Biomedical subjects

D M Clarke

Publications and source records attributed to D M Clarke.

At least 73 records · Page 4Linked to original sources

PubMed1995

Membrane topology of a cysteine-less mutant of human P-glycoprotein.

A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect to its ability to confer resistance to various drugs. The cysteine-less P-glycoprotein-A52 retained the ability to confer resistance to vinblastine, colchicine, doxorubicin, and actinomycin D with only a small decrease in efficiency relative to wild-type enzyme. Cysteine residues were then reintroduced into predicted extracellular or cytoplasmic loops of the cysteine-less P-glycoprotein-A52, and the topology of the protein was determined using membrane-permeant and impermeant thiol-specific reagents. It was found that 8 of 15 cysteine residues introduced into P-glycoprotein-A52 could be biotinylated, when cells expressing the mutant P-glycoprotein were incubated with membrane-permeant biotin maleimide. Biotinylation of a cysteine residue placed in predicted extracellular loops between transmembrane segment (TM) 5 and TM6, TM7 and TM8, or TM11 and TM12 was blocked by pretreatment of the cells with a membrane-impermeant maleimide, suggesting that these residues have an extracellular location. By contrast, biotinylation of cysteine residues located in the predicted cytoplasmic loops between TM2 and TM3, TM4 and TM5, TM8 and TM9, or TM10 and TM11 were not blocked by pretreatment with membrane impermeant maleimide, suggesting that these residues were in the cytoplasm. These results are consistent with the model of P-glycoprotein, which predicts six transmembrane segments in each of the two homologous halves of the molecule.

3T3 Cells↗

PubMed1995

The recognition of depression in patients referred to a consultation-liaison service.

The recognition of depression was examined in 987 medical and surgical patients referred to a consultation-liaison psychiatry service. Overall concordance of recognition of depression by the referring doctor and diagnosis of depression by the consultant psychiatrist was 74%; 41% false-positive rate, 15% false-negative rate. Concordance was higher in the Renal Unit and lower in the General Medical Unit. Patients for whom there was discordance were significantly older than those for whom there was concordance. Patients referred for depression but not diagnosed as such by psychiatrists received DSM-III-R diagnoses of Organic Mental Disorder, Somatoform and Related Disorders, Psychoactive Substance Use Disorders and Personality Disorders. On the other hand, patients diagnosed as having depression but not referred as such were referred instead for ill-defined reasons (suspected psychological component to illness, coping problems), suicide risk evaluation and routine pre-operative or pre-dialysis assessment. The results highlight the continuing misdiagnosis of psychiatric disorders, especially Organic Mental Disorders, as well as the mislabelling of the syndrome described by psychiatrists as depression. This is part of the wider problem of defining the boundaries of a clinical depressive syndrome in the physically ill.

Adolescent↗

PubMed1995

Consultation-liaison psychiatry in general medical units.

The activities of a consultation-liaison psychiatry service to general medical units in a university affiliated suburban teaching hospital are described, with a report from the MICRO-CARES clinical database on 165 consecutive referrals over a 12 month period. The referral rate was 4.2% of admissions. The data confirm the association of psychiatric referral and prolonged length of hospital stay (mean of 18 days for referred patients, 9 days for non-referred patients). The most common reasons for referral were depression, suicide risk evaluation, organic brain syndrome and suspected psychological component to illness. The most common psychiatric diagnoses were Mood Disorders (55%), Organic Mental Disorders (35%), Adjustment Disorders (19%), Somatoform and other Somatic Disorders (16%) and Personality Disorders (15%). Although 67% of patients received at least one confirmed diagnosis, 39% of all diagnoses remained "differential", or unconfirmed, at discharge. Concordance with drug recommendations was 97% and with non-drug recommendations 95%. Two groups of patients were prominent among the referrals: the young self-poisoning patient, and the older patient living alone. The issues involved in providing a liaison psychiatry service to general medical units with these characteristics are described.

Adaptation, Psychological↗

PubMed1994

Identification of an internal topogenic signal sequence in human Band 3, the erythrocyte anion exchanger.

The insertion of Band 3, the human erythrocyte anion exchanger, into microsomal membranes was studied in an in vitro reticulocyte lysate translation system. Band 3 consists of a 43-kDa amino-terminal cytosolic domain and a carboxyl-terminal 52-kDa membrane domain containing up to 14 transmembrane segments with a single N-glycosylation site at Asn-642. Insertion of truncated Band 3 molecules into microsomal membranes was assayed by glycosylation, resistance to alkaline extraction, and tryptic removal of the cytosolic domain. Truncations containing either the first four or the last eight putative transmembrane segments were stably integrated into microsomes showing that an intact membrane domain was not required for membrane integration. Furthermore, the extracytosolic domain following the seventh transmembrane segment was properly translocated across the microsomal membrane and glycosylated whether the seventh transmembrane segment was the first, last, or the only transmembrane segment in the construct. The ability of the entire membrane domain, the truncated domain beginning with the seventh transmembrane segment, or the seventh transmembrane segment to insert into microsomes was dependent on the presence of the signal recognition particle receptor. The seventh transmembrane segment in Band 3 therefore has the topogenic properties of an internal signal sequence.

