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P B Davis

Publications and source records attributed to P B Davis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor.

The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in alpha 1-antitrypain (alpha 1-AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human alpha 1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. This conjugate condensed DNA into spheroidal particles 18-25 nm in diameter. When transfected with the SECR-directed complex containing pGL3, Hep G2 cells that express the receptor, but not Hep G2 cells that do not, expressed a peak luciferase activity of 538,731 +/- 144,346 integrated light units/mg protein 4 days after transfection. Free peptide inhibited uptake and expression in a dose-dependent manner. Complexes of DNA condensed with polylysine or LC-sulfo-N-succinimidyl-3-(2-pyridyldithio)propionate-substituted polylysine were ineffective. Transfection with a plasmid encoding human factor IX produced expression in Hep G2 (high) and HuH7 cells that express SECR but not Hep G2 (low) cells that lack the receptor. Fluorescein-labeled C1315 peptide labeled 9-31% of Hep G2 (high), 10-14% of HuH7, and 0.6-3.4% of Hep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed beta-galactosidase. SECR-mediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.

Carcinoma, Hepatocellular↗
PubMed1997

Identifying treatments that halt progression of pulmonary disease in cystic fibrosis.

Rapid progress in cystic fibrosis research affords the possibility of halting the progress of the lung disease. We used data from 215 patients who had sputum cultures negative for Burkholderia cepacia, at least one outpatient pulmonary function test during 1990, and at least one test a year later to estimate the number of subjects and study duration required to demonstrate that a hypothetical treatment reduces the rate of decline of forced expiratory volume in 1 s (FEV1) to zero. Mean rate of decline of FEV1 (percent predicted) was about 2% predicted per year. Variability decreases with increasing time of observation. For a 1-y study, with alpha = 0.05 and beta = 0.20, over 550 patients must complete the study in each group to show that a treatment halts pulmonary decline. For a 2-y study, 86 subjects in each group are required, and for 4 y, 65. Increasing the number of data points used to determine the rate of decline of FEV1 had only small effect on sample size. Use of pulmonary function data collected at regular intervals for research purposes did not alter these conclusions. Higher initial FEV1 was associated with a greater rate of decline, and among patients with initial FEV1 > 60% predicted, younger subjects had a faster decline than did older subjects. Thus, fewer subjects will be required to detect a complete halt in progression of lung disease if the patients are young and have mild pulmonary disease.

Adolescent↗
PubMed1996

Phosphorylation-dependent block of cystic fibrosis transmembrane conductance regulator chloride channel by exogenous R domain protein.

The cystic fibrosis transmembrane conductance regulator (CFTR) constitutes a linear conductance chloride channel, which is regulated by cAMP-dependent protein kinase phosphorylation at multiple sites located in the intracellular regulatory (R) domain. Studies in a lipid bilayer system, reported here, provide evidence for the control of CFTR chloride channel by its R domain. The exogenous R domain protein (encoded by exon 13 plus 85 base pairs of exon 14) interacted specifically with the CFTR molecule and inhibited the chloride conductance in a phosphorylation-dependent manner. Only the unphosphorylated R domain protein blocked the CFTR channel. Such functional interaction suggests that the putative gating particle of the CFTR chloride channel resides in the R domain.

Base Sequence↗
PubMed1996

Rectification of cystic fibrosis transmembrane conductance regulator chloride channel mediated by extracellular divalent cations.

We report here distinct rectification of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel reconstituted in lipid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extracellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEPES), the inward currents (intracellular-->extracellular chloride movement) through a single CFTR channel were approximately 20% larger than the outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-voltage relationship of the channel could be restored through the addition of millimolar concentrations of MgCl2 or CaCl2 to the trans solution. The dose responses for [Mg]zero and [Ca]zero had half-dissociation constants of 152 +/- 72 microM and 172 +/- 40 microM, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid did not alter rectification of the CFTR channel. The nonlinear conductance property of the CFTR channel seemed to be due to negative surface charges on the CFTR protein, because in pure neutral phospholipid bilayers, clear rectification of the channel was also observed when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids on several extracellular loops joining the transmembrane segments, which could constitute the putative binding sites for Ca and Mg.

Buffers↗
PubMed1996

Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.

