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T Coleman

Publications and source records attributed to T Coleman.

At least 55 records · Page 3Linked to original sources
PubMed1995

COOH-terminal disruption of lipoprotein lipase in mice is lethal in homozygotes, but heterozygotes have elevated triglycerides and impaired enzyme activity.

The role of the enzyme lipoprotein lipase (LPL) in atherosclerosis is uncertain. To generate an animal model of LPL deficiency, we targeted the LPL gene in embryonic stem cells with a vector designed to disrupt the COOH terminus of the protein and used these cells to generate LPL-deficient mice. Germ line transmission of the disrupted LPL allele was achieved with two chimeric males, and offspring from each of these animals were phenotypically identical. Pups homozygous (-/-) for LPL deficiency died within 48 h of birth with extreme elevations of serum triglycerides (13,327 mg/dl) associated with essentially absent LPL enzyme activity in heart and carcass. Newborn heterozygous (+/-) LPL-deficient pups had lower LPL enzyme activity and higher triglycerides (370 versus 121 mg/dl) than wild type (+/+) littermates. Adult heterozygotes had higher triglycerides than wild type mice with ad libitum feeding (236 mg/dl for +/- versus 88 mg/dl for +/+) and after fasting for 4 h (98 mg/dl for +/- versus 51 for +/+) or 12 h (109 mg/dl for +/- versus 56 mg/dl for +/+). Triglycerides were present as very low density lipoprotein particles and chylomicrons, but high density lipoprotein cholesterol levels were not decreased in +/- animals. Plasma heparin-releasable LPL activity was 43% lower in +/- versus +/+ adult animals. LPL activity, mRNA, and protein were lower in the tissues of +/- versus +/+ mice. Homozygous LPL deficiency caused by disruption of the COOH terminus of the enzyme is lethal in mice. Heterozygous LPL deficiency caused by this mutation is associated with mild to moderate hypertriglyceridemia without affecting static HDL cholesterol levels. Heterozygous LPL-deficient mice could be useful for determining if hypertriglyceridemia, independently or in combination with other discrete defects, influences atherosclerosis.

Animals↗
PubMed1995

Novel centrosomal protein reveals the presence of multiple centrosomes in turkey (Meleagris gallopavo) bnbn binucleated erythrocytes.

The phenotype of the bnbn hemolytic anemia mutation in the domestic turkey is manifested as binucleation specifically in the definitive erythrocyte lineage, most likely as the consequence of anomolous centrosomal activity (Bloom et al., 1970; Searle and Bloom, 1979). Here we have identified in turkey two variants of the novel, centrosomally-associated erythroid-specific protein p23. One variant is Ca(2+)-sensitive and is highly homologous to its chick counterpart (Zhu et al., 1995, accompanying paper). The other, p21 is a truncated form resulting from a 62 amino acid deletion from the 3' end and a 40 amino acid insertion at the 5' end, and appears to lack Ca(2+)-sensitivity. These proteins are localized at the marginal band, centrosomes and nuclear membrane of differentiated erythrocytes. Anti-p23/p21 immunofluorescence revealed the presence of multiple centrosomes in bnbn erythrocytes. We therefore undertook a detailed genetic analysis to determine whether the p21 variant represented the bn mutation. Initial tests of normal BnBn and mutant bnbn individuals suggested that the p23/p21 proteins might be encoded by the Bn/bn genes. However, further genetic tests demonstrated independent segregation for these two genetic loci. Thus, these proteins are encoded by the heretofore undescribed genes, p23/p21, mapping to an autosomal locus in the turkey genome.

Amino Acid Sequence↗
PubMed1995

Human fatty acid synthase mRNA: tissue distribution, genetic mapping, and kinetics of decay after glucose deprivation.

To better understand the accelerated decay of fatty acid synthase (FAS) message that occurs after glucose deprivation (J. Biol. Chem. 1993. 268: 6961-6970), we characterized the 3' terminus of the human message and the kinetics of FAS mRNA decay in HepG2 cells. The FAS gene was localized to human chromosome 17q24-25 and to syntenic distal mouse chromosome 11. Expression of the FAS message in human tissues was ubiquitous with high levels in liver, lung, and intra-abdominal adipose tissue. The 806 nucleotide 3' untranslated region of the human mRNA contained two regions with the instability pentamer AUUUA. Unlike short-lived messages containing AUUUA motifs, FAS mRNA decay after glucose deprivation was not first order, and there were no detectable changes in the poly(A) tail. Glucose deprivation transiently caused FAS message to sediment more rapidly than control message in density gradients. In vivo treatment with different translational inhibitors showed that translation per se was not necessary for FAS mRNA decay; association of polysomes with FAS message protected it from decay. In cell-free decay experiments, FAS mRNA decay was more rapid using components from glucose-deprived than glucose-treated cells. These data suggest that glucose regulates cytoplasmic HepG2 FAS mRNA stability by partitioning the message between a translated pool not subject to degradation and a decay compartment, features reminiscent of regulated stability for other diet-responsive messages.

