Biomedical subjects

D Stevenson

Publications and source records attributed to D Stevenson.

At least 37 records · Page 2Linked to original sources

PubMed1999

Secreted human conjunctival mucus contains MUC5AC glycoforms.

This study addresses the extent of variation in secreted end-product mucins in human conjunctival mucus. The aim was to determine whether the variety of mucin species found was encompassed by the mucin genes which have been cloned to date. Extraction into guanidine hydrochloride and separation of mucin constituents, by a combination of cesium chloride density gradient centrifugation, size separation on Sepharose CL-2B, MonoQ ion exchange chromatography and agarose gel electrophoresis, demonstrates a complex mixture of mucins. Sample size limitations precluded compositional amino acid analysis. MUC 5AC and MUC1, 2, and 4 are all detected in the buoyant density range 1.3-1.5 g/ml by antibody binding. The mucins vary in size from >40 x 10(6)to <97 x 10(3)Da. A wide range of molecular size was confirmed using rate zonal centrifugation. The presence of smaller species contrasts with other mucous secretions similarly studied. In each size range are low, medium, and high charge mucins. Sialylation predominates in the medium charge and sulfate in the high charge. Only MUC5AC cross-reactivity is maintained throughout the analysis. It is detected in large and medium sized mucins but accounts for only the least mobile mucins within copurified species of similar density, size, and charge resolved using agarose electrophoresis. MUC5AC cross-reactivity is also detected in both medium and high charge species, indicating the presence of glycoforms.

Adolescent↗

PubMed1999

Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation.

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.

Amino Acid Sequence↗

PubMed1998

Determination of morphine in urine by solid-phase immunoextraction and high-performance liquid chromatography with electrochemical detection.

The analysis of morphine in biological fluids is of vital interest in monitoring opiate abuse and in drug abuse research. Although methods for analysis of morphine and its metabolites are well established, studies are still being carried out to improve sample preparation procedures as well as detection levels of morphine in biological samples. In this study, morphine-specific immunosorbents were developed to concentrate morphine prior to HPLC analysis. Urine (0.1 ml) was diluted 10-fold with phosphate-buffered saline, pH 7.4 (PBS), loaded onto a solid-phase immunoextraction column and washed with 15 ml PBS followed by elution with 2 ml of elution buffer (40% ethanol in PBS, pH 4). The eluted fraction was analysed for morphine by HPLC-electrochemical detection using a cyanopropyl (CN) analytical column with 25% acetonitrile in phosphate buffer-sodium lauryl sulphate, pH 2.4 as the mobile phase. Duration of the extraction procedure was approximately 40 min. Calibration graphs were linear from 100 ng ml-1 to 500 ng ml-1 in urine. The inter-assay R.S.D. was < 10% and the recovery of morphine from urine was > 98%. Immunocolumns demonstrated remarkably high specificity towards morphine showing minimal binding with other opiate metabolites such as codeine, normorphine, norcodeine, morphine-3-glucuronide, morphine-6-glucuronide.

Analgesics, Opioid↗

PubMed1998

Serum and urine levels of tamoxifen and its metabolites in patients with advanced breast cancer after a loading dose and at steady-state levels.

PURPOSE: To compare serum and urine levels of tamoxifen and metabolites after a loading dose and at the steady state. METHODS: A loading dose of 160 mg of tamoxifen was given to 14 patients with advanced breast cancer. Thereafter a regular daily dose of 30 mg of tamoxifen was given. Serum and urine levels of tamoxifen and metabolites were measured by high-performance liquid chromatography and compared with levels determined in 31 patients with advanced breast cancer at the steady state at a daily dose of 30 mg of tamoxifen. RESULTS: Serum and urine levels (24-h values) of tamoxifen and metabolites were lower (P < 0.05) after a loading dose than at the steady state. The difference was most pronounced for the metabolites, whereas the tamoxifen loading-dose level was near the steady state. CONCLUSION: Tamoxifen steady state can be reached in 1-2 days by the administration of a loading dose of 160 mg of tamoxifen for 2 days. Tamoxifen metabolite steady-state levels are reached regularly after 4 or more weeks during application of a loading dose. Very little tamoxifen or metabolites are excreted into the urine.

