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Biomedical subjects

D J Burke

Publications and source records attributed to D J Burke.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1993

Cloning and genetic characterization of a calcium- and phospholipid-binding protein from Saccharomyces cerevisiae that is homologous to translation elongation factor-1 gamma.

We have isolated a gene (CAM1) from the yeast Saccharomyces cerevisiae that encodes a protein homologous to the translational cofactor elongation factor-1 gamma (EF-1 gamma) first identified in the brine shrimp Artemia salina. The predicted Cam1 amino acid sequence consists of 415 residues that share 32% identity with the Artemia protein, increasing to 72% when conservative substitutions are included. The calculated M(r) of Cam1p (47,092 Da) is in close agreement with that of EF-1 gamma (M(r) = 49,200 Da), and hydropathy plots of each protein exhibit strikingly similar profiles. Disruption of the CAM1 locus yields four viable meiotic progeny, indicating that under normal growth conditions the Cam1 protein is non-essential. Attempts to elicit a translational phenotype have been unsuccessful. Since EF-1 gamma participates in the regulation of a GTP-binding protein (EF-1 alpha), double mutants with cam1 disruptions and various mutant alleles of known GTP-binding proteins were constructed and examined. No evidence was found for an interaction of CAM1 with TEF1, TEF2, SEC4, YPT1, RAS1, RAS2, CDC6, ARF1, ARF2 or CIN4. The possibility that Cam1p may play a redundant role in the regulation of protein synthesis or another GTP-dependent process is discussed.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of a cysteine proteinase from Saccharomyces cerevisiae.

We have isolated a gene from Saccharomyces cerevisiae that encodes a protein homologous to the mammalian cysteine proteinase bleomycin hydrolase. Sequence comparison between the yeast and rabbit proteins indicates an amino acid identity of 41.5% over 277 residues and a similarity of 78.3% when conservative substitutions are included. The apparent mass of the yeast protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 47 kDa, although sequence analysis indicates two potential initiator methionines that suggest calculated masses of either 51 or 55 kDa. The protein is nonessential in yeast as haploid mutants disrupted at several positions along the open reading frame remain viable. Furthermore, these mutants do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients, osmotic strength, or exogenous bleomycin. However, the purified protein does exhibit marked hydrolytic activity toward the substrate arginine 4-methyl-7-coumarylamide (Km = 12.8 microM, Vmax = 2.56 mumol mg-1 h-1), and yeast cells engineered to express this protein at higher levels maintain increased resistance to bleomycin compared to wild-type cells. Because this protein represents the first example of a cysteine proteinase identified in yeast, we have named it Ycp1 (yeast cysteine proteinase).

Amino Acid Sequence↗
PubMed1992

Some effects of isoflurane on I waves of the motor evoked potential.

We have investigated the effects of isoflurane anaesthesia on the motor evoked potential recorded in the extradural space during corrective spinal surgery in 15 patients. Isoflurane was added to a nitrous oxide in oxygen mixture supplemented with fentanyl and a neuromuscular blocking agent. Isoflurane was administered to achieve end-tidal concentrations of 2%, 1% and 0% in all patients, and also of 1.5% and 0.5% in nine patients. Transcranial electrical stimulation of the motor cortex was used to elicit descending volleys in corticospinal axons (the motor evoked potential). With stimuli of 450-750 V and no isoflurane, multiple I waves were always seen following the D wave. In all patients the number of I waves decreased and individual I waves became smaller in amplitude the greater the isoflurane concentration, but there were only minor changes in the D wave. The greatest depressant effect on I waves occurred at an end-tidal concentration of 0.5%. Given that I waves are an index of synaptic transmission, anaesthetic-induced changes in I waves may provide a useful model for the neuronal events underlying anaesthesia-induced unconsciousness.

Adolescent↗
PubMed1992

A delay in the Saccharomyces cerevisiae cell cycle that is induced by a dicentric chromosome and dependent upon mitotic checkpoints.