Amino Acid Sequence↗

PubMed1994

Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein.

Site-directed mutagenesis was used to investigate whether amino acids located in the predicted transmembrane segment, TM6 (residues 330-351), of human P-glycoprotein play essential roles in drug transport. Mutant cDNAs were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to cytotoxic drugs. Four mutations were found to strongly alter the drug resistance profile conferred by P-glycoprotein. Mutation of Val338 to Ala resulted in a mutant P-glycoprotein which conferred enhanced resistance to colchicine and reduced relative resistance to vinblastine. By contrast, mutant Gly341 to Val conferred little resistance to colchicine or doxorubicin, while its ability to confer resistance to vinblastine or actinomycin D was retained. A reduction in the ability of P-glycoprotein to confer resistance to all four drugs was observed for mutant Ala342 to Leu. Mutation of Ser344 to Ala, Thr, Cys, or Tyr resulted in mutant P-glycoproteins which were unable to confer drug resistance. Photolabeling of P-glycoprotein with azidopine in the presence of varying amounts of vinblastine showed that mutation of Ser344 to Tyr required approximately 15-fold more vinblastine to inhibit photolabeling when compared to wild-type enzyme. All of the Ser344 mutants were found to have reduced drug-stimulated ATPase activity relative to wild-type enzyme. These results, together with our previous demonstration that changes to Phe335 affected dissociation of vinblastine, suggest that TM6 may play an important role in drug--protein interaction and coupling of drug binding to ATPase activity.

3T3 Cells↗

PubMed1994

Prolonged association of temperature-sensitive mutants of human P-glycoprotein with calnexin during biogenesis.

Mutation of amino acids located within or immediately NH2-terminal to transmembrane segment 7 of human P-glycoprotein abolished the ability of the protein to confer resistance to cytotoxic drugs. Each of these mutant P-glycoproteins had an apparent mass of 150 kDa, compared with 170 kDa for wild-type P-glycoprotein, and the apparent mass was altered by endoglycosidase H digestion. These observations suggest that these mutant proteins were processed improperly, so that they were located in the endoplasmic reticulum and were not targeted correctly to the plasma membrane. Processing of the 150-kDa P-glycoprotein to the 170-kDa mature form of the enzyme for all of the mutants, except Glu707-->Ala and Tyr710-->Ala, was dramatically increased when the cells were grown at 26 degrees C. At the lower growth temperature, the mature protein was targeted to the plasma membrane, and drug efflux activity was restored. We also analyzed the mutants for possible molecular interactions that may contribute to their intracellular retention. We found that core-glycosylated forms of the wild-type and mutant P-glycoproteins were associated with the molecular chaperone calnexin. Only wild-type enzyme, however, was able to escape association with calnexin and be targeted to the plasma membrane. Prolonged association of the mutants with calnexin may be due to misfolding of the protein as evidenced by their relative short half-life of about 3 h, compared with 50 h for the wild-type enzyme. These results suggest that calnexin contributes to a quality control mechanism to retain misfolded forms of P-glycoprotein in the endoplasmic reticulum.

3T3 Cells↗

PubMed1994

Psychiatric disorders in and service use by young homeless people.

OBJECTIVES: To examine psychiatric morbidity, including substance use disorders, and service use in young people with experience of homelessness. DESIGN: A cross-sectional study of 34 new residents in a supported accommodation program in Melbourne. Current and lifetime psychiatric diagnoses were made using the Structured Clinical Interview for the Diagnostic and statistical manual of mental disorders, third edition, revised (DSM-III-R). Use of psychiatric and related services was also assessed. RESULTS: Of the 21 women and 13 men (mean age, 18.1 years; standard deviation, 2.2 years), 50% had a current major DSM-III-R diagnosis, and 82% had a lifetime DSM-III-R diagnosis. The most common diagnoses were alcohol dependence, depressive disorders and cannabis dependence. Co-morbidity was common. Few of the young people had sought or received any treatment for depressive or substance use disorders. CONCLUSIONS: Young people with experience of homelessness have a high prevalence of depressive disorders and substance use disorders, particularly alcohol and cannabis dependence. Despite this they have a low rate of service use. These findings suggest a need for closer interaction between mental health professionals and other agencies in the planning and provision of services to young homeless people.