The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild type CFTR cDNA, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0.142 for wild type (p = 0.073). The data obtained indicate that deleting 30 amino acids from the first intracellular loop of CFTR affects both processing and function of the CFTR chloride channel.

Blotting, Western↗
PubMed1996

Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel exhibits multiple subconductance states. To study the regulation of conductance states of the CFTR channel, we expressed the wild-type CFTR protein in HEK 293 cells, and isolated microsomal membrane vesicles for reconstitution studies in lipid bilayer membranes. A single CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub-open states with conductances of approximately 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on both sides of the channel. In 200 mM KCl, both H and L states could be measured in stable single-channel recordings, whereas M could not. Spontaneous transitions between H and L were slow; it took 4.5 min for L-->H, and 3.2 min for H-->L. These slow conversions among subconductance states of the CFTR channel were affected by extracellular Mg; in the presence of millimolar Mg, the channel remained stable in the H state. Similar phenomena were also observed with endogenous CFTR channels in T84 cells. In high-salt conditions (1.5 M KCl), all three conductance states of the expressed CFTR channel, 12.1 pS, 8.2 pS, and 3.6 pS, became stable and seemed to gate independently from each other. The existence of multiple stable conductance states associated with the CFTR channel suggests two possibilities: either a single CFTR molecule can exist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, and different conductance states are due to different aggregation states of the CFTR protein.

Adenosine Triphosphate↗
PubMed1996

Overexpression of R domain eliminates cAMP-stimulated Cl- secretion in 9/HTEo- cells in culture.

The intracellular hydrophilic region of the cystic fibrosis transmembrane conductance regulator (CFTR), the R domain, has been postulated to be a regulator of the Cl-channel. Under basal conditions R blocks the channel, but when phosphorylated, R undergoes conformational change to open the channel. Overexpression of R in 9/HTEo- cells, a human tracheal epithelial cell line with adenosine 3',5' -cyclic monophosphate (cAMP)-regulated Cl- conductance due to CFTR, caused reduced basal Cl- conductance and elimination of its response to isoproterenol, but ionomycin-stimulated Cl- efflux was preserved. Cells which overexpressed R showed no downregulation of endogenous CFTR mRNA and had normal cAMP production and protein kinase A (PKA) activity, so R did not act at these levels. Although the precise mechanism by which R affects CFTR conductance is undetermined, these cell lines could be useful in separating the cell biological consequences of impaired Cl- transport from those of mutant CFTR per se.

Base Sequence↗
PubMed1996

Cystic fibrosis.

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Cystic Fibrosis↗
PubMed1996

Immunologic responses to gene transfer into mice via the polymeric immunoglobulin receptor.

The respiratory epithelium is the primary target tissue for gene therapy of cystic fibrosis, and several methods of gene transfer permit the introduction of the gene encoding the normal cystic fibrosis transmembrane conductance regulator into cells of the respiratory tract in animals. DNA complexes based on Fab antibodies to secretory component have been used to mediate the delivery and uptake of expression plasmids into the respiratory tract via the polymeric immunoglobulin receptor both in vitro and in vivo. We evaluated the efficacy of gene transfer after several administrations of the DNA complexes, and examined the immunogenicity and toxicity of repetitive administration of anti-secretory component Fab-based complexes. Mice received single or multiple injections of the DNA complexes containing the plasmid pGL2 every 21 days after the initial treatment, and lysates from the lung and liver were assayed for luciferase expression. Luciferase activity was detected in the lungs of mice that received a single injection of the DNA complexes, whereas transgene expression was significantly lower in the mice that received three injections of the DNA complexes (17338 +/- 5469 integrated light units/mg and 3771 +/- 1778 integrated light units/mg, respectively). Serum samples from animals that underwent single or multiple injections were analyzed for a serologic response against the conjugate-DNA complexes by ELISA. No anticomplex antibodies were detected in the mice after a single injection. An escalating antibody response was noted with increasing number of treatments with the conjugate-DNA complexes. This serologic response was directed exclusively against the rabbit-derived, anti-secretory component (anti-SC) Fab antibody, and not against either the plasmid DNA or poly-L-lysine. Single injection of the conjugate-DNA complexes did not result in the consumption of circulating complement. Using direct immunofluorescence, perivascular deposits of immunoglobulin G were found in the liver of animals that received three treatments; no such deposition was detected in the lungs or kidneys. No increase in inflammatory cell infiltrates was observed in tissues after single and repeated injections of the DNA complexes. Thus, we conclude that repeated injections of the anti-SC Fab-based complexes evoked a humoral immune response against the heterologous Fab portion of the complex that was associated with reduced efficiency of gene transfer.

