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B L Kelly

Publications and source records attributed to B L Kelly.

At least 19 recordsLinked to original sources
PubMed2000

Coordinate regulation of the IL-4, IL-13, and IL-5 cytokine cluster in Th2 clones revealed by allelic expression patterns.

The cytokines IL-4, IL-13, and IL-5 are markers for the Th2 subset of effector T cells and are often expressed together. These cytokine genes are organized within 140 kb of orthologous DNA in both mouse and human. Using IL-4-expressing CD4+ T cell clones derived from F1 mice, we identified allelic polymorphisms for each of these cytokines and assessed the parental identity of the cytokine mRNAs. Both monoallelic and biallelic expression occurred for each gene and for an additional gene, IL-3, that lies with GM-CSF over 450 kb telomeric on the same chromosome. When coexpressed in T cell clones, IL-4 was expressed from the same allele as IL-13 or IL-5 in 81% of instances. In contrast, there was only 52% concordance of these three cytokines at the allelic level among clones that expressed IL-3. Independent expression of the cytokine alleles occurs commonly in T cells, but the clustered locus encompassing IL-4, IL-13, and IL-5 is subject to coordinate regulation.

Alleles↗
PubMed1998

Differentially expressed Leishmania major gp63 genes encode cell surface leishmanolysin with distinct signals for glycosylphosphatidylinositol attachment.

The Leishmania cell surface metalloproteinase, leishmanolysin or GP63, is expressed in all stages of Leishmania major. Initial studies reported that in L. major the gp63 genes were arranged as five homologous, tandemly repeated genes (gp63 genes 1-5) and a sixth, less conserved gp63 gene located 8 kb downstream of gp63 gene 5. This study compared the sequences of L. major gp63 gene 1 and gp63 gene 6 and identified a seventh L. major gp63 gene located downstream from gp63 gene 6. The L. major gp63 genes exhibited stage-specific differences in their expression: gp63 genes 1-5 were expressed in promastigotes only, gp63 gene 6 was expressed in promastigotes and amastigotes, while gp63 gene 7 was expressed predominantly in stationary phase promastigotes and in amastigotes. Analysis of the predicted protein sequence of gp63 gene 6 (GP63-6) and gp63 gene 1 (GP63-1) showed that these two proteins were homologous in terms of overall predicted domain structure. L. major GP63-1 has been reported to contain a glycosylphosphatidylinositol (GPI) membrane anchor while sequence analysis predicted that GP63-6 contained a different hydrophobic C-terminus that may act as a transmembrane region. Transfection studies using L. major gp63 gene 1 and gp63 gene 6 expressed in L. donovani promastigotes showed that GP63-6 was expressed at the cell surface and that the distinct GP63-6 C-terminus was capable of mediating GPI anchor attachment.

Amino Acid Sequence↗
PubMed1998

Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein.

Considerable advances have been made in characterizing the cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through the cell cycle, but there has been relatively lesser success in identifying the specific biochemical pathways and cell cycle events that are directly under CDK control. To identify physiologically significant CDK substrates we generated mutations in cyclin E that altered the ability of the cyclin to direct the cyclin-CDK holoenzyme to specific in vivo substrates. We show that one of these mutations defines a domain in cyclin E necessary for phosphorylation of the retinoblastoma protein (Rb). These observations confirm the idea that cyclins contribute to substrate recognition by cyclin-CDK complexes, demonstrate the utility of targeting mutants in the identification of essential cyclin-CDK substrates, and put cyclin E squarely into the family of proteins designed to regulate Rb.

3T3 Cells↗
PubMed1998

Purification and characterization of phosphatidylglycerolphosphate synthase from Schizosaccharomyces pombe.

The enzyme CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase (phosphatidylglycerolphosphate synthase; PGPS4; EC 2.7.8.5) is located in the mitochondrial inner membrane and catalyzes the committed step in the cardiolipin branch of phospholipid synthesis. Previous studies revealed that PGPS is the most highly regulated enzyme in cardiolipin biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. In this work, we report the purification to homogeneity of PGPS from S. pombe. The enzyme was solubilized from the mitochondrial membrane of S. pombe with Triton X-100. The solubilized enzyme, together with the associated detergent and intrinsic lipids, had a molecular mass of 120 kDa, as determined by gel filtration. The enzyme was further purified using salt-induced phase separation, gel filtration, and ionic exchange, hydroxylapatite, and affinity chromatographies. The procedure yielded a homogeneous protein preparation, evidenced by both SDS-polyacrylamide gel electrophoresis (PAGE) and agarose isoelectric focusing under nondenaturing conditions. The purified enzyme had an apparent molecular mass of 60 kDa as determined by SDS-PAGE. The enzyme showed a strong dependence on lipid cofactors for activity in vitro. While both phosphatidic acid and CDP-diacylglycerol appeared to be activators, the most significant activation was observed with cardiolipin. The possible physiological significance of the lipid cofactor effect is discussed. This is the first purification of a eucaryotic PGPS enzyme to date, and the first purification of a phospholipid biosynthetic enzyme from S. pombe.

Cations, Divalent↗
PubMed1995

Chromosomal organisation of a repeated gene cluster expressed in mammalian stages of Leishmania.

The genomic organisation of a large Leishmania gene cluster, expressed predominantly in intracellular, infective parasite stages, has been determined. Using cosmid cloning, parasite DNA fingerprinting, partial digestion and mapping with 'end-specific' probes, the Lm cDNA2 gene array has been localised to a 55-kb ClaI fragment within the L. major genome. Six tandemly linked gene copies are transcribed to produce an abundant 6-kb transcript; the seventh and last copy of the cluster is truncated at its 3' end. It is likely that these genes encode one or more proteins specific to infective stages of the parasite life cycle.

