor volley (by transcranial electrical stimulation) and the ascending somatosensory volley (by tibial nerve stimulation) at two spinal levels. RESULTS: Combined motor- and sensory-evoked potentials were recorded successfully in 138 of 160 children (81%). Changes in evoked potential waveforms were seen in eight patients (5%), but resolved or lessened in response to appropriate measures. Curve correction was satisfactory, and there were no new postoperative deficits or worsening of preexisting deficits in any patient. CONCLUSION: A spinal cord monitoring system is described that is safe, reliable, accurate, and makes it unnecessary to resort to the "wake-up" test.

Adolescent↗
PubMed1996

Variability of motor-evoked potentials recorded during nitrous oxide anesthesia from the tibialis anterior muscle after transcranial electrical stimulation.

When recorded as a compound muscle action potential (CMAP), the motor-evoked potential (MEP) is affected by volatile anesthetics and nitrous oxide. However, MEPs recorded using epidural electrodes in the presence of nitrous oxide are highly reproducible from trial to trial. We wished to establish the reproducibility over time of the CMAP produced by supramaximal transcranial electrical stimulation of the human motor cortex. Cascades of 100 successive CMAPs were recorded from the tibialis anterior muscles of six anesthetized patients undergoing scoliosis surgery, in response to transcranial electrical stimuli of > 500 V. Satisfactory CMAPs could be recorded in the presence of nitrous oxide, but not isoflurane. Latencies and amplitudes were reproducible in repeated sequences of 100 responses. However, amplitude and, to a lesser extent, latency, were highly variable within a sequence. In addition, occasional individual stimuli, although rarely successive ones, failed to evoke a CMAP. CMAPs have a much higher trial-to-trial variability than corticospinal volleys recorded from the epidural space. Using the present methodology it would be difficult to rely on CMAP recordings as an indicator of corticospinal function in the clinical monitoring situation.

Adolescent↗
PubMed1995

Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae.

Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure.

Cell Division↗
PubMed1994

Functional domains of recombinant bactericidal/permeability increasing protein (rBPI23).

The 23-kDa recombinant amino-terminal bactericidal/permeability increasing protein fragment (rBPI23) has all of the antibacterial and antiendotoxin properties of the holoprotein. In the current studies, we have identified multiple active domains within rBPI23 with chemical and proteolytic cleavage fragments and with synthetic overlapping peptides. We also demonstrate a novel, high affinity heparin binding property for rBPI23, in addition to its established bactericidal and lipopolysaccharide binding properties. Cleavage fragments and synthetic, overlapping peptides of rBPI23 were analyzed for inhibition of the lipopolysaccharide-induced Limulus amebocyte lysate reaction, for bactericidal activity, and for heparin binding. Three separate, active domains were identified in amino acid regions 17-45, 65-99, and 142-169. A single synthetic peptide (85-99) was bactericidal. These results indicate that rBPI23 is comprised of three separate functional domains which contribute to the high affinity interaction of rBPI23 with Gram-negative bacteria. The individual activity of each domain and the cooperative interaction among domains provide the basis for developing rBPI23 analogues with increased biologic efficacy.

Amino Acid Sequence↗
PubMed1994

Hybridoma stability.

Hybridoma stability issues include mutations, chromosome losses, and the potential effects of process variables on the yield, quality and homogeneity of the Monoclonal Antibody (MAb) product. MAb production by murine hybridomas is typically unstable in the early stages after fusion but repeated cloning normally produces stable clones. The stability of hybridomas and the consistency of the MAbs produced during extended high density perfusion cultures at Xoma Corporation were evaluated. Cell stability was assessed by recovering cells from the bioreactors at different intervals and comparing their growth and product formation kinetics and yields to those of cells started fresh from the corresponding Manufacturer's Working Cell Banks. Product consistency was evaluated in the crude harvests and in the corresponding purified MAb lots by biochemical and functionality tests including: SDS-PAGE (reducing and non-reducing), IEF, HPLC (size exclusion and cation exchange), peptide mapping, N-terminal sequencing, carbohydrate composition and binding assays. Several murine hybridomas were studied during runs lasting several months and found to be stable by all criteria employed. Such results support the viability of extended hollow fiber perfusion cultures for reproducible production of murine MAbs. Selecting stable clones and understanding the effects of process variables on the quantity and quality of the MAbs are keys to controlling hybridoma stability during the manufacturing process.

Animals↗
PubMed1993

Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae.

We have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated M(r) 90,098 Da. This protein consists of an N-terminal region that shares significant homology with the catalytic domains of several serine- and threonine-specific protein kinases, as well as a large, unique, C-terminal domain of unknown function. Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength. Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.

Amino Acid Sequence↗