Adolescent↗

PubMed1994

Functional consequences of glycine mutations in the predicted cytoplasmic loops of P-glycoprotein.

Site-directed mutagenesis was used to investigate whether glycine residues in the predicted cytoplasmic loops play essential roles in the structure and function of human P-glycoprotein. Mutant cDNAs in which codons for each of the 20 glycine residues were changed to valine, were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutation of Gly-251, -268, -269, or -781 yielded mutant proteins which were unable to confer drug resistance in transfected cells. Each of these mutant P-glycoproteins had an apparent mass of 150 kDa, compared with 170 kDa for wild-type P-glycoprotein and the apparent mass was altered by endoglycosidase H digestion. These observations suggest that these mutant proteins were improperly processed so that they were located in the endoplasmic reticulum and were not targeted correctly to the plasma membrane. The in vivo processing of mutants Gly-269 to Val and Gly-781 to Val was temperature-sensitive. When cells expressing these mutants were grown at a lower temperature (26 degrees C), the mature 170-kDa form of P-glycoprotein was the major product. Substitution of glycine with alanine at positions 251, 268, 269, or 781 yielded mutants with structural and functional characteristics similar to wild-type enzyme. Mutation of Gly-141, 187, 288, 812, or 830 to Val, altered the drug resistance profile conferred by P-glycoproteins expressed in transfected cells. All five mutations increased relative resistance to colchicine or adriamycin, without altering relative resistance to vinblastine. These results demonstrate that glycines located in the cytoplasmic loops play important roles in structure and function of P-glycoprotein.

3T3 Cells↗

PubMed1994

Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides.

P-glycoprotein consists of two homologous halves, each composed of six potential transmembrane sequences and an ATP-binding domain. The cDNA coding for human P-glycoprotein was divided in half and subcloned into separate plasmids in order to express each half as a separate polypeptide and to characterize its contribution to function. Expression of cDNAs coding for either the NH2- or COOH-terminal half-molecules in HEK 293 cells yielded products of 88 and 64 kDa, respectively. The NH2-terminal half-molecule was glycosylated, since its size decreased from 88 to 79 kDa when expressed in the presence of tunicamycin. No change was observed in the size of the COOH-terminal half-molecule when it was expressed in the presence of tunicamycin, indicating that it was not glycosylated. The cDNAs coding for each half of P-glycoprotein were transfected into NIH-3T3 cells to test for biological activity. No drug-resistant colonies were obtained when cells were transfected with cDNA coding for each half-molecule or when cells were co-transfected with both cDNAs, although stable expression of each half-molecule was detected. The inability to confer drug resistance was likely due to a defect in targeting of the half-molecules to the cell surface. Each half-molecule was then expressed in Sf9 insect cells using a baculovirus vector to allow measurement of partial function. The half-molecules exhibited ATPase activity, but their activities were not stimulated by drug substrates. Drug-stimulatable ATPase activity was present, however, when the half-molecules were expressed together. These results suggest that coupling of ATPase activity to drug binding requires interaction between both halves of P-glycoprotein.

3T3 Cells↗

PubMed1994

Interaction of rat kidney P-glycoprotein with a urinary component and various drugs including cyclosporin A.

The interaction of rat renal P-glycoprotein with various drugs and a hydrophobic component found in rat urine was studied to gain an understanding of both its transport function in kidney and its potential role in drug secretion and drug-induced nephrotoxicity. Rat kidney brush-border membranes (BBM) were photolabeled with [3H]azidopine, a calcium-channel blocker that covalently labeles P-glycoprotein. P-glycoprotein was immunoprecipitated with a rabbit polyclonal antibody against the human MDR1 protein (multidrug resistance gene class 1). The amount of [3H]azidopine incorporated into P-glycoprotein was quantitated following gel electrophoresis and fluorography. Photolabeling inhibition assays were conducted with a panel of drugs known to interact with P-glycoprotein in multidrug-resistant cells. Verapamil or quinidine [half-maximal inhibition constant (K0.5) = 1 microM], vinblastine (K0.5 = 3 microM), and doxorubicin or daunomycin (K0.5 = 10 microM) all blocked [3H]azidopine photolabeling of renal P-glycoprotein. Of the drugs tested, the immunosuppressant drug, cyclosporin A, interacted with kidney P-glycoprotein with the highest affinity (K0.5 = 50 nM). However, the cardiac glycoside, digoxin, failed to inhibit P-glycoprotein photolabeling. A hydrophobic rat urine extract prepared by reverse-phase chromatography also blocked photolabeling of renal P-glycoprotein. Our current hypothesis is that various drugs may inhibit urinary excretion of an endogenous substrate by virtue of their ability to bind with high affinity to P-glycoprotein. A hypothesis of drug-induced nephrotoxicity based on the interaction of various compounds like cyclosporin A with P-glycoprotein is presented.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Functional expression of human renal Na+/Ca2+ exchanger in insect cells.

