Biomedical subjects

R Ratcliff

Publications and source records attributed to R Ratcliff.

At least 37 records · Page 2Linked to original sources

PubMed1997

Chloride secretion in the trachea of null cystic fibrosis mice: the effects of transfection with pTrial10-CFTR2.

1. An improved novel plasmid backbone, pTrial10, has been developed. We have used this vector to deliver the cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR) to cells, both in vitro and in vivo, complexed with cationic liposomes. 2. Human 293 kidney epithelial cells (HEK 293) showed expression of an immunoprecipitable 165 kDa protein corresponding to CFTR when transfected in vitro with pTrial10-CFTR2, but not when the vector pTrial10 was used. 3. HEK 293 cells transfected with pTrial10-CFTR2, but not pTrial10, demonstrated a cAMP-dependent anion conductance, measured by fluorescence microscopy using a halide-sensitive probe, SPQ. 4. The CFTR-dependent, cAMP-sensitive chloride secretory response in murine tracheal epithelium could be measured if the calcium-dependent chloride secretory process was first maximally stimulated with a mixture of the Ca(2+)-ATPase inhibitor, TBHQ, and the calcium ionophore, A23187. With these conditions wild-type and CF-null (transgenic animals in which the cystic fibrosis (CF) gene has been disrupted so that no CFTR is produced) murine tracheas could be distinguished. The difference between the current elicited by forskolin in wild-type and CF tracheas was highly significantly different (P < 0.001), giving a CFTR-dependent current of 11.2 microA cm-2. 5. Transfection of the airways with pTrial10-CFTR2, but not pTrial10, significantly (P < 0.01) increased the CFTR-dependent chloride secretory current in CF tracheas. The degree of correction was greater when intra-tracheal installation rather than nasal insufflation was used to deliver the plasmids.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Cystic fibrosis transmembrane conductance regulator mediates the cyclic adenosine monophosphate-induced fluid secretion but not the inhibition of resorption in mouse gallbladder epithelium.

We have studied the physiological role of the cystic fibrosis (CF) gene product (cystic fibrosis transmembrane conductance regulator [CFTR]) in gallbladder epithelium using a knockout mouse model for CF. We found that normal mouse gallbladder epithelium expresses functional CFTR as shown by reverse-transcription polymerase chain reaction (RT-PCR) analysis and Ussing chamber experiments. Gallbladders from Cftr -/- mice were structurally intact as shown by microscopic and physiological parameters but lacked the cyclic adenosine monophosphate (cAMP)-induced chloride current observed in normal gallbladders. In fluid transport measurements, normal and Cftr -/- gallbladders were equally active in basal resorption. The addition of forskolin, which activates CFTR anion channel activity through the cAMP system, resulted in net fluid secretion in normal gallbladders. In contrast, Cftr -/- gallbladders were unable to secrete fluid while a complete inhibition of resorption by forskolin was observed. We conclude that, in normal mouse gallbladder epithelium, cAMP-induced fluid secretion involves simultaneous inhibition of apical sodium chloride resorption and activation of CFTR. Our data support the hypothesis that gallbladder disease in CF is at least in part caused by a deficient secretory response to the endogenous cAMP-linked hormones VIP and secretin.

Animals↗

PubMed1997

The intrinsic Cl- conductance of mouse kidney cortex brush-border membrane vesicles is not related to CFTR.