Adult↗

PubMed1998

Hematological and lipid changes in newborn piglets fed milk-replacer diets containing erucic acid.

Canola oil is not presently permitted in infant formulations in the United States because of lack of information concerning the effects of feeding canola oil to the newborn. We have previously reported a transient decrease in platelet counts and an increase in platelet size in newborn piglets fed canola oil for 4 wk, and have confirmed this in the present study. In canola oil-fed piglets, changes in platelet size and number were overcome by adding either long-chain saturated fatty acids from cocoa butter (16:0 and 18:0), or shorter-chain saturates from coconut oil (12:0 and 14:0). Feeding a high erucic acid rape-seed (HEAR) oil, with 20% 22:1n-9, led to an even greater platelet reduction and increased platelet size throughout the 4-wk trial. Bleeding times were longer in piglets fed canola oil or HEAR oil compared to sow-reared and soybean oil-fed piglets. There were no other diet-related changes. Diet-induced platelet changes were not related to platelet lipid class composition, but there were fatty acid changes. The incorporation of 22:1n-9 into platelet phospholipids of piglets fed canola oil was low (0.2-1.2%), and even for the HEAR oil group ranged from only 0.2% in phosphatidylinositol to 2.4% in phosphatidylserine. A much greater change was observed in the concentration of 24:1n-9 and in the 24:1n-9/24:0 ratio in platelet sphingomyelin (SM). The 24:1n-9 increased to 49% in the HEAR oil group compared to about 12% in animals fed the control diets (sow-reared piglets and soybean oil-fed group), while the 24:1n-9/24:0 ratio increased from about 1 to 12. Even feeding canola oil, prepared to contain 2% 22:1n-9, led to a marked increase in 24:1n-9 to 29% and had a 24:1n-9/24:0 ratio of 5. The canola oil/cocoa butter group, which also contained 2% 22:1n-9, showed a lower level of 24:1n-9 (20%) and the 24:1n-9/24:0 ratio (3) compared to the canola oil group. The results suggest that the diet-related platelet changes in newborn piglets may be related to an increase in 24:1n-9 in platelet SM, resulting from chain elongation of 22:1n-9. The inclusion of canola oil as the sole source of fat in the milk-replacer diets of newborn piglets resulted in significant platelet and lipid changes.

Animals↗

PubMed1998

Acute promyelocytic leukaemia with t(11;17)(q23;q12-21) and a good initial response to prolonged ATRA and combination chemotherapy.

Acute myeloid leukaemia (AML) of FAB subtype M3 is associated with t(15;17)(q22;q21) and a relatively good prognosis when treated with all-trans retinoic acid (ATRA) and combination chemotherapy. Rarely, alternative balanced translocations have been described in this subtype of AML. The translocation t(11;17)(q23;q21) leading to a PLZF/RARalpha rearrangement has been described in a very small number of cases and has been associated with a poor response to ATRA and an adverse prognosis. We describe a case of AML FAB type M3 with this translocation who entered morphological and cytogenetic complete remission after concurrent prolonged ATRA and one course of induction chemotherapy and remains in morphological and molecular remission at 10 months after presentation. This diagnosis therefore may not always be associated with a poor initial response to treatment.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

Role of oligosaccharides in the pharmacokinetics of tissue-derived and genetically engineered cholinesterases.

To understand the role of glycosylation in the circulation of cholinesterases, we compared the mean residence time of five tissue-derived and two recombinant cholinesterases (injected intravenously in mice) with their oligosaccharide profiles. Monosaccharide composition analysis revealed differences in the total carbohydrate, galactose, and sialic acid contents. The molar ratio of sialic acid to galactose residues on tetrameric human serum butyrylcholinesterase, recombinant human butyrylcholinesterase, and recombinant mouse acetylcholinesterase was found to be approximately 1.0. For Torpedo californica acetylcholinesterase, monomeric and tetrameric fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase, this ratio was approximately 0.5. However, the circulatory stability of cholinesterases could not be correlated with the sialic acid-to-galactose ratio. Fractionation of the total pool of oligosaccharides obtained after neuraminidase digestion revealed one major oligosaccharide for human serum butyrylcholinesterase and three or four major oligosaccharides in other cholinesterases. The glycans of tetrameric forms of plasma cholinesterases (human serum butyrylcholinesterase, fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase) clearly demonstrated a reduced heterogeneity and higher maturity compared with glycans of monomeric fetal bovine serum acetylcholinesterase, dimeric tissue-derived T. californica acetylcholinesterase, and recombinant cholinesterases. T. californica acetylcholinesterase, recombinant cholinesterases, and monomeric fetal bovine serum acetylcholinesterase showed a distinctive shorter mean residence time (44-304 min) compared with tetrameric forms of plasma cholinesterases (1902-3206 min). Differences in the pharmacokinetic parameters of cholinesterases seem to be due to the combined effect of the molecular weight and charge- and size-based heterogeneity in glycans.