The cDNA coding for the human renal isoform of the Na+/Ca2+ exchanger was cloned from a human embryonic kidney cell line (HEK 293) using a polymerase chain reaction strategy. It was found by sequence analysis that the cDNA encoded for a human kidney isoform of the Na+/Ca2+ exchanger. The kidney isoform is nearly identical to the human cardiac Na+/Ca2+ exchanger except for a segment of amino acids within the predicted cytoplasmic loop region of the molecule. Within this region, the kidney isoform contains a deletion of 36 amino acids, as well as a segment of 33 amino acids that is only 33% identical to the equivalent region of the cardiac exchanger. The cDNA was then tested for function by expression in insect cells. We constructed a recombinant baculovirus containing renal Na+/Ca2+ exchanger cDNA under control of the polyhedrin promoter. High levels of expression and Na+/Ca2+ exchange activity were found in Sf9 insect cells infected with the recombinant virus. Our results indicate that human kidney expresses a functional and alternatively spliced variant of the human cardiac Na+/Ca2+ exchanger.

Amino Acid Sequence↗

PubMed1993

Deletion of NH2- and COOH-terminal sequences destroys function of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum.

Deletion mutants of the Ca2+ ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) were constructed and expressed in COS-1 cells. The mutants were expressed at levels 7- to 15-fold lower than the wild-type and were inactive. In vitro transcription-translation-insertion experiments showed that deletion of transmembrane sequences M1 and M2, but not of M8, M9, M10 or the NH2-terminal 30 amino acids inhibited the stable insertion of the enzyme into the membrane. Thus there was no correlation between loss of function and membrane insertion. A signal sequence for membrane insertion may exist in M1 and M2.

Animals↗

PubMed1993

Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein.

Site-directed mutagenesis was used to investigate whether phenylalanine residues in predicted transmembrane sequences play essential roles in the function of human P-glycoprotein. Mutant cDNAs, in which codons for each of the 31 phenylalanine residues were changed to alanine, were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutation of either Phe-335 to Ala in transmembrane segment 6, or Phe-978 to Ala in transmembrane segment 12, drastically altered the drug resistance profile conferred by the mutant P-glycoprotein in transfected cells. Mutant Phe-335-->Ala conferred little resistance to vinblastine or actinomycin D but retained the ability to confer resistance to colchicine and adriamycin. The mutant also showed increased binding of azidopine, which could be inhibited by lower levels of vinblastine, relative to the wild-type enzyme. By contrast, mutant Phe-978-->Ala conferred little or no resistance to colchicine or adriamycin, while its ability to confer resistance to vinblastine or actinomycin D was retained. These results suggest that Phe-335 and Phe-978 play important roles in the recognition and transport of specific substrates by P-glycoprotein. Mutation of Phe-777 to Ala affected the biosynthesis of the transporter. Mutation of the other 28 phenylalanine residues yielded protein products with structural and functional characteristics that were indistinguishable from the wild-type enzyme.

3T3 Cells↗

PubMed1993

Functional consequences of alterations to hydrophobic amino acids located in the M4 transmembrane sector of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Those hydrophobic residues between Ile298 and Ile315 in transmembrane segment M4 of the Ca(2+)-ATPase of sarcoplasmic reticulum, not previously mutated, were mutated systematically in ways that would alter their size or polarity, and functional consequences were measured. Fourteen residues in this sequence are organized as juxtapositions of large, hydrophobic (Val, Leu, Ile) and small (Ala, Gly) residues, and these were altered so that large residues were substituted for small and vice versa. Several mutants exhibited diminished Ca2+ transport, but mutants A305V and A306V lost all Ca2+ transport function. In both cases, the mutants were phosphorylated with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that the Ca(2+)-binding sites were intact. Reduced Ca2+ affinity, as measured by Ca2+ dependence of phosphorylation from ATP, was observed for mutant A305V. In both mutants, the ADP-insensitive phosphoenzyme intermediate (E2P) decayed slowly relative to the wild-type enzyme, suggesting that the E2P to E2 conformational transition was impaired, slowing the rate of the phosphatase reaction. Double mutants which reversed the order of Val304 and Ala305 and Ala306 and Ile307, resulted in the same phenotype as the single Ala mutations. These results, combined with our previous demonstration that Glu309 is a Ca2+ binding residue, that Pro312 is involved in E1P to E2P conformational changes, and that Gly310 is involved in E2P to E2 conformational changes, support the hypothesis that transmembrane segment M4 plays a key role in the Ca2+ transport function of the Ca(2+)-ATPase through its involvement in both the binding of Ca2+ and the subsequent conformational changes which bring about the translocation of Ca2+ to the lumen of the membrane.