Brush-border membrane vesicles (BBMV) were prepared from whole Balb/c mice kidneys by a Mg2+ precipitation technique. The presence of an intrinsic Cl- conductance co-expressed with Na+/glucose cotransport was inferred by the anion dependence of [14C]glucose uptake and overshoot with inward Na(+)-anion gradients. In Na(+)-equilibrated conditions, an inside-negative membrane potential difference (p.d.) produced by an inward Cl- gradient alone was capable of driving intravesicular [14C]glucose accumulation. The apparent anion conductance had a selectivity of Br- = I- = Cl- > F- > > gluconate, was inhibited by 0.5 mM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) but was unaffected by 0.5 mM 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS). BBMV were isolated from mice in which the CFTR gene had been disrupted by a termination mutation (-/-) and compared with normal litter mates (+/+) and heterozygotes (-/+)[18]. [14C]Glucose uptake in NaCl media was significantly greater than glucose uptake in Na gluconate media for all three genotypes measured at 20 s: for homozygous -/- animals [14C]glucose uptake was increased by 2.80 +/- 0.53 fold in Cl- media compared to gluconate media, n = 6; for wild-type +/+, by 2.16 +/- 0.53 fold, n = 8; and for heterozygous +/- animals, by 2.17 +/- 0.45 fold, n = 8. The observation of a Cl-(-)dependent component in BBMV isolated from homozygous -/- mutant animals shows that the chloride conductance in these vesicles cannot be due to CFTR expression.

Animals↗

PubMed1997

Bias in auditory priming.

Priming for previously studied words in an implicit auditory memory task has been interpreted as evidence for a presemantic perceptual representation system that encodes acoustic representations of words (B. A. Church & D. L. Schacter, 1994). In this article, 3 experiments provided evidence that such priming may result instead from a bias to respond with studied words. In forced-choice identification with similar alternative choices, there was no overall improvement in performance due to prior study. Benefits for studied test words were offset by costs for similar but nonstudied test words. Prior study had no effect when forced-choice alternatives were dissimilar. The data are discussed in relation to current models of auditory information processing and a new model (R. Ratcliff & G. McKoon, in press) for priming in visual word identification.

Acoustic Stimulation↗

PubMed1997

A counter model for implicit priming in perceptual word identification.

A model for the identification of briefly presented words is presented. The model accounts for data from naming and forced-choice experiments in which factors such as similarity of alternatives and stimulus presentation time are varied. The model assumes that counts are accumulated in counters that correspond to words and that a word is chosen as a response when the number of counts in its counter exceeds the maximum of the numbers of counts in other counters by a criterial value. Prior exposure to a word causes its counter to attract more counts than it otherwise would, and this yields priming effects. Ten experiments are presented, and the model provides correct predictions for the data. Implications of the model for research in implicit memory are considered.

Analysis of Variance↗

PubMed1997

A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis.

Cystic fibrosis (CF) is a common, serious, inherited disease. The major cause of mortality in CF is lung disease, due to the failure of airway epithelial cells to express a functional product of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A potential treatment for CF lung disease is the expression of CFTR in the airways following gene transfer. We have undertaken a double-blinded, placebo-controlled, clinical study of the transfer of the CFTR cDNA to the nasal epithelium of 12 CF patients. Cationic liposomes complexed with plasmid containing the human CFTR cDNA were administered to eight patients, whilst four patients received placebo. Biopsies of the nasal epithelium taken 7 days after dosing were normal. No significant changes in clinical parameters were observed. Functional expression of CFTR assessed by in vivo nasal potential difference measurements showed transient correction of the CF chloride transport abnormality in two patients (15 days after dosing in one patient). Fluorescence microscopy demonstrated CFTR function ex vivo. In cells from nasal brushings. In total, evidence of functional CFTR gene transfer was obtained in six out of the eight treated patients. These results provide proof of concept for liposome-mediated CF gene transfer.

Adolescent↗

PubMed1997

A second dose of a CFTR cDNA-liposome complex is as effective as the first dose in restoring cAMP-dependent chloride secretion to null CF mice trachea.

