Search PubMed⌕ Search

Biomedical subjects

D M Clarke

Publications and source records attributed to D M Clarke.

At least 55 records · Page 3Linked to original sources
PubMed1997

Disease-associated mutations in cytoplasmic loops 1 and 2 of cystic fibrosis transmembrane conductance regulator impede processing or opening of the channel.

Since little is known about the contribution to function of the N-terminal cytoplasmic loops (CL1, residues 139-194; CL2, residues 242-307) of cystic fibrosis transmembrane conductance regulator (CFTR), all nine point mutations identified in CLs 1 and 2 from patients with cystic fibrosis were reconstructed in the expression vector pcDNA3-CFTR and expressed transiently in COS-1 and HEK-293 cells and stably in Chinese hamster ovary (CHO) cells. Four amino acid substitutions retarded production of mature, fully glycosylated CFTR, suggesting that misprocessing of the channel causes the disease symptoms in the affected patients. Protein maturation could not be promoted by cell culture conditions of reduced temperature (26 degrees C). When properly processed mutants were evaluated for functional defects by the iodide efflux method, the G178R- and E193K-CFTR-expressing cell lines showed impaired anion translocation activities. Patch-clamp studies of single channels revealed that E193K variants had a significantly decreased open probability, which resulted from an increase in the mean closed time of the channels. This contrasted with a previous study of disease-associated point mutations in CL3 that mainly affected the mean open time. None of the maturation-competent CL 1 and 2 mutants had altered conductance. Thus, the N-terminal CLs appear not to contribute to the anion translocation pathway of CFTR; rather, mutations in CL1 can impede transition to the open state. Interestingly, the ability of the non-hydrolyzable ATP analogue adenylyl imidodiphosphate (AMP-PNP) to lock the channel into open bursts was abolished by the I148T and G178R amino acid substitutions.

Adenylyl Imidodiphosphate↗
PubMed1997

Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12.

Transmembrane segments (TM) 6 and 12 are directly connected to the ATP-binding domain in each homologous half of P-glycoprotein and are postulated to be important for drug-protein interactions. Cysteines introduced into TM6 (L332C, F343C, G346C, and P350C) were oxidatively cross-linked to cysteines introduced into TM12 (L975C, M986C, G989C, and S993C, respectively). The pattern of cross-linking was consistent with a left-handed coiled coil arrangement of the two helices. To detect conformational changes between the helices during drug-stimulated ATPase activity, we tested the effects of substrates and ATP on cross-linking. Cyclosporin A, verapamil, vinblastine, and colchicine inhibited cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C. By contrast, ATP promoted cross-linking between only L332C/L975C. Enhanced cross-linking between L332C/L975C was due to ATP hydrolysis, since cross-linked product was not observed in the presence of ATP and vanadate, ADP, ADP and vanadate, or AMP-PNP. Cross-linking between P350C/S993C inhibited verapamil-stimulated ATPase activity by about 75%. Drug-stimulated ATPase activity, however, was fully restored in the presence of dithiothreitol. These results show that TM6 and TM12 undergo different conformational changes upon drug binding or during ATP hydrolysis, and that movement between these two helices is essential for drug-stimulated ATPase activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Accurate confidence intervals for measures of test performance.

The use of confidence intervals to estimate population parameters is briefly reviewed. Exact binomial confidence intervals can be calculated through the use of tables or statistical software packages. As an alternative, a microcomputer program to calculate sensitivity and specificity, point estimates and binomial confidence intervals for false-negative and -positive rate, positive and negative predictive power, prevalence of cases and non-cases, correct classification rate, and misclassification rate has also been developed. Characteristics of the computer program, 'AccuCon', which is available from the authors, are described.

Confidence Intervals↗
PubMed1997

Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.

There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein.

The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large superfamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bidirectional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.

Catalysis↗
PubMed1996

Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates.

