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Biomedical subjects

G M Wilson

Publications and source records attributed to G M Wilson.

At least 19 recordsLinked to original sources
PubMed1995

An episomal expression vector system for monitoring sequence-specific effects on mRNA stability in human cell lines.

A plasmid expression system has been developed which allows sequence-specific effects on mRNA degradation rates to be determined. This system uses stable, nonintegrating vectors that provide consistent levels of mRNA expression without the position effects common to integrating vectors. cDNAs encoding putative instability elements may be subcloned into the 5' untranslated region (5'UTR), the coding region, or the proximal 3'UTR of a beta-globin cDNA reporter. The effects of these sequences on mRNA stability may then be determined by actinomycin time course analyses of the fusion mRNAs and recombinant beta-globin mRNA in human cell lines. To demonstrate the utility of the vector system we fused an 820-bp fragment of the cDNA encoding the proximal 3'UTR of human 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase to the 3'UTR of the beta-globin reporter and introduced the vector into the human hepatocarcinoma cell line, HepG2. The fusion mRNA was degraded at a rate 2- to 2.5-fold greater than that of beta-globin alone, at a rate similar to that reported for HMG CoA reductase mRNA in normal rat liver. Similar to a number of other relatively unstable mRNAs, the rate of fusion mRNA degradation was greatly decreased by treatment with cycloheximide.

Base Sequence↗
PubMed1994

Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells.

We have previously identified and characterized a novel member of the ATP-binding cassette superfamily of transport proteins, multidrug resistance protein (MRP), and subsequently demonstrated that its overexpression is sufficient to confer multidrug resistance on previously sensitive cells (Cole et al., Science (Washington DC), 258: 1650-1654, 1992; Grant et al., Cancer Res. 54: 357-361, 1994). In the present study, we have transfected two different eukaryotic expression vectors containing MRP complementary DNA into HeLa cells to study the pharmacological phenotype produced exclusively by overexpression of human MRP. The drug resistance patterns of the two MRP-transfected cell populations were similar. They were characterized by a moderate (5- to 15-fold) level of resistance to doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide, and a low (< or = 3-fold) level of resistance to taxol, vinblastine, and colchicine. The transfectants were not resistant to 9-alkyl anthracyclines, mitoxantrone, or cisplatin. The MRP-transfected cells were also resistant to some heavy metal anions including arsenite, arsenate, and trivalent and pentavalent antimonials but were not resistant to cadmium chloride. Accumulation of radiolabeled vincristine was reduced by 45% in the MRP-transfected cells and could be restored to the levels found in sensitive cells by depletion of ATP. Rates of vincristine efflux did not differ greatly in the sensitive and resistant cells. The cytotoxic effects of vincristine and doxorubicin could be enhanced in a dose-dependent fashion by coadministration of verapamil. Cyclosporin A also increased vincristine toxicity but had less effect on doxorubicin toxicity. The degree of chemosensitization by verapamil and cyclosporin A was similar in MRP-transfected cells and in cells transfected with the vector alone, suggesting that sensitization involved mechanisms independent of MRP expression. Verapamil and cyclosporin A caused a modest increase in vincristine accumulation in the resistant cells but did not restore levels to those of the sensitive cells. Taken together, these data indicate that drug-resistant cell lines generated by transfection with MRP complementary DNA display some but not all of the characteristics of MRP-overexpressing cell lines produced by drug selection in vitro. They further demonstrate that the multidrug resistance phenotype conferred by MRP is similar but not identical to that conferred by P-glycoprotein and includes resistance to arsenical and antimonial oxyanions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions.

The NS-1 protein of minute virus of mice (MVM) is required for viral DNA replication and transcriptional regulation. To define the domain structure of NS-1, we have generated point mutations in its putative NTP-binding/ATPase domain. We show that all mutants were unable to support replication of MVM DNA in a transient DNA replication assay. Furthermore, all mutants, except for the K405S substitution, were able to transactivate the P38 promoter in transient transfection experiments. NS-1 proteins bearing COOH-terminal deletions of 29 and 33 amino acid residues were also transcriptionally inert. Biochemical analysis of recombinant NS-1 expressed in insect cells shows that mutations in the putative NTP-binding/ATPase domain severely reduced helicase activity in vitro. However, affinity labeling experiments indicate that none of these mutations, except for K469T, impaired NTP-binding activity. Finally, all point mutants retained significant levels of ATPase activity, except for the E444Q mutant (1%). These findings suggest that the replication and transcription activities of NS-1 reside in separate functional domains. In addition, NS-1 proteins with mutations in the putative nucleotide binding fold have lost helicase activity, whereas most retain nucleotide binding and ATPase functions, suggesting that the mutations have uncoupled the ATPase and helicase activities.

Adenosine Triphosphatases↗
PubMed1992

Purification and initial characterization of the lymphocyte-specific protein-tyrosyl kinase p56lck from a baculovirus expression system.