Animals↗
PubMed1995

Intracellular loop between transmembrane segments IV and V of cystic fibrosis transmembrane conductance regulator is involved in regulation of chloride channel conductance state.

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two membrane-spanning domains; each consists of six transmembrane segments joined by three extracellular and two intracellular loops of different length. To examine the role of intracellular loops in CFTR channel function, we studied a deletion mutant of CFTR (delta 19 CFTR) in which 19 amino acids were removed from the intracellular loop joining transmembrane segments IV and V. This mutant protein was expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild-type CFTR cDNA, while cells transfected with the mutant gene expressed only a core-glycosylated form (approximately 140 kDa). The chloride efflux rate (measured by 6-methoxyl-N-(3-sulfopropyl) quinolinium SPQ fluorescence) from cells expressing wild-type CFTR increased 600% in response to forskolin. In contrast, delta 19 CFTR-expressing cells had no significant response to forskolin. Western blotting performed on subcellular membrane fractions showed that delta 19 CFTR was located in the same fractions as delta F508 CFTR, a processing mutant of CFTR. These results suggest that delta 19 CFTR is located in the intracellular membranes, without reaching the cell surface. Upon reconstitution into lipid bilayer membranes, delta 19 CFTR formed a functional Cl- channel with gating properties nearly identical to those of the wild-type CFTR channel. However, delta 19 CFTR channels exhibited frequent transitions to a 6-picosiemens subconductance state, whereas wild-type CFTR channels rarely exist in this subconductance state. These data suggest that the intracellular loop is involved in stabilizing the full conductance state of the CFTR Cl- channel.

Amino Acid Sequence↗
PubMed1995

Effect of high-dose ibuprofen in patients with cystic fibrosis.

BACKGROUND: Since the inflammatory response to chronic infection contributes to lung destruction in patients with cystic fibrosis, we hypothesized that anti-inflammatory therapy might slow the progression of lung disease. METHODS: In a double-blind trial, 85 patients, 5 to 39 years of age, with mild lung disease (forced expiratory volume in one second [FEV1], > or = 60 percent of the predicted value) were randomly assigned to receive ibuprofen or placebo orally twice daily for four years. Doses were adjusted individually to achieve peak plasma concentrations of 50 to 100 micrograms per milliliter. Changes in pulmonary function, the percentage of ideal body weight, the chest-radiograph score, and the frequency of hospitalization were assessed. RESULTS: Patients randomly assigned to ibuprofen had a slower annual rate of change in FEV1 than the patients assigned to placebo (mean [+/- SE] slope, -2.17 +/- 0.57 percent vs. -3.60 +/- 0.55 percent in the placebo group; P = 0.02), and weight (as a percentage of ideal body weight) was better maintained in the former group (P = 0.02). Among the patients who took ibuprofen for four years and had at least a 70 percent rate of compliance, the annual rate of change in FEV1 was even slower (-1.48 +/- 0.69 percent vs. -3.57 +/- 0.65 percent in the placebo group, P = 0.03), and this group of patients also had a significantly slower rate of decline in forced vital capacity, the percentage of ideal body weight, and the chest-radiograph score. There was no significant difference between the ibuprofen and placebo groups in the frequency of hospitalization. One patient was withdrawn from the study because of conjunctivitis, and one because of epistaxis related to ibuprofen. CONCLUSIONS: In patients with cystic fibrosis and mild lung disease, high-dose ibuprofen, taken consistently for four years, significantly slows the progression of the lung disease without serious adverse effects.

Adolescent↗
PubMed1995

Gender differences in cystic fibrosis: Pseudomonas aeruginosa infection.

