Biomedical subjects

D R Gill

Publications and source records attributed to D R Gill.

At least 55 records · Page 3Linked to original sources

PubMed1994

Tamoxifen blocks chloride channels. A possible mechanism for cataract formation.

Tamoxifen is an antiestrogen frequently used in the treatment of breast cancer and is currently being assessed as a prophylactic for those at high risk of developing tumors. We have found that tamoxifen and its derivatives are high-affinity blockers of specific chloride channels. This blockade appears to be independent of the interaction of tamoxifen with the estrogen receptor and therefore reflects an alternative cellular target. One of the clinical side effects of tamoxifen is impaired vision and cataract. Chloride channels in the lens of the eye were shown to be essential for maintaining normal lens hydration and transmittance. These channels were blocked by tamoxifen and, in organ culture, tamoxifen led to lens opacity associated with cataracts at clinically relevant concentrations. These data suggest a molecular mechanism by which tamoxifen can cause cataract formation and have implications for the clinical use of tamoxifen and related antiestrogens.

3T3 Cells↗

PubMed1994

Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling.

P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

3T3 Cells↗

PubMed1994

Effects of beta-hydroxy-beta-methyl butyrate on performance and carcass quality of feedlot steers.

Beta-hydroxy-beta-methyl butyrate (HMB), a compound formed during catabolism of leucine, was fed to 256 crossbred steers as 0 or .03% of diet dry matter. Effects on performance, carcass characteristics, and tissue composition were measured. Groups of 32 steers per diet were slaughtered after 105, 119, 133, and 147 d on feed. The HMB was fed to each group only during the final 82 d they were fed. Averaged across slaughter date, animal performance was not altered by HMB; however, an interaction between HMB and time on feed was detected. Feeding HMB increased (P < .01) daily gain of steers slaughtered at 105 d but decreased (P < .01) daily gain of steers slaughtered at 147 d. Steers fed HMB had numerically higher marbling scores that resulted in a trend toward higher carcass quality grades. Steers receiving HMB tended to have less (P < .08) s.c. fat and fewer steers with yield grades of 4 or greater (1.6 vs 4.7%). Supplementation of HMB to feedlot steers tended to increase (P < .07) the ratio of intramuscular fat to subcutaneous fat. Steers fed HMB had higher (P < .001) blood plasma concentrations of HMB (3.06 vs 1.70 mg/L) and lower (P < .03) blood plasma concentrations of cholesterol (108.4 vs 118.7 mg/dL). Feeding HMB tended to increase (P < .10) lipid content of the longissimus muscles of those steers slaughtered at 105 d.

Adipose Tissue↗

PubMed1993

Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy.

Cystic fibrosis (CF) is a lethal inherited disorder affecting about 1 in 2,000 Caucasians. The major cause of morbidity is permanent lung damage resulting from ion transport abnormalities in airway epithelia that lead to mucus accumulation and bacterial colonization. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cyclic-AMP-regulated chloride channel. Cyclic-AMP-regulated chloride conductances are altered in airway epithelia from CF patients, suggesting that the functional expression of CFTR in the airways of CF patients may be a strategy for treatment. Transgenic mice with a disrupted cftr gene are appropriate for testing gene therapy protocols. Here we report the use of liposomes to deliver a CFTR expression plasmid to epithelia of the airway and to alveoli deep in the lung, leading to the correction of the ion conductance defects found in the trachea of transgenic (cf/cf) mice. These studies illustrate the feasibility of gene therapy for the pulmonary aspects of CF in humans.

Animals↗

PubMed1993

Specific inhibitors distinguish the chloride channel and drug transporter functions associated with the human multidrug resistance P-glycoprotein.

Expression of the human multidrug resistance P-glycoprotein is associated with two activities, active drug transport and a volume-regulated chloride channel. In this study we define four classes of compound, based on their differential effects on these two activities. Class I compounds are substrates transported by P-glycoprotein. They also prevent channel activation when added to the cytoplasmic face of the membrane. Class II compounds include reversers of multidrug resistance such as verapamil. These compounds inhibit drug transport and block the chloride channel when added to the outer face of the membrane. Class III compounds include conventional channel blockers which block the chloride channel but do not influence drug transport. Class IV compounds, for example cyclosporin A, appear to inhibit drug transport but do not affect chloride channel activity. These findings have implications for the relationship between the channel and transporter functions associated with P-glycoprotein expression, and for the development of clinical agents which reverse multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein.

