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Biomedical subjects

A E Smith

Publications and source records attributed to A E Smith.

At least 19 recordsLinked to original sources
PubMed1992

Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane Cl- channel regulated by cyclic AMP-dependent phosphorylation and by intracellular ATP. Mutations in CFTR cause cystic fibrosis partly through loss of cAMP-regulated Cl- permeability from the plasma membrane of affected epithelia. The most common mutation in cystic fibrosis is deletion of phenylalanine at residue 508 (CFTR delta F508) (ref. 10). Studies on the biosynthesis and localization of CFTR delta F508 indicate that the mutant protein is not processed correctly and, as a result, is not delivered to the plasma membrane. These conclusions are consistent with earlier functional studies which failed to detect cAMP-stimulated Cl- channels in cells expressing CFTR delta F508 (refs 16, 17). Chloride channel activity was detected, however, when CFTR delta F508 was expressed in Xenopus oocytes, Vero cells and Sf9 insect cells. Because oocytes and Sf9 cells are typically maintained at lower temperatures than mammalian cells, and because processing of nascent proteins can be sensitive to temperature, we tested the effect of temperature on the processing of CFTR delta F508. Here we show that the processing of CFTR delta F508 reverts towards that of wild-type as the incubation temperature is reduced. When the processing defect is corrected, cAMP-regulated Cl- channels appear in the plasma membrane. These results reconcile previous contradictory observations and suggest that the mutant most commonly associated with cystic fibrosis is temperature-sensitive.

3T3 Cells↗
PubMed1992

Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers.

Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.

3T3 Cells↗
PubMed1992

Production of cystic fibrosis transmembrane conductance regulator in the milk of transgenic mice.

Here we describe the production of cystic fibrosis transmembrane conductance regulator (CFTR), the product of the gene associated with cystic fibrosis, in the milk of transgenic mice. Mammary specific expression was achieved by placing the CFTR cDNA under the control of the goat beta-casein gene promoter. By fractionation, CFTR was shown to be associated with the membranes that envelop milk fat globules as they are discharged from the apical surface of the mammary epithelia. Since milk fat globules may comprise up to 10% of whole milk, this represents a novel, inexpensive and efficient approach to produce CFTR and possibly other membrane-associated proteins. The availability of large quantities of CFTR could have important implications for the development of new therapies for cystic fibrosis.

Animals↗
PubMed1992

Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.

Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.

Animals↗
PubMed1992

Formaldehyde.

In 1979 formaldehyde was projected into the toxicological limelight when Kerns and his group from the American Chemical Industry Institute of Toxicology (CIIT) released preliminary results of an inhalation study on male and female rats. Those animals exposed to 14.3 p.p.m. for 8 h/day and 5 days/week for up to 2 years showed the development of squamous cell carcinomas in a high proportion (55 per cent) of the animals. Rats similarly exposed to 5.6 p.p.m. were slightly affected with 0.85 per cent producing tumours. The next 10 years became a frenzy of activity by toxicologists, epidemiologists and industry groups, all striving to answer the question: 'Is formaldehyde a human carcinogen?'. This paper will attempt to examine the extent to which the question has been answered. It will consider how the industry responded to the initial shock finding and review the subsequent accumulation of animal and human data. It will also address the regulatory position and compare the steps taken in different countries to control the risk, and again consider how industry has responded to such regulatory forces. This review will necessarily only be able to cover the main issues and major conclusions as the extent of the subject is enormous. There are, however, a number of comprehensive texts on the subject should more information be required.

Animals↗
PubMed1992

The effect of neglecting correlations when propagating uncertainty and estimating the population distribution of risk.

Interest in examining both the uncertainty and variability in environmental health risk assessments has led to increased use of methods for propagating uncertainty. While a variety of approaches have been described, the advent of both powerful personal computers and commercially available simulation software have led to increased use of Monte Carlo simulation. Although most analysts and regulators are encouraged by these developments, some are concerned that Monte Carlo analysis is being applied uncritically. The validity of any analysis is contingent on the validity of the inputs to the analysis. In the propagation of uncertainty or variability, it is essential that the statistical distribution of input variables are properly specified. Furthermore, any dependencies among the input variables must be considered in the analysis. In light of the potential difficulty in specifying dependencies among input variables, it is useful to consider whether there exist rules of thumb as to when correlations can be safely ignored (i.e., when little overall precision is gained by an additional effort to improve upon an estimation of correlation). We make use of well-known error propagation formulas to develop expressions intended to aid the analyst in situations wherein normally and lognormally distributed variables are linearly correlated.