Each homologous half of P-glycoprotein consists of a transmembrane domain with six potential transmembrane segments and an ATP-binding domain. Labeling studies with photoactive drug analogs show that labeling occurs within or close to predicted transmembrane segments (TM) 6 (residues 331-351) and TM12 (residues 974-994). To test if these segments are in near-proximity we generated 42 different P-glycoprotein mutants in which we re-introduced a pair of cysteine residues into a Cys-less P-glycoprotein, one within TM6 (residues 332-338) and one within TM12 (residues 975-980) and assayed for cross-linking between the cysteines. All the mutants retained verapamil-stimulated ATPase activity. We found that only the mutant containing Cys-332 and Cys-975 was cross-linked in the presence of oxidant as judged by its decreased mobility on SDS gels. Similar results were obtained when the same mutations were introduced into Cys-less NH2-terminal and COOH-terminal half-molecules of P-glycoprotein followed by coexpression and treatment with oxidant. Cross-linking between Cys-332 and Cys-975, however, was inhibited by verapamil or vinblastine but not by colchicine. These results suggest that residues Cys-332 and Cys-975, which occupy equivalent positions when TM6 and TM12 are aligned, are close to each other in the tertiary structure of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

The minimum functional unit of human P-glycoprotein appears to be a monomer.

Several studies have demonstrated the presence of oligomers of P-glycoprotein in multidrug-resistant cells. The minimum functional unit of P-glycoprotein, however, is not known. In order to determine whether the functional unit is an oligomer, we tested for associations between P-glycoproteins containing either a histidine tag or the epitope tag for monoclonal antibody A52 at the COOH-terminal end of the molecule. Both tagged molecules were active and had indistinguishable drug resistance profiles. The tagged P-glycoproteins were expressed contemporaneously in HEK 293 cells, purified by nickel-chelate chromatography followed by immunoblot analysis. We found that P-glycoprotein-A52 did not copurify with functionally active P-glycoprotein-(His)10, even when the former was overexpressed relative to the histidine-tagged protein. Similar results were obtained with phosphorylation-deficient mutants of P-glycoprotein. By contrast, we could purify and reconstitute drug-stimulated ATPase activity when the half-molecules NH2-terminal half-(His)10/COOH-terminal half-A52 or NH2-terminal half-A52/COOH-terminal half-(His)10 were coexpressed in HEK 293 cells. These results suggest that nickel-chelate chromatography may be a suitable method for studying protein-protein interactions in membrane proteins and that the minimal functional unit of P-glycoprotein is likely to be a monomer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity.

To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wild-type CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 degrees C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P0) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P0, whereas the H949Y mutation doubled the P0 relative to wild-type. The changes in P0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range +/-80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.

Animals↗
PubMed1996

Mutational analysis of the predicted first transmembrane segment of each homologous half of human P-glycoprotein suggests that they are symmetrically arranged in the membrane.

A recent study of P-glycoprotein membrane topology using phoA gene fusions provided evidence that the orientation of the first transmembrane segment of each homologous half of P-glycoprotein was different (Béjà, O., and Bibi, E. (1995) J. Biol. Chem. 270, 12351-12354). To test this hypothesis, we compared the functional consequences of mutations to residues in transmembrane segments (TMs) TM1 and TM7. Mutations to 3 residues occupying homologous positions in TM1 and TM7 resulted in mutant P-glycoproteins that were inactive. These mutants were found to be misprocessed. By contrast, mutations to other residues in TM1 resulted in functional P-glycoproteins. When putative TM1 was replaced with TM7 or TM7 with TM1 to yield TM7/TM7 or TM1/TM1 chimeras, both constructs yielded P-glycoproteins that were capable of conferring drug resistance in transfected cells. The purified P-glycoproteins from mutants TM7/TM7 and TM1/TM1 retained 59 and 28% of verapamil-stimulated ATPase activity, respectively. By contrast, interchanging TM6 and TM12 to yield TM6/TM6, TM12/TM12, or TM12/TM6 constructs resulted in P-glycoproteins that did not have any detectable ATPase activity and did not confer drug resistance in transfected cells. These results suggest that TM1 and TM7 likely have similar structural and functional roles in P-glycoprotein and that they have identical topologies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity.

A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating.

Amino Acid Sequence↗
PubMed1996

An expert system for psychiatric diagnosis using the DSM-III-R, DSM-IV and ICD-10 classifications.

