Biomedical subjects
Publications and source records attributed to D Simpson.
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The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 X 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.
Human lymphoblast mutants at the X-linked hprt locus have been examined by Southern blot, Northern blot and DNA sequence analysis. A previous study had shown that approximately a third of the spontaneously-arising mutants and half those induced by formaldehyde showed no alteration in restriction fragment pattern and thus were classified as point mutations. In this report, Northern blot analysis was used to show that these point mutants fall into 4 categories: normal size and amount of RNA, normal size but reduced amounts, reduced size of RNA or no RNA. Sequence analyses of cDNAs prepared from hprt mRNAs were performed on 1 spontaneous and 7 formaldehyde-induced mutants with normal Northern blots. The spontaneous mutant was caused by an AT----GC transition. 6 of the formaldehyde-induced mutants were base substitutions, all of which occurred at AT base-pairs. There was an apparent hot spot, in that 4/6 independent mutants were AT----CG transversions at one specific site. The remaining mutant had lost exon 8.
Bilateral chorda tympani section is an accepted treatment for troublesome sialorrhoea. Nevertheless the effects of this operation have been poorly studied. Twenty patients were studied with unilateral chorda tympani section and a healthy contralateral ear. The effects on ipsilateral and contralateral stimulated submandibular and parotid flow and taste recognition and detection thresholds were measured. Stimulated submandibular flow accounted for only 27% of the total salivary flow. Chorda tympani section had no effect on submandibular flow in seven patients and only reduced submandibular flow by approximately 54% in the remaining 13 patients. No significant effect was observed on ipsilateral parotid flow rate. Electrogustometric taste detection thresholds were more than twice the accepted upper limit of normal on the lesioned side and taste recognition thresholds were also markedly abnormal. Chorda tympani section alone is a poor method of reducing stimulated salivary flow.
The detection of drugs of abuse in urine by four commercial immunoassay systems (TDx, BCL and PFI-20 opiates, and PFI-20 morphine) and one commercial TLC system (Toxi-Lab) was investigated and results compared with those obtained by a dual-column capillary GC system. The TDx system was the most reliable method for preliminary screening of urines for opiates; all the commercial immunoassay systems gave some results which were at variance with those of the GC. The GC method proved to be more reliable than the commercial TLC system in discriminating between the different opiates and is recommended for identification of drugs of abuse in urine specimens in which positive results have been obtained with preliminary screening procedures.
Using Syva "EMIT" reagents and the Cobas Bio centrifugal analyzer, we have developed cost-effective assays for therapeutic drug monitoring of carbamazepine, phenobarbital, and phenytoin. With these optimized methods one can assay up to 2470 samples with a single 100-test EMIT kit with good precision and accuracy. Between-batch CVs, based on results of repeat analysis of routine patients' samples, were 4.6%, 7.0%, and 5.3%, respectively. Results correlated well with those by methods previously used [gas chromatography and fluorescence polarization (Abbott TDx)], but there was a negative bias (-10.9%) of our method for phenobarbital results in patients' samples, as compared with those obtained with the TDx.
A cost-effective immunoassay for the detection of opiates in urine has been developed using commercial reagents on a centrifugal analyser. Using this method it is possible to assay up to 2470 samples with a 100 test kit while maintaining good precision. A mean CV of 4.2% was obtained for the concentration range 123-573 micrograms/l. The assay is suitable for high-risk urines since rigorous heat treatment may be performed prior to analysis.
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A method has been developed for the specific amplification and cloning of human hprt cDNA which can be used for mutant sequence analysis. Messenger RNA is isolated from TK6 lymphoblasts and is used to produce a first strand cDNA with reverse transcriptase primed with oligo dT. Second strand synthesis and subsequent amplification of hprt sequences is accomplished using Thermus aquaticus DNA polymerase and hprt-specific primers in the polymerase chain reaction (PCR) procedure. Convenient restriction enzyme sites have been built into the 5' ends of the PCR primers to allow cloning of the hprt fragments in M13mp19. Dideoxy sequencing of hprt with specific primers can be carried out using either the PCR reaction product or fragments cloned in M13mp19 as substrate. This general cloning/sequencing method can be used to analyze hprt mutation in human cells obtained both in vitro and in vivo.
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Three solutions administered by continuous extradural infusion for postoperative analgesia were compared in a randomized, double-blind manner. All patients underwent major abdominal gynaecological surgery and received 0.125% bupivacaine in 0.9% saline, diamorphine in 0.9% saline (0.5 mg in 15 ml) or diamorphine mixed with 0.125% bupivacaine (0.5 mg in 15 ml), at a rate of 15 ml h-1. The bupivacaine-diamorphine mixture provided significantly superior analgesia compared with either bupivacaine or diamorphine alone. No major side effects were encountered.