Adenosine Triphosphate↗

PubMed1993

Functional consequences of proline mutations in the predicted transmembrane domain of P-glycoprotein.

Site-directed mutagenesis was used to investigate whether prolines in the predicted transmembrane domains play essential roles in the function of human P-glycoprotein. Mutant cDNAs in which codons for each of the 13 prolines were changed to alanine were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to various drugs. Mutations of either Pro223 in transmembrane segment 4 or Pro866 in transmembrane segment 10, drastically reduced the ability of the mutant proteins to confer resistance to colchicine, adriamycin, or actinomycin D, whereas the capacity to confer resistance to vinblastine was retained. These results strongly suggest that residues in putative transmembrane segments 4 and 10, which are found in identical positions when homologous, presumably duplicated, halves of the transporter are aligned, play important roles in recognition of colchicine, adriamycin, and actinomycin D. They may either interact to form a single drug-binding site or form part of two equivalent, but independent, drug-binding sites. The lack of detectable effect of either mutation on vinblastine transport, however, indicates that there are differences in the requirements for binding of various substrates to P-glycoprotein. Mutation of Pro709 in transmembrane segment 7 resulted in a protein unable to confer drug resistance. A change at this position was found to induce a structural aberration, since the major protein product observed in transfected cells had an apparent molecular weight of 150,000, whereas the wild-type enzyme had an apparent molecular weight of approximately 170,000. Mutation of the other 10 prolines yielded protein products with structural and functional characteristics indistinguishable from wild-type P-glycoprotein.

3T3 Cells↗

PubMed1993

Establishing a consultation-liaison psychiatry clinical database in an Australian general hospital.

This paper describes the institution of a clinical database in the consultation-liaison psychiatry service of an Australian general hospital psychiatry unit. One of the problems faced was that many researchers and clinicians in Australia use the ICD-9 rather than the DSM-III-R classification system. Nevertheless, it was possible to use the DSM-III-R-based MICRO-CARES clinical database management system in this project. The data obtained during the first 12 months of its use are presented. Despite differences in the patient demographic characteristics, the data obtained are within the ranges described for North American and European sites. The local benefits of such a clinical database are described, and it is argued that such a database is also necessary for interhospital and international collaborative studies and comparisons.

Adult↗

PubMed1993

Consultation-liaison psychiatry in Australia.

Consultation-liaison (C-L) psychiatry as a service entity has developed slowly and to a variable extent in Australia despite the presence of factors conducive to its development. These include a compulsory national health insurance system and a favorable disposition to the biopsychosocial model and to psychoanalytic ideas. C-L psychiatry, however, does enjoy a high profile in many undergraduate medical and postgraduate psychiatry teaching programs. Research on C-L psychiatry is emerging, complementing a strong history of research in psychosomatic medicine. An Australian and New Zealand C-L psychiatry interest group is being formed. Collaboration with the MICRO-CARES Consortium and the European Consultation-Liaison Workgroup is proving to be a great stimulus.

Australia↗

PubMed1993

A comparative study of screening instruments for mental disorders in general hospital patients.

OBJECTIVE: Self-administered questionnaires are frequently used in studies of hospitalized physically ill patients to identify and measure psychiatric morbidity. This study examines the validity of some commonly used questionnaires in this context. METHOD: One hundred and seventy-nine patients in a general hospital completed the GHQ, HAD, BDI and STAI and were interviewed using the SCID-R. The findings were evaluated against DSM-III-R diagnoses using the QROC curve. RESULTS: Thirty-eight percent of patients obtained one or more DSM-III-R diagnoses; 25 percent mood disorder, 12 percent anxiety disorder, 11 percent drug abuse or dependence, 2 percent a somatic syndrome. Eleven percent had more than one diagnosis. As screening instruments for general psychiatric morbidity there were no statistically significant differences between versions of the GHQ and the HAD. With respect to identifying depression, the GHQ tended to perform better than the other instruments. CONCLUSIONS: The questionnaires identified general morbidity and depression satisfactorily but anxiety and drug abuse and dependence syndromes poorly. The results also support the notion that the DSM-III-R classification has a number of deficiencies when used in this population.

Adult↗