Acetylcholinesterase↗

PubMed1998

Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menA gene from Escherichia coli.

A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1, 4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a menA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the orf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into the orf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.

Alkyl and Aryl Transferases↗

PubMed1998

Validating the Braden Scale for the prediction of pressure ulcer risk in blacks and Latino/Hispanic elders: a pilot study.

The purpose of this pilot study was to examine the Braden Scale for Predicting Pressure Sore Risk and determine which variables predict pressure ulcer risk in Black and Latino/Hispanic elders. A prospective clinical design was used to conduct the study. Among 36 patients, 60 years of age or older, 14 (38%) developed a pressure ulcer. A Fisher Exact test (2-tail) revealed that, at a cut-off score of 16, the Braden Scale significantly underpredicted those patients at risk for pressure ulcers (5.13 E-03). Sensitivity was 35 percent and specificity was 100 percent for a Braden Scale score of < or = 16.

Black or African American↗

PubMed1997

Bioanalysis of p-trifluoromethylmandelic acid and Mosher's acid by chiral gas chromatography and fluorine nuclear magnetic resonance to study chiral inversion: application to rat urine samples.

Methods for the nuclear magnetic resonance and gas chromatographic analysis of the enantiomers of p-trifluoromethylmandelic acid (p-TFM) and Mosher's acid (alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid) present in rat urine samples are described. Gas chromartography was performed using cyclodextrin capillary columns with both compounds analysed following derivatisation with methanolic HCl. Nuclear magnetic resonance was performed directly on the untreated urine samples following addition of beta-cyclodextrin. The methods were suitable for the determination of the individual enantiomers of the analytes in urine. Analysis of the rat urine samples indicated that the p-TFM had undergone a unidirectional enantiomeric interconversion in vivo, while the enantiomers of Mosher's acid were excreted unchanged.

Animals↗

PubMed1997

Anesthetic activity of novel water-soluble 2 beta-morpholinyl steroids and their modulatory effects at GABAA receptors.

(3 alpha,5 alpha)-3-Hydroxypregnan-20-ones and (3 alpha,5 alpha)-3-hydroxypregnane-11,20-diones bearing a 2 beta-morpholinyl substituent were synthesized, and the utility of these steroids as anesthetic agents was evaluated through determination of their potency and duration of hypnotic activity in mice after intravenous administration. Alkylation of the morpholinyl substituent or chlorination at C-21 afforded the novel amino steroids (2 beta,3 alpha,5 alpha)-3-hydroxy-2-(2,2-dimethyl-4-morpholinyl)-pregnane-11,20-dione (19) and (2 beta,3 alpha,5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one (37) that were more potent and advantageously produced shorter sleep times than related compounds which were previously reported. Furthermore, salts of these and other amino steroids generally retained good aqueous solubility. In a radioligand binding assay the compounds inhibited the specific binding of [35S]-tert-butyl bicyclophosphorothionate to rat whole brain membranes, and in an electrophysiological assay they potentiated GABAA receptor-mediated currents recorded from voltage-clamped bovine chromaffin cells. These in vitro results are consistent with the anesthetic activity of the amino steroids being related to their modulatory effects at GABAA receptors.

Anesthesia↗

PubMed1997

Aerosolization of infectious virus by excimer laser.