Amino Acid Sequence↗
PubMed1994

Intravenous pamidronate: infusion rate and safety.

In view of previous animal studies showing that pamidronate (Aredia) can cause renal damage, and human data indicating that pamidronate in doses of 60-90 mg is more effective in the control of tumor-induced hypercalcemia than when given at lower doses, we decided to investigate whether pamidronate 90 mg infused over 60 minutes at weekly intervals had any adverse effects on renal function in patients with bone metastases. Twelve patients, 7 female (all with breast cancer) and 5 male (4 with prostate cancer, 1 with bladder cancer) were entered into the trial. Each patient received weekly intravenous infusions of pamidronate 90 mg in 250 ml normal saline over 60 minutes for 4 weeks. 51Cr-EDTA clearances showed no significant changes in renal function. Urinary N-acetyl-B-D-glucosaminidase/creatinine ratios fluctuated considerably, but no consistent changes were found. No patient with a normal level of urinary beta 2-microglobulin had elevated levels at the end of the trial. Serum creatinine levels did not change significantly, though 1 patient had a corrected serum calcium level of < 2 mmol/L on a single occasion on day 8. No evidence of renal toxicity was detected. However, the possibility that neprohtoxicity would ultimately appear cannot be excluded, and these favourable short-term results cannot be extrapolated to patients with impaired renal function.

Adult↗
PubMed1993

Physiologic concentrations of glucose regulate fatty acid synthase activity in HepG2 cells by mediating fatty acid synthase mRNA stability.

Carbohydrate feeding of animals results in striking increases in hepatic fatty acid synthesis but much of this induction is presumed to be hormone mediated. To clarify the mechanisms responsible for the specific effect of carbohydrate on fatty acid synthesis, the effects of D-glucose on the expression of human fatty acid synthase (FAS) in HepG2 cells cultured in serum-free medium were studied. Northern blots of total RNA from these cells showed a single FAS mRNA band of 9.3 kilobases that was increased 2.7-5.4-fold in the presence of D-glucose. Lactate and citrate but not L-glucose mimicked this effect. Glucose induction of FAS mRNA was time- and concentration-dependent and most of the increase in FAS message was detected within the range of physiologic glucose concentrations. Glucose induction of FAS mRNA, protein synthetic rate, and enzyme activity were similar suggesting that glucose regulates FAS expression at a pretranslational level in this system. Transcription run-off experiments showed that glucose feeding was associated with no change in the FAS transcription initiation rate despite a 5-fold increase in FAS mRNA. FAS mRNA stability as determined by actinomycin D chase was 7-fold greater in the presence of glucose. Differences between FAS mRNA levels in cells grown in the presence versus the absence of glucose were inhibited by cycloheximide but not puromycin, suggesting that glucose regulation of FAS mRNA stability is not dependent on translation. Glucose, at physiologic concentrations and in the absence of hormones, appears to regulate FAS gene expression in HepG2 cells predominantly by mediating FAS mRNA stability.

Base Sequence↗
PubMed1992

A missense (Asp250----Asn) mutation in the lipoprotein lipase gene in two unrelated families with familial lipoprotein lipase deficiency.