1. Alpha-chloralose, formed by the condensation of glucose with chloral, is used as a rodenticide and in the control of bird pests. 2. In two cases of poisoning with alpha-chloralose the clinical features included coma and generalized convulsions resembling the effects of strychnine more than those of chloral hydrate. 3. The principles of management are the support of vital functions in the unconscious patient and removal of unabsorbed poison by gastric lavage. Diazepam may be used to control convulsions.
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Photosystem I (PSI) preparations with a chlorophyll a/b ratio of 6.0 were isolated from barley thylakoids using two different methods. The high-molecular-mass complex (CP1a) which is resolved by non-denaturing gel electrophoresis had the same properties as a PSI preparation (PSI-200) isolated by Triton X-100 solubilisation of thylakoids followed by sucrose gradient ultracentrifugation. This material had a chlorophyll:P700 ratio of 208:1 and was composed of three different chlorophyll-protein complexes which could be separated from each other by solubilising the PSI preparation in dodecyl maltoside followed by sucrose gradient ultracentrifugation. Approximately half of the chlorophyll, including all the chlorophyll b, was located in two antenna complexes designated LHCI-680 and LHCI-730, which were identified by their characteristic low-temperature fluorescence emission spectra. The rest of the chlorophyll a was associated with the PSI reaction centre, P700 Chla-P1, which fluoresced at 720 nm. Each chlorophyll-protein complex had a unique polypeptide composition and characteristic circular dichroic and absorption spectra. The use of dodecyl maltoside instead of dodecyl sulphate resulted in a less denatured form of LHCI-680, which fluoresced at 690 nm at 77 K. One of the sucrose gradient fractions contained a complex consisting of only LHCI-730 and P700 Chla-P1 which fluoresced at 731 nm, indicating that LHCI-730 is structurally associated with P700 Chla-P1 and quenches its fluorescence. Approximately three-quarters of the light-harvesting antenna chlorophyll was in LHCI-730, but only about one-quarter of the normal complement of LHCI-730 was required to quench the reaction centre. By reducing the amount of Triton relative to the chlorophyll concentration, a PSI preparation (chlorophyll a/b ratio of 3.5) with a chlorophyll:P700 ratio of 300:1 was isolated. It contained no photosystem II, but a significant amount of LHCII which was functionally connected to the PSI reaction centre. Reconstitution studies demonstrated that excitation energy transfer from LHCII to PSI requires the presence of LHCI-680, and we propose that, in PSI, the following linear excitation energy transfer sequence occurs: LHCII----LHCI-680----LHCI-730----P700 Chla-P1.
Since exposure to blood products occurs on a daily basis during hemapheresis, the acquired immunodeficiency syndrome (AIDS) epidemic has a serious impact both for patients undergoing apheresis procedures as well as for health professionals working in the field. We studied serum samples from 110 patients who underwent therapeutic plasmapheresis for a variety of diseases not related to AIDS for the presence of antibodies to human immunodeficiency virus (HIV). Exchange fluids used in the majority of the patients were plasma protein fraction and 5% human albumin. Four patients received only fresh-frozen plasma. Fifty-five patients also received IV gammaglobulin. The follow-up period exceeded 24 months. All patients who did not belong to any known high-risk group for AIDS were negative for HIV antibodies prior to treatment and remained negative at last follow-up. Seven patients were homosexual men. All seven were seropositive prior to plasmapheresis and remained so throughout the treatment period. Seven health professional working in a busy haemapheresis unit were followed for 2 1/2 years. All remain HIV seronegative with normal immune function. These data indicate that transmission of HIV is unlikely through haemapheresis procedures.
The prehospital index (PHI) is a triage-oriented trauma severity scoring system. This prospective multicenter validation of the PHI was undertaken in response to a favorable pilot study. We applied the PHI to 3,581 patients from 14 different institutions during the period from January 1985 to February 1986. The PHI was accurate in predicting the need for emergency life-saving surgery within four hours (P less than .0001) and mortality within 72 hours (P less than .0001) following traumatic injury. The curves were generated for PHI versus emergency surgery, mortality, surgery and mortality, injury severity score, and ICU admission rate. These data compare favorably with those from previously published, prospectively tested, triage-oriented trauma severity scoring systems.