The human multidrug resistance P-glycoprotein is an active transporter that pumps cytotoxic drugs out of cells. Expression of P-glycoprotein is also associated with a volume-activated chloride channel. Here we address the relationship between these two functions. Drug transport requires ATP hydrolysis while, in contrast, ATP binding is sufficient to enable activation of the chloride channel. The chloride channel and drug transport activities of P-glycoprotein appear to reflect two distinct functional states of the protein that can be interconverted by changes in tonicity. Transportable drugs prevent channel activation but have no effect on channel activity once it has been preactivated by hypotonicity. The transport and channel functions of P-glycoprotein have been separated by directed mutations in the nucleotide-binding domains of the protein. These data provide further evidence that P-glycoprotein is bifunctional with both transport and channel activities. Implications for the design of chemotherapeutic drugs and for the function of the related cystic fibrosis gene product, CFTR, are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein.

Expression of P-glycoprotein, the product of the MDR1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). P-glycoprotein (relative molecular mass 170,000) is an ATP-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. It is a member of the ABC (ATP-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major histocompatibility locus, and the product of the cystic fibrosis gene (the cystic fibrosis transmembrane regulator, CFTR). CFTR is located in the apical membranes of many secretory epithelia and is associated with a cyclic AMP-regulated chloride channel. At least two other chloride channels are present in epithelial cells, regulated by cell volume and by intracellular Ca2+, respectively. Because of the structural and sequence similarities between P-glycoprotein and CFTR, and because P-glycoprotein is abundant in many secretory epithelia, we examined whether P-glycoprotein might be associated with one or other of these channels. We report here that expression of P-glycoprotein generates volume-regulated, ATP-dependent, chloride-selective channels, with properties similar to channels characterized previously in epithelial cells.

3T3 Cells↗

PubMed1992

The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Localised mutagenesis of the fts YEX operon: conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients.

After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants. Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX, whereas others were defective in the heat shock regulator gene rpoH. Both missense and amber mutant alleles of these genes were produced. The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site. Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein. This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man. One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients. These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein.

ATP-Binding Cassette Transporters↗

PubMed1992

Evaluation of ultrasound for prediction of carcass fat thickness and longissimus muscle area in feedlot steers.

Four hundred fifty-two yearling steers from two experiments were measured for subcutaneous fat thickness and longissimus muscle area between the 12th and 13th ribs using real-time linear array ultrasound equipment. Ultrasonic predictions were compared to corresponding carcass measurements to determine accuracy of ultrasound measurements. In Exp. 1, 74% of the ultrasonic estimates of fat thickness were within 2.54 mm of carcass values (r = .81) and muscle area was predicted within 6.45 cm2 for 47% of all carcasses (r = .43). Although similar correlation coefficients between ultrasonic and carcass fat thickness were obtained in Exp. 2 (r = .82), estimates were more biased; only 62% of ultrasound estimates were within 2.54 mm of carcass measurements. Improvement in longissimus muscle area estimates was noted in Exp. 2, in which 54% of ultrasonic estimates were within 6.45 cm2 of carcass values (r = .63). The extremes for each trait proved most difficult to predict; fat thickness was underestimated on fatter cattle and muscle area was underpredicted on more heavily muscled steers. Ultrasonic measurements of fat thickness are precise and accurate in determining carcass fat thickness, but muscle area estimates are inconsistent and warrant further investigation.

Adipose Tissue↗

PubMed1992

Effect of days fed, carcass grade traits, and subcutaneous fat removal on postmortem muscle characteristics and beef palatability.

Angus x Hereford steers (n = 48) similar in frame size and in muscle thickness were allotted to eight groups (n = 6) of similar mean live weight for serial slaughter at 28-d intervals (0 to 196 d). Except for d-0 steers, which served as grass-fed controls, all steers were fed a high-concentrate diet during the finishing period. Upon slaughter, one side of each carcass was trimmed of subcutaneous fat in the wholesale rib region. Postmortem longissimus muscle (LM) temperature was monitored for each side during the 24-h chilling period. After quality and yield grade data were collected, rib steaks were removed and aged (7 d) and sensory traits of the steaks were evaluated. Most carcass grade traits increased linearly (P less than .01) with days on feed, whereas most sensory panel variables and marbling increased curvilinearly (P less than .05). Generally, after 56 d on feed, carcasses chilled at slower rates (P less than .05) with increased days fed. Taste panel tenderness, amount of perceived connective tissue, and shear force values peaked at 112 d and were slightly less desirable for cattle fed longer than 112 d (quadratic term, P less than .01). Postmortem muscle temperature at 2.5 h was the chilling time most highly correlated with tenderness values among untrimmed sides. Correlations for shear force with 2.5-h LM temperature, marbling score, days fed, fat thickness, and carcass weight were -.63, -.61, -.56, -.55, and -.53, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1990

Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport.

The ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization. This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export of yeast a-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR). Here we present a tertiary structure model of the ATP-binding cassettes characteristic of this class of transport system, based on similarities between the predicted secondary structures of members of this family and the previously determined structure of adenylate kinase. This model has implications for both the molecular basis of transport and cystic fibrosis and provides a framework for further experimentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Binding protein-dependent transport systems.