A baculovirus expression system has been used to express large quantities of the lymphocyte-specific protein-tyrosyl kinase p56lck. A series of chromatographic steps, including the novel application of metalchelate affinity chromatography for protein kinase purification, were employed to obtain p56lck in a highly active form. Recombinant p56lck was purified to apparent homogeneity as determined by polyacrylamide gel electrophoretic analyses and was found to migrate in SDS gels as two related species, both with apparent molecular masses close to 56 kDa. p56lck phosphorylated all assayed substrates exclusively on tyrosyl residues, and underwent autophosphorylation at one principal site, also on a tyrosyl residue. p56lck displayed a high affinity for a synthetic peptide corresponding to the cytoplasmic domain (residues 52-164) of the T-cell receptor zeta-chain (TCR-zeta) (Km approximately 6.5 microM) but a low affinity for a peptide corresponding to its own autophosphorylation site (Km approximately 900 microM). p56lck was also found to be highly active for a purified protein-tyrosyl kinase (Vmax greater than 400 pmol.min-1.micrograms-1 using the TCR-zeta (52-164) as a substrate). A variety of agents were tested for their ability to inhibit p56lck, with zinc ions (I50 approximately 1.7 mM) and staurosporine (I50 approximately 500 nM) proving the most potent.

Animals↗
PubMed1992

Severe anaemia and ileocolic anastomotic ulceration.

Two children are described with anaemia from ileocolic anastomosic ulceration as a late complication of surgery in the newborn period. The anastomosis was revised in each case but in one child there was early recurrence of ulceration.

Anastomosis, Surgical↗
PubMed1991

Expression of minute virus of mice major nonstructural protein in insect cells: purification and identification of ATPase and helicase activities.

The gene encoding the major nonstructural (NS-1) protein of minute virus of mice (MVM) has been expressed in insect cells using a baculovirus expression system. This 83-kDa polypeptide was found to be localized in the soluble (cytosolic) fraction in insect cells, in contrast with the nuclear localization of NS-1 expressed in MVM-infected mouse LA-9 cells. The protein was purified by immunoaffinity chromatography using a monoclonal antibody (MAb) prepared to an NS-1 fusion peptide [(Yeung et al., Virology 185, 35-45 (1991)]. Recombinant NS-1 was eluted using either low pH or a synthetic peptide corresponding to the epitope of the MAb. The peptide-eluted material is greater than 95% pure and biologically active in that it has ATPase activity and ATP-dependent helicase activity as determined by a strand displacement assay.

Adenosine Triphosphatases↗
PubMed1989

The effect of E1 mutations on biochemical transformation by an adenovirus carrying the herpes simplex virus thymidine kinase gene in region E3.

A series of human adenovirus type 5 (Ad5) vectors has been constructed in which a vector containing the human herpes simplex virus thymidine kinase (TK) gene has been recombined with several Ad5 early region 1 (E1) mutants. The resulting viruses were used to study host-virus interactions in TK- rat cells and to examine the importance of E1 functions in a biochemical transformation assay. One of the most important parameters affecting transformation efficiency in this system was the cytotoxicity of the transforming virus. Ad5 viruses expressing the E1a 289 amino acid protein were all highly cytotoxic and induced significantly fewer colonies than did less cytotoxic mutants which were defective in expression of the 289 amino acid product. When correction was made for differential cell viability the variation in transformation efficiencies was considerably reduced although some E1a mutants still demonstrated an enhanced ability to transform in comparison to wt virus. The significance of these results to morphological transformation by adenoviruses is discussed.

Adenoviruses, Human↗
PubMed1987

Asymptomatic degenerative disk disease and spondylosis of the cervical spine: MR imaging.

Evidence on magnetic resonance (MR) images of disk degeneration and herniation, as well as of cord and root impingement, may be regarded either as normal, age-related changes or as causative of symptoms. Individuals referred for MR examinations of the larynx without symptoms referable to the cervical spine were studied retrospectively (35 patients) or prospectively (65 patients) over a 2-year period. With a solenoid surface coil, 5-mm-thick sections were acquired in sagittal, axial, and coronal planes with T1-weighted spin-echo pulsing sequences. Disk protrusion (herniation/bulge) was seen in five of 25 (20%) patients aged 45-54 and 24 of 42 (57%) patients older than 64 years of age. Posterolateral protrusions were seen in only nine of 100 patients and occurred with greatest frequency in patients over 64 years of age. In no patient was obliteration of the intraforaminal fat seen. Spinal cord impingement was observed in nine of 58 (16%) patients under 64 years of age, and in 11 of 42 (26%) patients over 64 years of age. Cord compression was observed in seven of 100 patients and occurred solely secondary to disk protrusion in all cases. The percentage of cord area reduction never exceeded 16% and averaged approximately 7%.

Aged↗
PubMed1986

Influence of complex charge and size on the uptake of 99mTc-diphosphonates in osteogenic tissue.

