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Biomedical subjects

A J Warren

Publications and source records attributed to A J Warren.

At least 37 records · Page 2Linked to original sources
PubMed1995

Substrate-induced inactivation of a crippled beta-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role.

The beta-glucosidase from Agrobacterium sp. catalyzes the hydrolysis of beta-glucosides via a covalent alpha-D-glucopyranosyl-enzyme intermediate involving Glu358. Hydrolysis of 2,4-dinitrophenyl beta-D-glucopyranoside by the low activity Glu358Asp mutant of Agrobacterium beta-glucosidase is accompanied by time-dependent inactivation of the enzyme. Through kinetic studies, labeling, and sequence analysis, inactivation is shown to be a consequence of the occasional (1 time in 1100) attack of Tyr298 on the anomeric center of the substrate, in place of the catalytic nucleophile, with formation of a stable alpha-D-glucopyranosyl tyrosine residue. Tyr298 is conserved throughout family 1 of glycoside hydrolases, an indication of a possible role in catalysis. Results of a kinetic analysis of the Tyr298Phe mutant are consistent with a function of Tyr298 in both orienting the nearby nucleophile Glu358 and stabilizing its deprotonated state in the free enzyme.

Amino Acids↗
PubMed1995

The Hox11 gene is essential for cell survival during spleen development.

The HOX11 homeobox gene was identified via the translocation t(10;14) in T cell leukaemia. To determine the function of this gene in mice, null mutations were made using homologous recombination in ES cells to incorporate lacZ into the hox11 transcription unit. Production of beta-galactosidase from the recombinant hox11 allele in +/- mutants allowed identification of sites of hox11 expression which included the developing spleen. Newborn hox11 -/- mice exhibit asplenia. Spleen formation commences normally at E11.5 in hox11 -/- mutant embryos but the spleen anlage undergoes rapid and complete resorption between E12.5 and E13.5. Dying spleen cells exhibit molecular features of apoptosis, suggesting that programmed cell death is initiated at this stage of organ development in the absence of hox11 protein. Thus hox11 is not required to initiate spleen development but is essential for the survival of splenic precursors during organogenesis. This function for hox11 suggests that enhanced cell survival may result from the t(10;14) which activates HOX11 in T cell leukaemias, further strengthening the association between oncogene-induced cell survival and tumorigenesis.

Animals↗
PubMed1994

The LIM protein RBTN2 and the basic helix-loop-helix protein TAL1 are present in a complex in erythroid cells.

Chromosomal translocations in T-cell acute leukemias can activate genes encoding putative transcription factors such as the LIM proteins RBTN1 and RBTN2 and the DNA-binding basic helix-loop-helix transcription factor TAL1 associated with T-cell acute lymphocytic leukemia. While not expressed in normal T cells, RBTN2 and TAL1 are coexpressed in erythroid cells and are both important for erythroid differentiation. We demonstrate, using anti-RBTN2 and anti-TAL1 antisera, that the LIM protein RBTN2 is not phosphorylated and is complexed with the TAL1 phosphoprotein in the nucleus of erythroid cells. A complex containing both RBTN1 and TAL1 also occurs in a T-cell acute leukemia cell line. Since both RBTN2 and TAL1 are crucial for normal erythropoiesis, these data have important implications for transcription networks therein. Further, since both proteins can be involved in leukemogenesis, these data provide a direct link between proteins activated by chromosomal translocations in T-cell acute leukemia.

Adaptor Proteins, Signal Transducing↗
PubMed1994

The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development.

The LIM domain protein rbtn2 is associated with T cell acute leukemias. We demonstrate that rbtn2 is a nuclear protein expressed in the erythroid lineage in vivo, and using homologous recombination, we show that it is essential for erythroid development in mice. The homozygous rbtn2 null mutation leads to failure of yolk sac erythropoiesis and embryonic lethality around E10.5. Moreover, in vitro differentiation of yolk sac tissue from homozygous mutant mice and sequentially targeted double-mutant ES cells demonstrates a block to erythroid development. This shows a pivotal role for a LIM domain protein in lineage specification during mammalian development and suggests that RBTN2 and GATA-1 are critical at similar stages of erythroid differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed1994

An internal cellulose-binding domain mediates adsorption of an engineered bifunctional xylanase/cellulase.