Phase I clinical trials have provided encouraging data suggesting that gene transfer could provide a treatment for cystic fibrosis (CF). However, for all the current viral and nonviral vectors used to deliver the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the duration of CFTR expression is limited, necessitating a repeat dosing regimen to provide a long-term treatment. This study was performed to determine whether a second delivery of a CFTR cDNA-liposome complex could result in a similar level of functional CFTR expression observed after a single delivery and to assess whether the deliveries produced adverse inflammatory responses. CFTR functional expression was assessed by short circuit current measurements of tracheas taken from CF null mice (Cftrtm1Cam) treated with a CFTR cDNA-liposome complex in the upper airways. Mice receiving two deliveries of this complex, the second after the response to the first had declined, showed cAMP-stimulated chloride currents which were not significantly different from normal tracheas or tissues assayed after a single dose of the complex. This double treatment was well tolerated with no discernible inflammation of lung tissue.

Animals↗

PubMed1997

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice.

The cystic fibrosis (Cftr and multidrug resistance (Mdr1) genes encode structurally similar proteins which are members of the ABC transporter superfamily. These genes exhibit complementary patterns of expression in vivo, suggesting that the regulation of their expression may be co-ordinated. We have tested this hypothesis in vivo by examining Cftr and Mdr1 expression in cystic fibrosis knockout transgenic mice (Cftr(tm1CAM)). Cftr mRNA expression in Cftr(tm1CAM)/Cftr(tm1CAM) mice was 4-fold reduced in the intestine, as compared with littermate wild-type mice. All other Cftr(tm1CAM)/Cftr(tm1CAM) mouse tissues examined showed similar reductions in Cftr expression. In contrast, we observed a 4-fold increase in Mdr1 mRNA expression in the intestines of neonatal and 3- to 4-week-old Cftr(tm1CAM)/Cftr(tm1CAM) mice, as compared with age-matched +/+ mice, and an intermediate level of Mdr1 mRNA in heterozygous Cftr(tm1CAM) mice. In 10-week-old, Cftr(tm1CAM)/Cftr(tm1CAM) mice and in contrast to the younger mice, Mdr1 mRNA expression was reduced, by 3-fold. The expression of two control genes, Pgk-1 and Mdr2, was similar in all genotypes, suggesting that the changes in Mdr1 mRNA levels observed in the Cftr(tm1CAM)/Cftr(tm1CAM) mice are specific to the loss of Cftr expression and/or function. These data provide further evidence supporting the hypothesis that the regulation Cftr and Mdr1 expression is co-ordinated in vivo, and that this co-ordinate regulation is influenced by temporal factors.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1997

Phenotypic and molecular characterization of three clinical isolates of Mycobacterium interjectum.

Introduction of molecular biology-based technology into an Australian mycobacterial reference laboratory has resulted in the identification of three isolates of Mycobacterium interjectum in the past 12 months. Conventional phenotypic methods failed to identify the species of these isolates, and high-performance liquid chromatography found that only one of the three isolates had a mycolic acid pattern similar to that of the type strain. In contrast, all three isolates were rapidly identified as M. interjectum by 16S rRNA gene sequence analysis. Two isolates were recovered from the lymph nodes of children with cervical lymphadenitis, confirming the pathogenicity of this organism. However, the third isolate was obtained from the sputum of an elderly male with chronic lung disease without evidence of clinical or radiological progression, suggesting that isolation of M. interjectum should not imply disease. With the increasing use of molecular biology-based technology in mycobacterial laboratories, M. interjectum may be recognized more frequently as a pathogen or commensal organism.

Aged↗

PubMed1996

Separating implicit from explicit retrieval processes in perceptual identification.

Light and Kennison (this issue) proposed that bias effects in the forced-choice perceptual identification of words result from a strategy engaged in by subjects to retrieve explicit information about the words. This article enumerates several problems with this proposal and presents new experimental data against it. It is concluded that subjects do not ordinarily employ an explicit retrieval strategy. The data are discussed in the context of the general problem of separating implicit from explicit influences on performance.

Adolescent↗

PubMed1996

Testing models of decision making using confidence ratings in classification.