Dicentric chromosomes are genetically unstable and depress the rate of cell division in Saccharomyces cerevisiae. We have characterized the effects of a conditionally dicentric chromosome on the cell division cycle by using microscopy, flow cytometry, and an assay for histone H1 kinase activity. Activating the dicentric chromosome induced a delay in the cell cycle after DNA replication and before anaphase. The delay occurred in the absence of RAD9, a gene required to arrest cell division in response to DNA damage. The rate of dicentric chromosome loss, however, was elevated in the rad9 mutant. A mutation in BUB2, a gene required for arrest of cell division in response to loss of microtubule function, diminished the delay. Both RAD9 and BUB2 appear to be involved in the cellular response to a dicentric chromosome, since the conditionally dicentric rad9 bub2 double mutant was highly inviable. We conclude that a dicentric chromosome results in chromosome breakage and spindle aberrations prior to nuclear division that normally activate mitotic checkpoints, thereby delaying the onset of anaphase.

Cell Cycle↗
PubMed1991

Monitoring spinal cord function during scoliosis surgery with Cotrel-Dubousset instrumentation.

Cotrel-Dubousset instrumentation has been a major advance in spinal surgery but, at least theoretically, it carries a higher risk of spinal cord damage. This paper describes our experience in monitoring the function of either ascending sensory pathways or both ascending sensory and descending motor pathways in the spinal cord during such procedures. Seventy-nine juvenile and adult patients are presented in whom either somatosensory recording (n = 39) or simultaneous corticospinal and somatosensory monitoring (n = 40) was attempted by means of epidural electrodes during corrective surgery with Cotrel-Dubousset instrumentation. Abnormal evoked responses were seen in two cases but these resolved in both before the operation was completed. No new neurological deficits occurred nor were any pre-existing deficits increased.

Adolescent↗
PubMed1991

Random segregation of chromatids at mitosis in Saccharomyces cerevisiae.

Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.

Antibodies, Monoclonal↗
PubMed1991

The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes.

Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation. Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication. Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle. Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence. The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies. cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis. This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules. Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.

Amino Acid Sequence↗
PubMed1991

Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.

Cell Cycle↗
PubMed1989

Glutamine-supplemented total parenteral nutrition improves gut immune function.

Glutamine has been demonstrated to be an important source of fuel for the gut. The purpose of this study was to evaluate the effect of glutamine-supplemented hyperalimentation on gut immune function. Thirty-six female Fischer rats were randomized into three groups: group 1 (chow) was fed rat chow and water ad libitum, group 2 (total parenteral nutrition) received a standard hyperalimentation formula, and group 3 (total parenteral nutrition-glutamine) received a hyperalimentation solution that contained 2% glutamine. Animals were maintained on their respective diets for 2 weeks and then killed. Mesenteric lymph nodes were harvested for culture, bile was assayed for secretory IgA, and bowel was excised to assay bacterial adherence. Results indicated that glutamine-supplemented total parenteral nutrition protects against bacterial translocation from the gut seen with standard formulas. This effect may be mediated by the secretory IgA immune system.

Animals↗
PubMed1988

Genomic organization of major sperm protein genes and pseudogenes in the nematode Caenorhabditis elegans.

The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3' untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3' untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.

Animals↗
PubMed1986

Rapid cation-exchange chromatography of hemoglobins and other proteins.

A new, weak cation exchanger (MA7C) is useful for the very rapid resolution of protein mixtures. MA7C columns (30 X 4.6 mm I.D.) are packed with non-porous spherical particles (7-micron in diameter) having carboxylic acid ion-exchange groups. These columns can readily separate human hemoglobin A1c from other hemoglobin species in 7 min. Quantitative results agree well with those of accepted techniques. Other separations are possible with flow-rates up to 5 ml/min. Chromatography on MA7C columns is characterized by very narrow bandwidths, even at high flow-rates.

Buffers↗
PubMed1986

Rapid protein profiling with a novel anion-exchange material.