A chimeric xylanase/endoglucanase (XynCenA) with an internal cellulose-binding domain was constructed by fusing the Bacillus subtilis xyn gene fragment to the 5'-end of the Cellulomonas fimi cenA. A polyhistidine-encoding sequence was also fused to the 5'-end of the xyn gene. The gene fusion was overexpressed in Escherichia coli and the fusion polypeptide purified from the cell extracts using the polyhistidine tail. The hybrid protein behaved like the parental endoglucanase or xylanase when assayed on a number of soluble and insoluble cellulosic substrates or xylans. The presence of two distinct active sites and the internal cellulose-binding domain did not significantly affect the hydrolysis of any of these substrates. However, the fusion protein exhibited a strong affinity for both microcrystalline cellulose (Avicel) and regenerated chitin. Like the parental endoglucanase, bound XynCenA could not be eluted from these polysaccharides with either low or high salt buffer or distilled water. More stringent conditions, such as 1% SDS or 8 M guanidinium hydrochloride, fully desorbed the protein. The fusion protein did not adsorb significantly to insoluble xylan.

Adsorption↗
PubMed1993

Fee structure for investigational drug studies.

The development and implementation of a fee structure for a pharmacy-coordinated investigational drug service is described. A pilot task and time study established specific time and cost elements for investigational drug services provided by the pharmacy department. To fully assess the costs in dispensing investigational drugs, each research study that used the investigational drug service was broken down into five phases of service. Each phase was further categorized by specific tasks or activities, and a time element to perform each activity or task was determined. Since some studies could require more elaborate randomization of patients, more extensive review of protocols, or more individualized dispensing procedures than others, a range of charges was derived: $2800 for standard protocols to $5700 for more complicated studies. An institutional drug therapy newsletter describing the services and costs was distributed to all medical staff members and principal investigators. The development of a fee structure for an investigational drug service coordinated by the pharmacy department has ensured that pharmacy services are adequately reimbursed and has allowed the department to allocate appropriate personnel to provide the services.

Drug Evaluation↗
PubMed1991

Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein.

Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp. The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose. Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8. The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8. Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C. At higher temperatures, the bound enzyme lost activity. The thermal stability of the fusion protein was greatly improved by immobilization. Immobilization did not alter the pH stability. Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme.

Adsorption↗
PubMed1986

Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi.

Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli.

Amino Acid Sequence↗
PubMed1984

Human bubonic plague transmitted by a domestic cat scratch.

Bubonic plague was transmitted to a 10-year-old girl in Oregon by a scratch wound inflicted by a domestic cat. The cat probably was infected by contact with infected wild rodents or their fleas. Yersinia pestis was identified in Diamanus montanus fleas collected from an abandoned burrow near the patient's home. Domestic cats may infect humans with Y pestis by inoculation from a scratch.

Animals↗
PubMed1982

Carcinogenic epoxides of benzo[a]pyrene and cyclopenta[cd]pyrene induce base substitutions via specific transversions.

We have determined the spectrum of base-pair substitution mutations induced in the lacI gene of a uvrB- strain of Escherichia coli by two polycyclic aromatic hydrocarbons--(+/-)7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10 tetrahydrobenzo[a]pyrene (BPDE), and 3,4-epoxycylopenta[cd]pyrene (CPPE). Approximately 10% of all lacI mutations induced by either BPDE or CPPE are nonsense mutations, suggesting that base-pair substitutions are a large fraction of the mutational events induced by these agents in the uvrB- bacteria. Both carcinogens specifically induced the G . C leads to T . A and, to a lesser extent, the A . T leads to T . A transversions. One possible mechanism for transversion induction at G . C sites by BPDE might involve carcinogen binding to the exocyclic amino group of guanine in the template strand followed by a rotation of the modified base around its glycosylic bond from the anti to the syn conformation. This could allow specific pairing of modified bases with an imino tautomer of adenine.