Making a systematic and comprehensive psychiatric evaluation of mental disorders in a patient can be a rather complex and involving process. We describe an expert system, MILP, which is designed to produce such systematic diagnoses of mental disorders using selected categories from the classification and diagnostic guidelines published in DSM-III-R, DSM-IV and ICD-10. An innovative part of the MILP design is the incorporation of constraint-based reasoning as a key part of the system. We believe that the MILR design gives a flexible framework which is suitable in general for the automated diagnoses of large classes of mental disorders.

Diagnosis, Computer-Assisted↗
PubMed1995

Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity.

The ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide (NEM), which is postulated to modify cysteine residues within either of the homology A consensus sequences for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively) (Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992). To test this postulate as well as determine the contribution of either nucleotide-binding domain to function, a Cys-less mutant was constructed, and then a single cysteine residue was reintroduced back into each nucleotide-binding consensus sequence. We then tested the sensitivity of the ATPase activity of each mutant to covalent modification by NEM. It was found that covalent modification of a single cysteine residue within either nucleotide-binding consensus sequence (Cys-431 and Cys-1074, respectively) with NEM inhibited drug-stimulated ATPase activity of P-glycoprotein. The concentrations of NEM required for half-maximal inactivation of ATPase activity were 7 and 35 microM for mutants Cys-431 and Cys-1074, respectively. In both cases, inactivation of ATPase activity by NEM was prevented by ATP. These results suggest that both nucleotide-binding domains may need to bind ATP to couple drug binding to ATPase activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Recognising drug and alcohol problems in patients referred to consultation-liaison psychiatry.

OBJECTIVE: To study the extent of recognition by referring doctors of drug and alcohol problems in patients referred to consultation-liaison psychiatry for any reason, and the factors associated with non-recognition. DESIGN: Comparison of referring doctors' reasons for referral and psychiatrists' DSM-III-R diagnoses in 2347 inpatients referred consecutively over three years, by means of the prospective MICROCARES clinical database used by the consultation-liaison psychiatry service of each hospital involved. SETTING: Four general teaching hospitals of Monash University. RESULTS: Psychiatrists considered a psychoactive substance use disorder diagnosis likely in 336 (14%) of the referred patients; referring doctors missed 188 (56%) of these, referring instead for "depression", "anxiety" and "suicide attempt evaluation". The factors correlating with this discordance included younger age, male sex, having a personality disorder diagnosed, and having attempted self-harm. CONCLUSIONS: The extent of the failure to identify drug and alcohol problems in patients in whom some psychological problem worthy of referral to psychiatry had been detected is symptomatic of the extent of this failure in medicine generally. Education and the use of screening procedures may lead to development of coherent drug and alcohol protocols in individual institutions.

Alcoholism↗
PubMed1995

Rapid purification of human P-glycoprotein mutants expressed transiently in HEK 293 cells by nickel-chelate chromatography and characterization of their drug-stimulated ATPase activities.

P-glycoprotein containing 10 tandem histidine residues at the COOH end of the molecule was transiently expressed in HEK 293 cells and purified by nickel-chelate chromatography. The purified protein had an apparent mass of 170 kDa, and its verapamil-stimulated ATPase activity in the presence of phospholipid was 1.2 mumol/min/mg of P-glycoprotein. We then characterized P-glycoprotein mutants that exhibited altered drug-resistant phenotypes and analyzed the contribution of the two nucleotide binding folds to drug-stimulated ATPase activity. Mutation of residues in either nucleotide binding fold abolished drug-stimulated ATPase activity. The pattern of drug-stimulated ATPase activities of mutants, which conferred increased relative resistance to colchicine (G141V, G185V, G830V) or decreased relative resistance to all drugs (F978A), correlated with their drug-resistant phenotypes. By contrast, the ATPase activity of mutant F335A was significantly higher than that of wild-type enzyme when assayed in the presence of verapamil (3.4-fold), colchicine (9.1-fold), or vinblastine (3.7-fold), even though it conferred little resistance to vinblastine in transfected cells. These results suggest that both nucleotide-binding domains must be intact to couple drug binding to ATPase activity and that the drug-stimulated ATPase activity profile of a mutant does not always correlate with its drug-resistant phenotype.