Classification implies decision making (or response selection) of some kind. Studying the decision process using a traditional signal detection theory analysis is difficult for two reasons: (a) The model makes a strong assumption about the encoding process (normal noise), and (b) the two most popular decision models, optimal and distance-from-criterion models, can mimic each other's predictions about performance level. In this article, the authors show that by analyzing certain distributional properties of confidence ratings, a researcher can determine whether the decision process is optimal, without knowing the form of the encoding distributions. Empirical results are reported for three types of experiments: recognition memory, perceptual discrimination, and perceptual categorization. In each case, the data strongly favored the distance-from-criterion model over the optimal model.

Adult↗

PubMed1996

Bias effects in implicit memory tasks.

A major focus of recent research in memory has been performance on implicit tasks. The phenomenon of most interest has been repetition priming, the effect that prior exposure to a stimulus has on later perception of the stimulus or on a later decision about the stimulus. Picture naming, word identification, and word production in stem- and fragment-completion tasks all show repetition priming effects. The separation of implicit from explicit memory systems provides one account of this data, but a different theoretical view is proposed here: Repetition-priming effects come about because the processes that perform a task are biased products, temporary modifications of the processes, which influence later processing. The aim of this article is to demonstrate the potential of this view for developing new theories and for prompting new empirical questions.

Auditory Perception↗

PubMed1996

Reliability of prosodic cues for resolving syntactic ambiguity.

Although previous research has shown that listeners can use prosody to resolve syntactic ambiguities in spoken sentences, it is not clear whether naive, untrained speakers in experimental situations ordinarily produce the prosodic cues necessary for disambiguating such sentences. In a series of experiments, the authors found that neither professional nor untrained speakers consistently produced such prosodic cues when simply reading ambiguous sentences in a disambiguating discourse context. Speakers who were aware of the ambiguities and were told to intentionally pronounce the sentences with one meaning of the other, however, did produce sufficient prosodic cues for listeners to identify the intended meanings.

Adult↗

PubMed1996

Sentential context effects on lexical decisions with a cross-modal instead of an all-visual procedure.

J. L. Nicol and D. Swinney (1989) reported facilitation in a cross-modal lexical-decision task as evidence that implicit objects of verbs (WH-traces) are reinstated during comprehension. G. McKoon and R. Ratcliff (1994) found the same priming effects in the absence of implicit objects, suggesting that the effects are attributable to some factor other than a syntactic process that would fill in implicit objects. J. L. Nicol, J. D. Fodor, and D. Swinney (1994) questioned the relevance of McKoon and Ratcliff's findings because they were obtained with all-visual rather than cross-modal presentation. In 2 experiments, the authors replicated McKoon and Ratcliff's results using cross-modal lexical decision.

Decision Making↗

PubMed1996

CFTR expression and mucin secretion in cultured mouse gallbladder epithelial cells.

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in humans is frequently associated with progressive liver disease, which appears to result from obstruction of biliary ducts with mucous material. CFTR in the liver is expressed in the biliary epithelium. With the use of a mouse model for cystic fibrosis (CF) we have studied the relationship between CFTR expression and glycoprotein secretion in primary culture of mouse gallbladder epithelial cells (MGBC) MGBC in culture maintain a well-differentiated phenotype as shown by microscopy. The cells produce CFTR mRNA to levels comparable to the intact tissue. With patch-clamp analysis we could frequently observe a linear protein kinase A-regulated Cl- channel that shows all the major characteristics of human CFTR, although its conductance is lower (5 pS compared with 8 pS). MGBC in culture produce and secrete high molecular weight glycoproteins (HMG) in a time-dependent and temperature-sensitive manner. Secretion of HMG was not stimulated significantly by either adenosine 3',5'-cyclic monophosphate (cAMP), Ca2+, or protein kinase C agonists in this system. High concentrations (3 mM) of extracellular ATP stimulated secretion threefold, but low concentrations (0.3 mM) had no effect. Approximately one-third of the HMG produced and secreted consisted of mucin. Cultured MGBC from CFTR-deficient mice produced and secreted mucin to a similar extent as normal cells. We conclude that cultured mouse gallbladder cells are a convenient model to study both CFTR function and mucin secretion. In this system, we found no evidence for a direct link between mucin secretion and CFTR activity, as has been suggested for other cell types.