Benzopyrenes↗
PubMed1976

Genetic and morphological study of aggregation in the cellular slime mold Polysphondylium violaceum.

A system for genetic analysis in the cellular slime mold P. violaceum has been developed. Two growth-temperature-sensitive mutants were isolated in a haploid strain and used to select rare diploid heterozygotes arising by spontaneous fusion of the haploid cells. A recessive mutation to cycloheximide resistance in one stain enables selection of segregants, which often appear to be aneuploid.-Aggregation-defective (ag-) mutants having a wide range of phenotypes were isolated in both temperature-sensitive strains after nitrosoguanidine treatment, and complementation tests were performed between pairs of these mutants. Of 380 diploids isolated, 32 showed defective aggregation and were considered to contain 2 noncomplenting ag- mutations. Among noncomplementing mutants interallelic complentation is common. Noncomplementing mutants fall into 4 complementation groups, and those within each complementation group are phenotypically similar. Statistical analysis of the results suggests that the number of complementation units involved in aggregation is about 50.

Alleles↗
PubMed1969

Variations in genetic recombination due to amber mutations in T4D bacteriophage.

Recombination experiments were performed to assess the affect of amber mutations in 12 genes of T4D bacteriophage on genetic recombination. Crosses were performed in various suppressor-containing bacterial hosts to permit the production of progeny phage. Amber mutations in genes 32, 46, and 47 caused decreased recombination, amber mutations in genes 30, 41, 42, 43, 56, 61, and 62 caused increased recombination, whereas mutations in genes 63 and 37 showed no demonstrable effect on recombination.

Coliphages↗
PubMedDate not supplied

Processing of fusion proteins with immobilized factor Xa.

Factor Xa, with a cellulose-binding domain (CBD) fused to the C-terminus of the heavy chain (FXa-CBD), is active in solution and when immobilized on cellulose. A second derivative of factor Xa in which a hexahistidine tail is fused to the C-terminus of the heavy chain (FXa-H6) also retains activity when immobilized, in this case on Ni(2+)-NTA agarose. The stabilities and activities of of FXa-CBD and FXa-H6 immobilized on cellulose and Ni(2+)-NTA agarose, respectively, are similar. Immobilized factor Xa derivatives can be used to remove affinity tags from appropriate fusion proteins without contaminating the desired product with factor Xa.

Affinity Labels↗
PubMedDate not supplied

Synthesis and structural characterization of the N2G-mitomycin C-N2G interstrand cross-link in a model synthetic 23 base pair oligonucleotide DNA duplex.

Mitomycin C (MMC) is a genotoxic cancer chemotherapeutic agent that reacts principally at the N2 position of guanine to form one of two predominant monoadducts, or a G-G interstrand cross-link at CpG sites, or a G-G intrastrand cross-link at GpG sites. Previous studies of MMC adduction have principally used very short duplex oligonucleotides (5-15 bp) or very long native duplex DNAs. We examined the formation and structural features of the MMC CpG interstrand cross-link on a model 23 bp synthetic oligonucleotide duplex having the (upper strand) sequence 5'-ATAAATACGTATTTATTTATAAA-3'. MMC was reacted with the duplex oligonucleotide in the presence of sodium dithionite at ratios of 6 mM dithionite: 1.5 mM MMC:0.03 mM duplex. The yield of cross-link in the reaction was determined to be approximately 4.8% by denaturing gel electrophoresis, which represented approximately 75% of the total bound MMC. The cross-linked DNA was isolated to greater than 97% purity in a single step by high temperature size exclusion column chromatography. Characterization of the purified product confirmed that the complex contained exclusively the N2G-MMC-N2G cross-link at the single central CpG site. CD spectroscopy demonstrated a negative band at approximately 290-320 nm which has previously been shown to be characteristic of the MMC cross-link. The relative intensity of this band compared to those reported for shorter duplexes suggested that the majority of the duplex is in a normal B-DNA helical configuration. Base-specific chemical footprinting techniques also indicated that there were subtle but distinct structural perturbations principally within the central four to six base pairs containing and adjacent to the cross-link.

Base Composition↗