The biodistributions of six chromatographically pure 99mTc-HEDP complexes have been determined in soft tissues, normal bone and osteogenic lesions (induced with a Walker 256 tumor) in Fisher 344 rats. The physical properties of each 99mTc-HEDP complex including anionic charge, partial molar volume, molecular weight and spectral characteristics are known; thus allowing structure-activity relationships to be drawn. The results indicate that the smallest, low charged, mononuclear 99mTc-HEDP complexes have the greatest uptake in bone lesions, and the highest lesion to muscle and lesion to normal bone ratios.

Animals↗
PubMed1978

Measurement of thyroid size by ultrasound, palpation and scintiscan.

Thyroid gland size was calculated from grey-scale ultrasound images in twenty patients with goitre. Results were compared with measurements by palpation and in some cases with measurements by scintiscan and at operation. There was a good correlation between ultrasound measurements and both the size of surgical specimens and clinicians' estimations, although clinicians under-estimated the size of large (greater than 40 ml) goitres compared with ultrasound and surgical specimens. Gland size calculated from scintiscan did not correspond well with measurements by ultrasound or palpation.

Goiter↗
PubMed1976

Total thyroidal content of iodine in thyrotoxic patients measured by in vivo neutron activation analysis.

This paper describes an in vivo method for measuring total thyroidal iodine stores by activation analysis, its evaluation and measurements in thyrotoxic patients. There was good correlation between measurements of solutions of iodine and post-mortem thyroids by activation analysis and chemical analysis. Measurements in thyrotoxic patients showed low levels in untreated and treated (antithyroid drugs) patients and a marked increase in patients studied whilst in clinical remission. The practical importance of this method of measurement of thyroidal iodine stores is that it is a reliable in vivo measurement obtained at a single visit and should enable the definition of the relationship of thyroidal iodine stores to pathophysiology and prognosis.

Activation Analysis↗
PubMed1976

Prevention of bone loss following oophorectomy in premenopausal women: a retrospective assessment of the effects of oophorectomy and a prospective controlled trial of the effects of mestranol therapy.

Prospective studies of bone mass in women following oophorectomy for benign conditions were done by the double-blind technique. Skeletal response to treatment was measured by photon absorption densitometry. Untreated patients were found to lose bone mass rapidly during the first two years after oophorectomy. When estrogen replacement was started within two months of oophorectomy, it was found to be effective in preventing subsequent bone tissue loss. Three years following oophorectomy, untreated women who had already lost bone tissue, and who were then started on estrogen replacement, showed a highly significant increase in their bone mass. The women in whom this treatment was delayed for six years did not respond. No untoward effects were noted in these women, perhaps, in part, because they had undergone hysterectomy. Long-term effects of this treatment are now being evaluated.

Adult↗
PubMed1975

General professional training in medicine.

A programme of general professional training in medicine based on the Royal Commission report has been in operation at the Western Infirmary, Glasgow, and associated hospitals since 1969. It involves a two-year rotation through general medicine, dermatology, psychiatry, paediatrics, geriatrics, and a variety of medical specialties. A third year is spent as far as possible in one unit of the doctor's choice. The scheme has been popular with trainees and senior staff. A high pass-rate has been achieved in the M.R.C.P.--23 out of 26 being successful within the three-year period. The trainees have subsequently taken up a wide variety of hospital posts within the broad division of medicine or entered general practice. The concept of a broadly based three-year period of general professional training in medicine has proved both practical and useful.

Curriculum↗
PubMed1975

Effect of teaching on students' attitudes to self-poisoning.

The attitudes of students, resident house physicians, and medical social workers towards 10 medical conditions were assessed in relation to both personal attitudes and the opinions expressed of the attitudes of the medical profession. Final-year students and house physicians showed unfavourable attitudes towards self-poisoning in contrast to fourth-year students and medical social workers. The fourth-year students were given the opportunity to admit patients referred to hospital with self-poisoning and visited the family doctor and the patient after discharge. After this exposure there was a subjective impression that the students became more interested in the problems of use self-poisoned patients, and this was supported by a review of their attitudes at the end of the teaching project.

Attitude of Health Personnel↗
PubMed1975

Assessment of clinical competence using objective structured examination.

To avoid many of the disadvantages of the traditional clinical examination we have introduced the structured clinical examination. In this students rotate round a series of stations in the hospital ward. At one station they are asked to carry out a procedure, such as take a history, undertake one aspect of physical examination, or interpret laboratory investigations in the light of a patient's problem, and at the next station they have to answer questions on the findings at the previous station and their interpretation. As they cannot go back to check on omissions multiple-choice questions have a minimal cueing effect. The students may be observed and scored at some stations by examiners using a check list. In the structured clinical examination the variables and complexity of the examination are more easily controlled, its aims can be more clearly defined, and more of the student's knowledge can be tested. The examination is more objective and a marking strategy can be decided in advance. The examination results in improved feed-back to students and staff.

Clinical Laboratory Techniques↗