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Biomedical subjects

D M Clarke

Publications and source records attributed to D M Clarke.

At least 109 records · Page 6Linked to original sources
PubMed1987

Nucleotide sequence and in vitro expression of rubella virus 24S subgenomic messenger RNA encoding the structural proteins E1, E2 and C.

The complete nucleotide sequence of the 24S subgenomic mRNA of wild-type M33 strain rubella virus has been determined. This RNA is 3,383 nucleotides in length excluding the 3'-terminal poly(A) tract. After the three multiple in-phase termination codons clustered in the 5' terminus of this RNA, there are 81 nucleotides of nontranslated nucleic acid followed by a reading frame of 2,978 nucleotides that encodes the 110 kD precursor of the structural proteins. The 3'-untranslated region is 263 nucleotides. The 110 kD polyprotein is processed to produce nucleocapsid C, the glycoproteins E2 and E1 in that order. Sites of post-translational cleavage to produce E2 and E1 were located using available N-terminal amino acid sequences. RNAs synthesized by transcription in vitro are effective messengers in the rabbit reticulocyte cell-free translation system. Post-translational processing of the structural proteins was observed in the cell-free system supplemented with microsomes from dog pancreas.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli.

A 3240-base-pair DNA fragment spanning the pyridine nucleotide transhydrogenase (pnt) genes of Escherichia coli has been sequenced. The sequence contains two open-reading frames, pntA and pntB of 1506 and 1386 base pairs, coding for the transhydrogenase alpha and beta subunits, respectively. The coding sequences are preceded by a promoter-like structure and are most likely co-transcribed. Each coding sequence is preceded by a Shine-Dalgarno sequence. The amino-terminal amino acid sequences were determined from the purified alpha and beta subunits of the transhydrogenase. These sequences agree with those predicted from the nucleotide sequences of the pntA and pntB genes. The predicted relative molecular masses of 53906 (alpha) and 48667 (beta) are close to the values obtained by analysis of the subunits by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed in each subunit. These results indicate that transhydrogenase is an intrinsic membrane protein.

Amino Acid Sequence↗
PubMed1986

Expression of the cloned subunits of Escherichia coli transhydrogenase from separate replicons.

The pntA and pntB genes of Escherichia coli, encoding the alpha- and beta-subunits of the pyridine nucleotide transhydrogenase, were cloned individually in two different compatible plasmids into Escherichia coli mutants lacking transhydrogenase activity. Energy-linked and non-energy-linked transhydrogenase activities were produced only in cells carrying both plasmids thus showing that the products of both genes are required for the formation of an active enzyme. ATP-energized transhydrogenase activity was not increased in cells containing amplified levels of the transhydrogenase when the cell membrane ATPase was also amplified. It is suggested that the excess transhydrogenase is effectively uncoupled from the ATPase by compartmentalization in the cell.

Cell Membrane↗
PubMed1986

Isolation of a fourth cysteinyl-containing peptide of the alpha-subunit of the F1 ATPase from Escherichia coli necessitates revision of the DNA sequence.

The rapid determination of cysteinyl residues by Creighton's method [(1980) Nature 284, 487-489] led to the discovery of a discrepancy between protein and DNA sequence data in the alpha-subunit of the F1 ATPase from Escherichia coli [(1984) Arch. Biochem. Biophys. 229, 320-328]. We have isolated a cysteinyl-containing decapeptide from the alpha-subunit with a protein sequence (AGCAMGEYFR) which is only partially recognizable from DNA data. Re-sequencing of DNA in the region coding for the peptide has resulted in two corrections: insertion of a cytosine before position 715 and deletion of a thymine at position 731 of the uncA gene.

Amino Acid Sequence↗
PubMed1986

Detection of antibodies to individual proteins of rubella virus.

Individual rubella virus structural polypeptides were electroeluted from SDS-polyacrylamide gels. The eluted polypeptides were used, without further purification, as antigens in ELISA assays for the detection of rubella-specific antibodies in patients' sera. This provided a more sensitive detection method than that involving classical serological assays such as HI or VN or that using immunoprecipitation. Antisera against individual viral polypeptides were raised in mice. No haemagglutination inhibition activity was observed in any of these sera and weak virus neutralizing activity was only detected with antiserum to the E1 protein. Antisera to either the E1 or E2(a,b) complex proteins cross-reacted with both the E1 and E2(a,b) complex proteins.

Animals↗
PubMed1985

Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli.

The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, alpha and beta, of apparent Mr 50000 and 47000. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [14C]Dicyclohexylcarbodiimide preferentially labelled the 50000-Mr subunit of the transhydrogenase enzyme. The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.

Allosteric Site↗
PubMed1985

Cloning and expression of the transhydrogenase gene of Escherichia coli.

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity.