PURPOSE: To determine the potential for aerosolization of infectious virus present within the tear film during excimer laser photoablation of the cornea. METHODS: Cell monolayers infected with herpes simplex virus or adenovirus, simulating virus-infected corneas, were ablated with the 193-nm excimer laser. Adjacent dishes containing noninfected cell monolayers were subsequently assayed for viral infection. RESULTS: Viral spread to sentinel dishes occurred with both herpes simplex and adenovirus. The titer of virus present in the infected cell monolayers influenced the likelihood of spread to adjacent dishes. The presence of a vacuum aspiration system appeared to influence the direction of virus spread, with dishes located in the direction of the vacuum most likely to contain virus. CONCLUSIONS: The potential for aerosolization of infectious virus exists with photoablation using a large-diameter excimer laser beam. Our experimental design, however, does not prove that spread of infectious virus is likely to occur in the clinical setting. Appropriate measures should be taken to reduce the possibility of the spread of virus from the patient to the surgeon, other medical staff, or other patients.

Adenovirus Infections, Human↗

PubMed1997

Optimisation of the enantiomeric separation of 12 2-aminotetralin analogues using Chiral AGP high-performance liquid chromatography by simultaneous factorial design.

A method for the simultaneous optimisation of mobile phase composition for the resolution of pairs of enantiomers of 12 2-aminotetralin analogues is presented. The selectivity necessary to discriminate between 12 analytes was obtained by using mass selective detection. The ability to examine more than a few analytes at a time extends the otherwise limited applicability of a factorial design strategy to the rapid development of chiral assays.

Chromatography, High Pressure Liquid↗

PubMed1997

Enzyme immunoaffinity chromatography--a rapid semi-quantitative immunoassay technique for screening the presence of isoproturon in water samples.

Immunochromatography devices based on the principles of affinity chromatography and enzyme immunoassay have been developed to illustrate the possibility of providing extra-laboratory 'in the field' tests for pesticide monitoring. Isoproturon was chosen in this study as an example though other pesticides could have been used provided a suitable antiserum existed. The test system was prepared by immobilising isoproturon antibodies to porous silica which were then packed into disposable columns (immunoaffinity columns). The addition of chromagen-substrate 3,3',5,5'-tetramethylbenzidine into the immunoaffinity columns, after the application of a mixture containing an equal volume of isoproturon samples or isoproturon standard solutions with a fixed concentration of isoproturon labelled with horseradish peroxidase, allowed the development of a colorimetric portable assay capable of screening samples qualitatively for the presence of isoproturon in water. Samples with contamination levels of 0.12 microgram l-1 isoproturon and above were visually identified as positive samples in comparison to the zero standard sample. However, samples with concentrations below this level would be considered as negative (no isoproturon present at this limit of detection). The duration of the sample screening procedure on the column was less than 25 min making the technique highly suitable for a rapid estimate of isoproturon concentrations. Furthermore, the system could estimate isoproturon concentrations in water from various sources with no requirement for sample preparation. Therefore, the technique is ideally suited for monitoring the presence of pesticides in water. Further refinement of the assay could result in a test suitable for use by non-skilled personnel in extra-laboratory locations (e.g., a mobile or field laboratory).

Chromatography, Affinity↗

PubMed1997

Mixed immunosorbent for selective on-line trace enrichment and liquid chromatography of phenylurea herbicides in environmental waters.

An immunosorbent containing antiisoproturon and antichlortoluron antibodies immobilised on aldehyde-activated silica was employed for the on-line preconcentration and liquid chromatography-diode array detection of several phenylureas. The efficiency of the coupling of the immunosorbent to the liquid chromatographic system and the characteristics of the immunosorbent were evaluated. The on-line system allowed the selective trace enrichment of chlortoluron, isoproturon, metobromuron, linuron and chlorbromuron at the 0.05-0.5 microgram l-1 level in ground and river waters and provided detection limits in the range 0.01-0.03 microgram l-1 by percolating only 10 ml of water sample. The proposed method was validated by analysing freeze-dried tap water samples with a high content in pesticides of different chemical functionalities. The results obtained were compared with the mean value obtained in an interlaboratory exercise.

Chromatography, Liquid↗

PubMed1997

Adenovirus infection of the cornea causes histopathologic changes in the lacrimal gland.