We have identified the molecular basis for familial lipoprotein lipase (LPL) deficiency in two unrelated families with the syndrome of familial hyperchylomicronemia. All 10 exons of the LPL gene were amplified from the two probands' genomic DNA by polymerase chain reaction. In family 1 of French descent, direct sequencing of the amplification products revealed that the patient was heterozygous for two missense mutations, Gly188----Glu (in exon 5) and Asp250----Asn (in exon 6). In family 2 of Italian descent, sequencing of multiple amplification products cloned in plasmids indicated that the patient was a compound heterozygote harboring two mutations, Arg243----His and Asp250----Asn, both in exon 6. Studies using polymerase chain reaction, restriction enzyme digestion (the Gly188----Glu mutation disrupts an Ava II site, the Arg243----His mutation, a Hha I site, and the Asp250----Asn mutation, a Taq I site), and allele-specific oligonucleotide hybridization confirmed that the patients were indeed compound heterozygous for the respective mutations. LPL constructs carrying the three mutations were expressed individually in Cos cells. All three mutant LPLs were synthesized and secreted efficiently; one (Asp250----Asn) had minimal (approximately 5%) catalytic activity and the other two were totally inactive. The three mutations occurred in highly conserved regions of the LPL gene. The fact that the newly identified Asp250----Asn mutation produced an almost totally inactive LPL and the location of this residue with respect to the three-dimensional structure of the highly homologous human pancreatic lipase suggest that Asp250 may be involved in a charge interaction with an alpha-helix in the amino terminal region of LPL. The occurrence of this mutation in two unrelated families of different ancestries (French and Italian) indicates either two independent mutational events affecting unrelated individuals or a common shared ancestral allele. Screening for the Asp250----Asn mutation should be included in future genetic epidemiology studies on LPL deficiency and familial combined hyperlipidemia.

Amino Acid Sequence↗
PubMed1991

Molecular cloning, expression, and sequence of the pilin gene from nontypeable Haemophilus influenzae M37.

Nontypeable Haemophilus influenzae M37 adheres to human buccal epithelial cells and exhibits mannose-resistant hemagglutination of human erythrocytes. An isogenic variant of this strain which was deficient in hemagglutination was isolated. A protein with an apparent molecular weight of 22,000 was present in the sodium dodecyl sulfate-polyacrylamide gel profile of sarcosyl-insoluble proteins from the hemagglutination-proficient strain but was absent from the profile of the isogenic hemagglutination-deficient variant. A monoclonal antibody which reacts with the hemagglutination-proficient isolate but not with the hemagglutination-deficient isolate has been characterized. This monoclonal antibody was employed in an affinity column for purification of the protein as well as to screen a genomic library for recombinant clones expressing the gene. Several clones which contained overlapping genomic fragments were identified by reaction with the monoclonal antibody. The gene for the 22-kDa protein was subcloned and sequenced. The gene for the type b pilin from H. influenzae type b strain MinnA was also cloned and sequenced. The DNA sequence of the strain MinnA gene was identical to that reported previously for two other type b strains. The DNA sequence of the strain M37 gene is 77% identical to that of the type b pilin gene, and the derived amino acid sequence is 68% identical to that of the type b pilin.

Amino Acid Sequence↗
PubMed1989

New fluorescence assay for the quantitation of fungi.

Quantitative determination of fungal mass is easily achieved with a new procedure that detects particle epifluorescence. Fungi are detected after exposure to a fluorescent stain (Fungiqual; CIBA-GEIGY Corp., Summit, N.J.) by using a fluorescence particle concentration analyzer. This report describes a simple fluorescence method for quantitation of either yeast or mycelial forms of fungi. The nature of the staining reaction was studied, and a practical application of this procedure for determination of fungal susceptibility to an antifungal agent is presented.

Amphotericin B↗
PubMed1988

Antibody to sigma 32 cross-reacts with DnaK: association of DnaK protein with Escherichia coli RNA polymerase.

A polyclonal antibody to sigma 32, the heat shock sigma factor, has been used to show the presence of low levels of sigma 32 in Escherichia coli RNA polymerase preparations (E sigma 70), which explains the observed in vitro activity of E sigma 70 towards heat shock genes. The sigma 32 antibody cross-reacts with DnaK, and DnaK has been found associated with purified preparations of both E sigma 70 and the heat shock RNA polymerase, E sigma 32.

Bacterial Proteins↗
PubMed1987

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents.

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes.

S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system. The S-30 extracts prepared from cells that have been shifted to 45 degrees C express HS genes in vitro approximately 8 times better than extracts from cells at 33 degrees C. In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% of that seen in extracts from cells at 33 degrees C. These results correlate well with the levels of HS sigma factor and normal sigma factor bound to RNA polymerase. Thus, there was an 8-fold increase in the HS sigma factor and a 60% decrease in the normal sigma factor associated with RNA polymerase at the higher temperature. Part of the increase in the level of the HS sigma factor could be accounted for by a 3-fold increase in the level of HS sigma factor mRNA during heat induction.

DNA-Directed RNA Polymerases↗