Animals↗
PubMed1994

Expression in yeast of an Escherichia coli gene encoding a phospholipid biosynthetic enzyme.

Cardiolipin (CL) is a structurally unique phospholipid having important functional roles in both prokaryotic and eukaryotic cells. The genes encoding CL biosynthetic enzymes have been identified and extensively studied in Escherichia coli, and manipulation of CL biosynthesis in this organism has elucidated a great deal about CL function in prokaryotes. In contrast, little is known about CL biosynthesis or its regulation in eukaryotic cells. We sought to determine whether we could utilize E. coli genes to manipulate expression of CL biosynthetic enzymes and CL content in yeast. The E. coli pgsA gene encodes phosphatidylglycerophosphate synthase (PGPS), catalyzing the first step in the CL biosynthetic pathway. We constructed plasmids with pgsA under the control of the yeast CUP1 promoter. Extracts of Saccharomyces cerevisiae cells transformed with this plasmid contained high levels of E. coli PGPS activity. However, when compared to cells transformed with a control plasmid, pgsA-transformed cells did not exhibit differences in phospholipid composition. The most likely explanation is that the in vitro activity of the E. coli pgsA product is not indicative of its activity in vivo, due to mislocalization of the enzyme and/or inaccessibility of the enzyme to the substrates. To our knowledge, this is the first demonstration of expression of a bacterial phospholipid biosynthetic enzyme in yeast.

Bacterial Proteins↗
PubMed1991

Phosphatidylglycerolphosphate synthase expression in Schizosaccharomyces pombe is regulated by the phospholipid precursors inositol and choline.

The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the committed step in the cardiolipin biosynthetic pathway. To study the regulation of PGPS in Schizosaccharomyces pombe, we characterized the enzyme biochemically. Maximum activity occurred in the presence of 6 mM Triton X-100 at pH 7.5. The apparent Km values for CDP-diacylglycerol and glycerol 3-phosphate were 130 and 26 microM, respectively. Optimal activity was at 35 degrees C, and enzyme activity was labile above 40 degrees C. Thioreactive agents were inhibitory to PGPS activity. To determine whether S. pombe PGPS is regulated by phospholipid precursors, we examined the time-dependent expression of PGPS upon inositol and choline starvation. Starvation for inositol resulted in a threefold increase in PGPS expression in wild-type cells. In cho1 and cho2 mutants, which are blocked in phosphatidylcholine synthesis, starvation for choline resulted in transient derepression of PGPS expression. In choline auxotrophs starved for inositol, PGPS was derepressed 2.5- to 3-fold in the presence of choline and less or not at all in the absence of choline. This is the first description of PGPS regulation in S. pombe and the first demonstration of inositol-mediated regulation in the inositol-requiring yeast species.

Choline↗
PubMed1990

Characterization and regulation of phosphatidylglycerolphosphate phosphatase in Saccharomyces cerevisiae.

Phosphatidylglycerophosphatase (EC 3.1.3.27) activity was characterized in mitochondrial extracts from Saccharomyces cerevisiae. The enzyme has a pH optimum of 5.5. Maximum activity occurs in the presence of Triton X-100 (5 mM) and cobalt or magnesium ions (5 mM). The apparent Km for PGP is 14.6 microM. The temperature optimum is between 50 degrees C and 60 degrees C. The enzyme is labile above 50 degrees C. The presence of inositol in growth media results in a slight but reproducible increase in PGPase activity in mitochondrial extracts from glucose-grown cells but not glycerol-grown cells. The inositol effect is not seen in crude cell extracts. Carbon source does not affect PGPase activity in mitochondrial extracts or in crude cell extracts.

Cobalt↗
PubMed1986

Antibiotic susceptibilities of swine Salmonella isolants from 1979 to 1983.

Two hundred seventy-seven Salmonella (27 serotypes) were isolated from Kansas swine over a 5-year period (1979 to 1983). Salmonella cholerae-suis was the predominant isolant from all tissues and made up 94.4% of the Salmonella isolants from the lungs. The percentage of Salmonella isolants susceptible to various antimicrobial agents was determined. The percentage of Salmonella susceptible to carbadox decreased from 1980 to 1983.

Animals↗
PubMed1982

Using enzyme-linked immunosorbent assay to detect Escherichia coli K88 pili antigens from clinical isolates.

Colonization of the small intestine is a prerequisite for enterotoxigenic Escherichia coli (ETEC) to cause diarrheal disease. Colonization is dependent on the capability of ETEC to adhere to the villous epithelium of the small intestine. This adherence attribute is conferred by pili structures produced by ETEC. The present study compares the efficiencies of the standard agglutination test, Y-1 mouse adrenal cell test, and infant-mouse gastric test with the efficiency of the enzyme-linked immunosorbent assay (ELISA) for the detection of the K88 pilus antigen and enterotoxin-producing E coli. The ELISA, a double antibody sandwich assay utilizing specific anti-K88 pilus antiserum, was used. Identification of isolates from clinical samples was accomplished on suspensions of bacteria. The sensitivity of the assay was in the nanogram per milliliter range, as determined by measuring purified pili. Results could be determined visually, but quantitative results indicated a positive optical density to negative optical density rate of 1.9 to greater than or equal to 3.0 on samples submitted to a clinical laboratory. The development of this assay indicates the application of such an ELISA for rapid identification of ETEC possessing K88 pili.

Agglutination Tests↗