PURPOSE: To explore the effects on the lacrimal gland of adenovirus infection of the cornea. METHODS: Rabbit corneas were inoculated with human adenoviruses Ad5, Ad14, or a rabbit adapted form of Ad 5, and in some instances booster inoculations were given. Sections of lacrimal glands removed 21-59 days post-inoculation were immunostained using antibodies against rabbit Class I and Class II MHC molecules, CD4, CD8, CD18, and rabbit thymic lymphocyte antigen (RTLA). Relative numbers of positively stained cells were quantified with a Metamorph image analysis system. RESULTS: RTLA and CD18 antigens were expressed on many interstitial cells in the normal lacrimal gland, but few expressed CD4 or CD8. The number of RTLA+ cells increased by 60-100% after inoculation of Ad5 and after boosting, and CD18+ cells increased from 33-100% after inoculation of Ad5 and after boosting. Booster inoculations also caused focal lymphocytic infiltration. MHC Class I was expressed on interstitial cells and duct epithelium, but not acinar cells, and there was no detectable difference after viral infection. In controls, MHC Class II was localized to a population of interstitial cells and a few acinar cells. A single inoculation of the Ad5 virus did not result in an increase in the total number of MHC Class II-positive cells at 21 days, but inoculation with the rabbit-adapted Ad 5 and booster inoculations caused a 30% increase. CONCLUSIONS: Ad5 and rabbit-adapted Ad5 infection of the cornea induce lymphocytic infiltration in the lacrimal gland, and the effect is enhanced by boosting. There is also an increase in expression of MHC Class II after inoculation with rabbit-adapted Ad5 and with booster inoculations.

Adenoviridae↗

PubMed1996

Stereoselective metabolism of two keto-pyrrol analogues of 8-hydroxy-2-dipropyl-aminotetralin by freshly isolated rat hepatocytes.

In vitro metabolism models have been used to determine the relative metabolic stability of novel 2-aminotetralin analogues for the treatment of CNS diseases. Few of these new compounds had been produced as stereochemically pure materials and the achiral analytical techniques, used initially, measured the average metabolic clearance of the two enantiomers of the racemic mixtures. A chiral HPLC assay, using a Chiral AGP column, was developed for two of these racemic analogues and was used to measure the clearance of the enantiomers from suspensions of freshly isolated rat hepatocytes. Robust separations were obtained for both compounds and a number of metabolic products. The enantiomers of only one analogue were subject to different rates of metabolism. The extent of the difference was dependent upon the initial starting concentration of the incubation. The identity of certain metabolites was investigated using LC/MS. The enantio-selectivity appears to have arisen from the restricted hydroxylation of one analogue compared to that of the other.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1996

Evaluation of Cidofovir (HPMPC, GS-504) against adenovirus type 5 infection in vitro and in a New Zealand rabbit ocular model.

The antiviral inhibitory activity of Cidofovir [1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine dihydrate, HPMPC, GS-504] against adenovirus type 5 (Ad5) in the New Zealand rabbit ocular replication model was evaluated. The 50% inhibitory dose (ID50) of Cidofovir was determined to be 4.7-9.5 micrograms/ml against four adenoviruses (two Ad5, Ad8 and Ad14) by plaque reduction assay in A549 cells. Twenty-four New Zealand rabbits received intrastromal inoculation and topical application of 2 x 10(6) plaque-forming units (PFU) per eye of Ad5 McEwen, a clinical isolate. Cidofovir was administered topically at three different concentrations twice per day, beginning 16 h postinoculation and continuing for 20 consecutive days. The inhibitory effects were determined by measuring suppression of virus replication and by observation of the clinical effects. Compared to the placebo group, the 1% and 0.5% Cidofovir-treated groups showed significantly reduced Ad5 ocular titers, fewer days of viral shedding and less severe subepithelial opacities (P = 0.0001). The 1% Cidofovir group had the lowest humoral antibody titer against adenovirus antigens, but the difference was not significant (P = 0.24). Cidofovir proved to have potent antiviral activity against adenovirus replication and may have great promise for the treatment of adenovirus infection. Further investigation is recommended.

Adenoviridae Infections↗