The median survival age for females with cystic fibrosis (CF) is approximately 3 years younger than for males. We tested whether earlier acquisition of Pseudomonas aeruginosa (PA) by female CF patients or the greater impact of this organism on their lung disease, or both, contribute to their poorer survival. PA infection status, survival, pulmonary function tests, and chest X-ray scores from patients who were followed at our center for at least 2 years with a minimum of three respiratory cultures per year were analyzed (n = 848). The median age of chronic infection with mucoid PA was 1.7 years earlier in females than in males. Patients infected with mucoid PA had poorer survival, chest X-ray scores, and pulmonary function tests than patients who had either no Pseudomonas species or only the nonmucoid phenotype. Acquisition of mucoid PA was associated with an accelerated rate of decline in pulmonary function. However, the rate of change of pulmonary function after mucoid PA infection was similar for males and females. Moreover, even among patients who had only the mucoid form or only the nonmucoid form, males had better percent predicted forced expiratory volume in 1 sec and better survival. Therefore, factors in addition to earlier acquisition of mucoid PA may contribute to the poorer survival of female CF patients.

Adolescent↗
PubMed1995

Heterosexual behaviour and HIV risk in New Zealand: data from a national survey.

The pattern of any future major heterosexual epidemic of acquired immunodeficiency syndrome (AIDS) will depend partly on sexual behaviour and condom use among heterosexuals. This survey was designed to provide information on patterns of sexual behaviour in New Zealand. A national sample aged 18 to 54 was selected using a random method and telephone interviews were administered to 2361 people, using a questionnaire based on the protocol developed by the Global Program on AIDS of the World Health Organization. The reported mean lifetime number of partners increased with age up to 25 to 29 years for women and 30 to 34 years for men, and declined at older ages. Fifteen or more lifetime partners were reported by 17 per cent of men and 4 per cent of women. Multiple partnerships in the previous 12 months were commonest in those aged 20 to 24. In this age group, 32 per cent of men and 20 per cent of women reported two or more partners. Recent condom use for contraception was reported by 23 per cent of men and 19 per cent of women. Use was highest amongst those aged 18 to 24, and decreased sharply with age. The true proportion of the population with many sexual partners may be higher than reported. These data will be useful in modelling approaches to estimate the likelihood of future heterosexual spread of AIDS. The data on lifetime numbers of partners suggest that sexual decisions depend not just on age and sex but also on the era, and thus on changing social values about sexual behaviour.

Adolescent↗
PubMed1995

Dietary changes improve survival of CFTR S489X homozygous mutant mouse.

Over 90% of untreated CFTR S489X homozygous (CF) mutant mice reportedly die of intestinal obstruction by 40 days of age, significantly limiting their usefulness as a model for the human disease. Because the period of highest mortality is during the week after weaning, we hypothesized that providing a low-residue liquid diet would improve survival and growth. When 99 CF mice that survived to 10 days of age were fed Peptamen (Clintec Nutrition), an elemental liquid diet, and housed on corn-cob bedding, 88% of them survived to maturity (50 days). The diet causes only minor histologic and ion transport changes in the intestines of normal mice and does not reduce growth rate or size. CF mice raised on Peptamen continue to display severe pathological changes in the intestine and completely lack a adenosine 3',5'-cyclic monophosphate-inducible chloride current in the cecum. This combination of dietary and bedding changes provides a reliable method for keeping CF mice alive well into adulthood and will be useful for the evaluation of the effect and duration of potential therapies for CF.

Animals↗
PubMed1995

Progression of pulmonary disease after disappearance of Pseudomonas in cystic fibrosis.

Once cystic fibrosis (CF) patients become chronically colonized, eradication of Pseudomonas aeruginosa (PA) is rare. We report five patients, each colonized for at least 6 yr, whose subsequent cultures did not reveal PA or any other gram-negative pathogen for at least 2 yr. Two patients harbored yeast, normal throat flora, and occasional colonies of Aspergillus fumigatus, but no PA, Haemophilus influenzae, or other gram-negative pathogens. In two patients, sputum cultures revealed Staphylococcus aureus. Sputum smear showed no gram-negative organisms in any patient. Antipseudomonal antibody titers in all patients decreased during the noncolonized period. However, titers increased in four other CF patients who had continued PA colonization. Despite disappearance of PA, four of five patients had clinical progression of lung disease with deterioration of FEV1. We conclude that (1) PA, even after it has chronically colonized the airways of CF patients, can occasionally disappear; and (2) lung disease can progress despite the disappearance of PA. Thus, the elimination of a milieu favorable to PA, such as might be anticipated with therapy directed at the basic defect, may not be sufficient to halt the pulmonary disease progression in CF.

Adult↗
PubMed1995

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

Animals↗