Bacterial binding protein-dependent transport systems are the best characterized members of a superfamily of transporters which are structurally, functionally, and evolutionary related to each other. These transporters are not only found in bacteria but also in yeasts, plants, and animals including man, and include both import and export systems. Although any single system is relatively specific, different systems handle very different substrates which can be inorganic ions, amino acids, sugars, large polysaccharides, or even proteins. Some are of considerable medical importance, including Mdr, the protein responsible for multidrug resistance in human tumors, and the product of the cystic fibrosis locus. In this article we review the current state of knowledge on the structure and function of the protein components of these transporters, the mechanism by which transport is mediated, and the role of ATP in the transport process.

Adenosine Triphosphate↗

PubMed1990

The identification of the Escherichia coli ftsY gene product: an unusual protein.

The ftsYEX operon in Escherichia coli encodes three proteins, two of which (FtsE and FtsX) are known to be required for cell division. Although FtsE and FtsX have been identified using SDS-PAGE, the FtsY protein has not. We have used in vitro insertion mutagenesis to identify FtsY as a 92 kD polypeptide in maxicell experiments, although predictions from the DNA sequence estimated FtsY to be 54 kD. Results suggest that this disparity could be due to the unusually high percentage of acidic residues within the protein. Complementation tests indicated the presence of a promoter within ftsY required for expression of ftsE and ftsX. The FtsY protein exhibits sequence homology with the SR alpha protein of eukaryotes which is involved in protein secretion. The essential nature of the ftsY gene was also demonstrated.

Bacterial Proteins↗

PubMed1990

Sequence of the peh gene of Erwinia carotovora: homology between Erwinia and plant enzymes.

Polygalacturonase (Peh) and other pectolytic enzymes play a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have sequenced the peh gene of Erwinia carotovora subsp. carotovora, and identified its product as a precursor of molecular weight 42,639, and a mature protein of molecular weight 42,200. A putative KdgR-binding site was identified in the region 5' to the peh gene. The Peh protein showed significant homology with Peh from tomato. In addition, we have found homologies between pectin methylesterase and pectate lyase from Erwinia and their counterparts in tomato. These homologies are described, and their significance discussed.

Amino Acid Sequence↗

PubMed1990

Efficacy of laidlomycin propionate for increasing rate and efficiency of gain by feedlot cattle.

One thousand twenty steers and heifers were used in six feeding trials to examine the influence of laidlomycin propionate on feedlot performance and to determine the most efficacious dietary concentrations of that ionophore. Cattle were fed diets ranging in energy content from 1.08 to 1.49 Mcal NEg/kg of DM. Laidlomycin propionate improved rate of gain and feed conversion in both steers and heifers. Improvements in performance were not evident when laidlomycin propionate was fed at only 3 mg/kg. However, both average daily gain and feed conversion were improved by laidlomycin propionate within the range of 6 to 12 mg/kg of DM (P less than .001). Feed consumption was not substantially affected by inclusion of laidlomycin propionate in the diet. Improvements in ADG and feed conversion were greater on lower-energy diets than on higher-energy diets, but both these performance characteristics were improved regardless of the type of diet fed. Average daily gain was maximized with laidlomycin propionate at 6 mg/kg, whereas improvements in feed conversion were sustained through 12 mg/kg. Carcasses of cattle fed diets containing 6 to 12 mg/kg of laidlomycin propionate weighed 7.3 kg more (P less than .001) than carcasses of cattle fed the control diets. Yield grade and quality grade were not affected by laidlomycin propionate (P greater than .05).

Animal Feed↗

PubMed1989

Extracellular and periplasmic isoenzymes of pectate lyase from Erwinia carotovora subspecies carotovora belong to different gene families.

Pectate lyase (Pel) plays a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have characterized the four Pel isoenzymes of Erwinia carotovora subspecies carotovora strain SCRI193. In this paper we concentrate on two isoenzymes which have different locations in SCRI193: PLb is periplasmic and PLc is extracellular. Comparison of the gene products and nucleotide sequences of pelB and pelC allowed us to assign them to different gene families. In addition, we have identified a number of conserved amino acid residues that are common to all extracellular Pel isoenzymes.

Amino Acid Sequence↗

PubMed1987

The Escherichia coli cell division proteins FtsY, FtsE and FtsX are inner membrane-associated.

The cell division genes ftsY, ftsE and ftsX form an operon mapping at 76 min on the Escherichia coli chromosome. The protein products of these genes have been identified previously. We have studied the cellular location of the radiolabelled Fts proteins using maxicells and standard fractionation procedures. Previous protein sequence homologies suggested an inner membrane location for FtsE. We have confirmed this predicted location and have shown that FtsY and FtsX are also inner membrane-associated. These results are in agreement with the hypothesis that FtsE may act at the inner membrane, in a "septalsome" complex, by coupling ATP hydrolysis to the process of bacterial cell division.

Bacterial Proteins↗