3T3 Cells↗
PubMed1995

P-glycoprotein. Associations between domains and between domains and molecular chaperones.

P-glycoprotein consists of two homologous halves, each composed of a transmembrane domain and a nucleotide-binding domain. In order to understand how the domains interact in P-glycoprotein, we expressed each domain as a separate polypeptide and tested for associations using coimmunoprecipitation assays. We found that the interactions between the two halves of P-glycoprotein were mediated through associations between the two transmembrane domains as well as through the nucleotide-binding domains. In addition, the nucleotide-binding domain also associated with the transmembrane domain in each half of the molecule. By contrast, we could not detect any association either between the first nucleotide-binding domain and the second transmembrane domain, or between the second nucleotide-binding domain and the first transmembrane domain. We then tested whether individual domains associated with molecular chaperones, since biogenesis of P-glycoprotein appears to involve the chaperones calnexin and Hsc70. We found that calnexin associated only with the transmembrane domains, while Hsc70 associated only with the nucleotide-binding domains. These results suggest that noncovalent interaction between the domains of P-glycoprotein can contribute to structure and function of P-glycoprotein and that chaperones may participate in the folding of each domain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Expression of a functionally active human renal sodium-calcium exchanger lacking a signal sequence.

The Na+-Ca2+ exchanger is an unusual membrane transport protein as it contains an NH2-terminal signal sequence which is co-translationally removed in the endoplasmic reticulum during synthesis. To determine if the signal sequence was essential for biosynthesis, mutations were introduced in the NH2 terminus of the cDNA coding for the human renal Na+-Ca2+ exchanger in order to alter processing of the protein. To prevent cleavage of the signal sequence during biosynthesis, the last residue of the consensus signal sequence, Ala-1, was changed to Phe. Deletion mutants were also constructed to encode for exchangers which lacked the signal sequence, the signal sequence and the first extracellular loop, or all of the NH2 terminus including the first transmembrane segment of the mature protein. These mutants were expressed in HEK 293 cells and assayed for Na+-Ca2+ exchange activity. Mutants lacking either a signal sequence or containing a noncleavable signal sequence were still targeted to the plasma membrane, where they exhibited Na+-Ca2+ exchange activity. By contrast, the mutants which had more than the signal sequence deleted did not demonstrate any exchange activity. These mutants were, however, still integrated into the membrane and were resistant to alkali extraction. These results show that the signal sequence is not essential for biogenesis of the Na+-Ca2+ exchanger and suggests that the molecule contains one or more internal signal sequences for insertion into the membrane during biosynthesis.

Amino Acid Sequence↗
PubMed1995

Failure of P-glycoprotein (MDR1) expressed in Xenopus oocytes to produce swelling-activated chloride channel activity.

1. P-glycoprotein, the protein product of the multidrug resistance (MDR1) gene, has ATP-dependent transporter activity. It has been suggested that P-glycoprotein may also function as a volume-regulated chloride channel or chloride channel regulator. To assess the chloride channel function of P-glycoprotein, we examined swelling-activated chloride conductances in Xenopus oocytes injected with human MDR1 cRNA. 2. Functional expression of P-glycoprotein in Xenopus oocytes was confirmed using Western blot analysis and by assessing transport of the P-glycoprotein substrate, calcein AM. 3. Endogenous, swelling-activated chloride conductances were virtually absent by the time P-glycoprotein expression was confirmed. Thus, this expression system afforded the advantage of assessing putative MDR1-associated chloride currents in the absence of background currents. 4. The currents activated by hypotonic shock (50%) in both MDR1-injected and control (water-injected) oocytes were not significantly different. The swelling response was due in part to the activation of a potassium-selective conductance which could be inhibited by barium. No chloride-selective currents were activated by hypotonic shock in the presence or absence of barium. Therefore, we conclude that P-glycoprotein expression does not produce a swelling-activated chloride conductance in the Xenopus oocyte expression system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