Analysis of Variance↗
PubMed1992

Gene probe analysis of soil microbial populations selected by amendment with 2,4-dichlorophenoxyacetic acid.

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monoxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils.(ABSTRACT TRUNCATED AT 250 WORDS)

2,4-Dichlorophenoxyacetic Acid↗
PubMed1992

Concomitance of psoriasis and atopic dermatitis.

There is a widespread belief that psoriasis (Ps) and atopic dermatitis (AD) are clinically mutually exclusive. A prospective study was undertaken to record the concurrent and/or consecutive coincidence of the two conditions and any shared clinical features. Patients attending a dermatology clinic were systematically examined for the presence of Ps and/or AD. Nine hundred and eighty-three patients were studied--428 with Ps, 224 with AD, 45 with both Ps and AD, and 286 controls. Of AD patients 16.7% had Ps, and 9.5% of Ps patients had AD. In consecutive occurrences, Ps generally followed AD. The ratio of concurrent to consecutive incidences was 3:1. The two diseases are shown not to be mutually exclusive and may coexist in the same individual.

Adolescent↗
PubMed1992

Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.

Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.

Antibodies, Monoclonal↗
PubMed1992

Communication status following laryngectomy: the Iowa experience 1984-1987.

Data regarding treatment and outcome for a consecutive series of 73 total laryngectomy patients were collected from clinical records with a follow-up period of 30 to 78 months postoperatively. Notable findings were that 27% were women; 75% reported hoarseness or a voice change as an early symptom; and 22% reported combined heavy use of both cigarettes and alcohol. Of the 73 patients, 38 (52%) died during the follow-up period; 18 of the 38 died within 1 year postoperatively. Thirty-nine (53%) of the 73 had received a surgical tracheoesophageal fistula (TEF) for voice restoration, as either a primary or a secondary procedure. Of the 39, 75% were using the TEF at last examination, with no failures attributed to sphincter spasm. Of the total group, the primary communication modality was reported to be use of a TEF by 44% and use of an electrolarynx by 50%. Limitations of the study and directions for future investigations are discussed.

Aged↗
PubMed1992

The amino-terminal half of pp59c-fyn contains sequences necessary for formation of a 75 kDa form and also repressive elements absent in pp60c-src.

Members of the src family of tyrosine kinases are composed of amino acid sequences which can be divided into three regions: the unique amino-terminal 80 residues; the next 180 residues of conserved but non-catalytic sequence; and the catalytic carboxy-terminal half of the molecule. To characterize the elements that regulate the catalytic and transforming activities of two members of this family, pp59c-fyn and pp60c-src, we generated six chimeric proteins by interchanging the three regions of the 531F mutant of pp59c-fyn and of the 527F mutant of pp60c-src. Our data indicate that substituting all or part of the amino-terminal 263 residues of pp59c-fyn for those of 527F inhibited the kinase and transforming activities of 527F. Conversely, substituting the amino-terminal half of pp60c-src for that of 531F resulted in an increase in the transforming potential of 531F. These results suggest that the amino-terminal half of pp59c-fyn contains elements which act to suppress the catalytic and transforming activities of the enzyme and that these suppressive elements are either absent or inactive in pp60c-src. These differences argue that the src family of tyrosine kinases are regulated differently in the cell. In vitro translation of some of the chimeras in rabbit reticulocyte lysates generated a 75 kDa protein in addition to the expected 59 kDa product. This 75 kDa species is analogous to the p75 protein previously detected in wild-type pp59c-fyn translation products. Interestingly, formation of p75 required the presence of DNA sequences encoding the unique amino-terminal residues of pp59c-fyn.

Animals↗
PubMed1992

Reimbursement for clinical nutrition services: a 10-year experience.

Charges for nutrition products and services grew more than 800% between 1980 and 1990 at Children's Memorial Medical Center, Chicago, Ill. Three factors contributed to the growth in charges: distinguishing special services from those included in the bed charge, requiring rigorous documentation of services ordered and provided, and building administrative support for the program. Of the charges, 91% were related to nutrition support. The hospital converted 99% of the charges to private payers into actual revenue. The charge program provides the structure, discipline, and data to demonstrate the value of nutrition services for improving patient outcomes and may help sustain financial support even if the health care industry continues to move away from direct payment for services. Charging for nutrition products and services is a useful way for dietitians to document and be compensated for the value of their services in a hospital setting.

Documentation↗
PubMed1991

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.

Adenosine Triphosphate↗
PubMed1991

Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel.

CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in cAMP-responsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR Cl- channel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is sufficient for regulation of Cl- channel activity.

Chloride Channels↗
PubMed1991

Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity.

Amino Acid Sequence↗