A new anion-exchange material has been developed which allows very rapid resolution of protein mixtures. The Microanalyzer MA7P matrix consists of small (7 micron), spherical, non-porous, polymethacrylate beads with polyethyleneimine (PEI) covalently coupled to the surface. When packed 30 X 4.6 mm I.D. columns, this matrix is particularly well suited for applications in which 1-300 micrograms of a protein mixture must be resolved in a minimum of time. Recoveries of injected proteins are usually quantitative, even when the amounts of individual proteins are in the submicrogram range. Chromatography on Microanalyzer MA7P columns is characterized by very narrow bandwidths, even at relatively high flow-rates. This is due to the combined effects of short column length, high selectivity, and the lack of velocity-dependent bandbroadening attributable to diffusion into and out of pores. These columns have no discernable gel filtration effects in the molecular weight range from 10(3) to 10(6) daltons. Columns are very rapidly equilibrated with new solvents, further reducing cycle-to-cycle times.

Animals↗
PubMed1985

Interactions of aminoacyl-tRNA synthetases in high-molecular-weight multienzyme complexes from rat liver.

The functional interaction of Arg-, Ile-, Leu-, Lys- and Met-tRNA synthetases occurring within the same rat liver multienzyme complex are investigated by examining the enzymes catalytic activities and inactivation kinetics. The Michaelis constants for amino acids, ATP and tRNAs of the dissociated aminoacyl-tRNA synthetases are not significantly different from those of the high-Mr multienzyme complex, except in a few cases where the Km values of the dissociated enzymes are higher than those of the high-Mr form. The maximal aminoacylation velocities of the individual aminoacyl-tRNA synthetases are not affected by the presence of simultaneous aminoacylation by another synthetase occurring within the same multienzyme complex. Site-specific oxidative modification by ascorbate and nonspecific thermal inactivation of synthetases in the purified rat liver 18 S synthetase complex are examined. Lys- and Arg-tRNA synthetases show remarkably parallel time-courses in both inactivation processes. Leu- and Met-tRNA synthetases also show parallel kinetics in thermal inactivation and possibly oxidative inactivation. Ile-tRNA synthetase shows little inactivation in either process. The oxidative inactivation of Lys- and Arg-tRNA synthetases can be reversed by addition of dithiothreitol. These results suggest that synthetases within the same high-Mr complex catalyze aminoacylation reactions independently; however, the stabilities of some of the synthetases in the multienzyme complex are coupled. In particular, the stability of Arg-tRNA synthetase depends appreciably on its association with fully active Lys-tRNA synthetase.

Amino Acids↗
PubMed1985

Functional analysis of fractionated Drosophila Kc cell tRNA gene transcription components.

We have previously described the partial purification of RNA polymerase III transcription components from Drosophila Kc cell extracts (Burke, D. J., Schaack, J., Sharp, S., and Söll, D. (1983) J. Biol. Chem. 258, 15224-15231). Here we report further physical and functional analysis of the two transcription-required fractions designated as factors B and C. Using stable complex formation-competition experiments with ordered addition of these transcription components, we have shown that factor C associates rapidly with the tRNA gene, but will not form a stable complex with the DNA in the absence of factor B. Reconstitution experiments of the individual Drosophila Kc cell factors with those from human HeLa cells demonstrated that the HeLa factor B, combined with Kc factor C, can form active transcription complexes. These now exhibit a property of the Drosophila system, a strong dependence on sequences in the 5'-flanking regions of tRNA genes. However, the Kc factor B (which has an apparent molecular weight of 260,000 as judged by gel filtration analysis) is not compatible with the HeLa factor C. These results, together with the isolation of a large complex containing all necessary transcription components, emphasize the importance of the interaction of these components for both stable complex formation and transcription initiation.

Animals↗
PubMed1985

Structure and transcription of eukaryotic tRNA genes.

The availability of cloned tRNA genes and a variety of eukaryotic in vitro transcription systems allowed rapid progress during the past few years in the characterization of signals in the DNA-controlling gene transcription and in the processing of the precurser RNAs formed. This will be the subject matter discussed in this review.

Animals↗