Animals↗

PubMed1995

The genetic advantage hypothesis in cystic fibrosis heterozygotes: a murine study.

1. The delta F508 mutation of the cystic fibrosis (CF) gene is of high frequency in man (1 in 25) and in homozygotes causes cystic fibrosis. It is suggested that cystic fibrosis heterozygotes withstand secretory diarrhoea better than normal individuals and so are genetically advantaged. This hypothesis has been examined by measuring electrogenic chloride secretion in gut epithelia of normal and heterozygous CF mice. 2. Chloride secretory responses of normal and heterozygous colonic epithelia to forskolin, vasoactive intestinal polypeptide (VIP), isoprenaline, cholera toxin, heat-stable enterotoxin (STa), guanylin, carbachol and lysylbradykinin were examined. No significant differences in responses of tissues of the two genotypes were found. 3. Responses of normal and heterozygous ileal epithelia to forskolin and glucose were investigated. Heterozygous tissues responded as well as normal tissues. 4. Frusemide (furosemide) caused virtually identical inhibition of the chloride secretory responses to forskolin in colonic epithelia of both genotypes. 5. No evidence to support the genetic advantage hypothesis in ileal or colonic epithelia of the null CF mouse has been found, at least for acute responses. If the hypothesis is true then either (a) other non-cystic fibrosis transmembrane conductance regulator (non-CFTR) transport processes are involved, (b) prolonged exposure to secretagogues is required, or (c) delta F508 CFTR is responsible for the protective effect.

Animals↗

PubMed1995

Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice.

Calcium-activated anion secretion is elevated in the pancreatic ductal epithelium of transgenic cf/cf mice which lack the cystic fibrosis transmembrane conductance regulator (CFTR). To elucidate whether this effect is due to increased activity of calcium-activated chloride channels, we have studied the relationship between CFTR and calcium-activated chloride currents in pancreatic duct cells isolated from Cambridge cf/cf mice. CFTR chloride currents activated by cAMP were detected in 59% (29/49) of wild-type cells and in 50% (20/40) of heterozygous cells. However, we could not detect any CFTR currents in the homozygous cf/cf cells (0/25). The maximum CFTR current density measured at a membrane potential of 60 mV was 23.5 +/- 2.8 pA/pF (n = 29) in wild-type cells, and about half that value, i.e. 12.4 +/- 1.6 pA/pF (n = 20) in heterozygotes (P = 0.004). Calcium-activated chloride currents were detected in 73% (24/33) of wild-type, 75% (21/28) of heterozygous and in 58% (7/12) of homozygous cf/cf cells. There was no significant difference between the steady-state calcium-activated current densities in the three genotypic groups; the current measured at 60 mV being 527 +/- 162 pA/pF (n = 24) from wild-type, 316 +/- 35 pA/pF (n = 21) from heterozygote and 419 +/- 83 pA/pF (n = 7) from homozygous cells. Our data suggest that lack of CFTR does not enhance the calcium-activated chloride conductance in murine pancreatic duct cells.

Animals↗

PubMed1995

Process dissociation, single-process theories, and recognition memory.

According to the assumptions of L. L. Jacoby's (1991) process dissociation method, performance in recognition memory is determined by the combination of an unconscious familiarity process and a conscious intentional recollection process. The process dissociation method is used to produce estimates of the contributions of the 2 components to recognition performance. This article investigates whether the method provides the correct estimates of components if performance actually depends on only a single process or on 2 processes different from those assumed by the method. The SAM model (G. Gillund & R. M. Shiffrin, 1984) was used to produce simulated data based on a single process. Variants of SAM with 2 processes and R. C. Atkinson and J. F. Juola's (1973) 2-process model were used to produce data based on 2 processes.

Cognition↗