Chromosome Mapping↗
PubMedDate not supplied

A caution on the use of cut-points applied to screening instruments or diagnostic criteria.

Although cut-points or threshold scores are commonly used in screening instruments as well as in psychiatric diagnostic practice, much is still to be learned about their functional characteristics. This study demonstrates that, for a cut-off of y items on a screening instrument, there is great variability in the identification of caseness between different combinations of y items. The results suggest that the use of cut-points is perhaps not as reliable as is sometimes thought.

Adjustment Disorders↗
PubMedDate not supplied

Comparing correlated kappas by resampling: is one level of agreement significantly different from another?

Researchers comparing diagnostic systems or screening tests frequently need to compare indices of agreement such as the kappa coefficient. While asymptotic methods exist for comparing kappas derived from independent samples, no satisfactory approach exists for comparing kappas derived from the same or related samples. Resampling methods for comparing kappa coefficients obtained from the same sample are presented. Easily undertaken using readily available software, these methods are illustrated by application to a small sample of psychiatric data, as well as to several thousand samples of simulated data. An acceptable type I error rate was exhibited. Resampling techniques-easily implemented and making few assumptions-deserve wider application in psychiatric research.

Adult↗
PubMedDate not supplied

Monash Interview for Liaison Psychiatry (MILP). Development, reliability, and procedural validity.

The Monash Interview for Liaison Psychiatry (MILP) is a structured interview designed for use with patients who have physical and psychiatric comorbidity. Linked to a computerized diagnostic algorithm, the MILP is able to establish diagnoses according to DSM-III-R, International Classification of Diseases--10th Edition (ICD-10), and DSM-IV criteria, as well as a range of other criteria relevant to consultation-liaison psychiatry. Interrater reliability was assessed with 54 joint interviews, in which the mean kappa for agreement of items was 0.83 and of diagnoses was 0.68. Comparative procedural validity was tested against DSM-III-R decision-tree diagnoses, ICD-10 checklist diagnoses, and Structured Clinical Interview for DSM-III-R interview diagnoses on another sample of 54 patients. Mean kappas for these comparisons were 0.61, 0.56, and 0.31, respectively. As predicted, the MILP more fully covered the spectrum of somatizing disorders, compared with the other methods for establishing diagnoses.

Adult↗
PubMedDate not supplied

Consultation-liaison psychiatrists management of depression.

Prospective data on 1,360 consecutive inpatients referred to the consultation-liaison psychiatry service of 2 metropolitan general teaching hospitals and diagnoses as having a Depressive Illness Spectrum Disorder were collected by using the MICRO-CARES clinical database system. The distribution of DSM-III-R diagnoses was major depression (MD) 49%; dysthymia (DYS) 15%; organic or substance-induced mood disorder or depressive disorder not otherwise specified (ORG/NOS) 14%; and adjustment disorder with depressed mood (AD) 29%s. Antidepressants were prescribed in 59% of the MD cases, 40% of the DYS cases, 36% of the ORG/NOS cases, and 17% of the AD cases. In confirmed MD, antidepressants were prescribed in 69%, and significantly more often in those who were older, female, had a prior history of physical illness, had a neoplasm or a disorder of the nervous or musculoskeletal systems, had higher Axis IV scores, or were referred because of pain or terminal illness. The patients with confirmed MD prescribed antidepressants had a longer length of stay and were referred later than those not prescribed antidepressants. The results illustrate the importance of all the forms of depression in consultation-liaison psychiatry and the vigor with which all forms are treated.

Adult↗
PubMedDate not supplied

Psychiatric disturbance and acute stress responses in surgical patients.

BACKGROUND: A number of reports have appeared of post-traumatic stress symptoms occurring in physically ill patients. This study sought to measure the extent of psychiatric disturbance and acute post-traumatic stress reactions occurring in surgical inpatients, and to examine their relationship with the stress of illness and hospitalization and the personal coping style of the patient. METHODS: Thirty-seven admissions to a general surgical unit were assessed on admission and immediately prior to discharge. Measures were made of depression, anxiety, cognitive impairment, post-traumatic stress symptoms, severity of illness, level of physical functioning, degree of hospital stress and coping style. RESULTS: There was a significant reduction in anxiety scores postoperatively. Twenty-seven per cent of patients developed high levels of acute post-traumatic stress symptoms and these correlated with depression at admission and intra-hospital stress. Depression at discharge was related to depression at admission, physical functioning and coping style. In general, avoidance and acceptance-resignation were associated with a poorer psychiatric outcome. CONCLUSIONS: Postoperative psychiatric disturbance, including acute stress symptoms, can be understood as a reaction to the 'trauma' of illness and hospitalization, predisposed to by pre-existing depression. Interventions could be directed at screening for at-risk patients, minimizing stress, and encouraging adaptive mechanisms of coping in patients.

Acute Disease↗