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Biomedical subjects

B A Mitchell

Publications and source records attributed to B A Mitchell.

At least 19 recordsLinked to original sources
PubMed2001

Current progress in isolation and characterization of toxins isolated from Pfiesteria piscicida.

The isolation and partial purification of toxic substances derived from Pfiesteria piscicida Steidinger & Burkholder extracts is described. Four distinct bioassay systems were used to monitor bioactivity of the P. piscicida extracts, including a high throughput cell cytotoxicity assay and a reporter gene assay as well as assays using brine shrimp and fish. Using these bioassays to guide fractionation, we have isolated two distinct, active fractions from Pfiesteria culture medium and cell mass extracts on the basis of their solubility characteristics. We have identified and characterized a bioactive lipophilic substance from Pfiesteria-derived extracts as di(2-ethylhexyl)phthalate, a commonly used plasticizer. The source of this typically man-made substance has been identified as originating from Instant Ocean (Aquarium Systems, Mentor, OH, USA), a commercially available seawater salt mixture used to prepare our mass culture growth medium. We have developed chromatographic methodology to isolate a bioactive polar compound isolated from extracts of Pfiesteria culture and presently report the characterization of the activity of this substance. The molecular structural analysis of the polar active component(s) using mass spectrometry and nuclear magnetic resonance spectroscopy is currently under way.

Animals↗
PubMed1999

Bioenergetics of the staphylococcal multidrug export protein QacA. Identification of distinct binding sites for monovalent and divalent cations.

The multidrug efflux pump QacA from Staphylococcus aureus confers resistance to an extensive range of structurally dissimilar compounds. Fluorimetric analyses demonstrated that QacA confers resistance to the divalent cation 4',6-diamidino-2-phenylindole, utilizing a proton motive force-dependent efflux mechanism previously demonstrated for QacA-mediated resistance to the monovalent cation ethidium. Both the ionophores nigericin and valinomycin inhibited QacA-mediated export of ethidium, indicating an electrogenic drug/nH+ (n >/= 2) antiport mechanism. The kinetic parameters, Km and Vmax, were determined for QacA-mediated export of four fluorescent substrates, 4',6-diamidino-2-phenylindole, 3', 3'-dipropyloxacarbocyanine, ethidium, and pyronin Y. Competition studies showed that QacA-mediated ethidium export is competitively inhibited by monovalent cations, e.g. benzalkonium, and non-competitively inhibited by divalent cations, e.g. propamidine, which suggests that monovalent and divalent cations bind at distinct sites on the QacA protein. The quaternary ammonium salt, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, was used as a membrane-specific fluorescence probe and demonstrated that the amount of substrate entering the inner leaflet was significantly reduced in QacA-containing strains, supporting the notion that the substrate is extruded directly from the membrane.

Bacterial Proteins↗
PubMed1998

QacA multidrug efflux pump from Staphylococcus aureus: comparative analysis of resistance to diamidines, biguanidines, and guanylhydrazones.

The staphylococcal multidrug efflux pump QacA mediates resistance to a broad spectrum of monovalent and divalent antimicrobial cations. Resistance toward various classes of these compounds identified features of the substrate that may be important for interaction with QacA. Analysis of combinations of two substrates suggested that the same mechanism is used for the extrusion of different classes of compounds.

Bacterial Proteins↗
PubMed1998

Intergenerational exchanges and perceptions of support within "boomerang kid" family environments.

Drawing on the social exchange perspective, we examine: 1) the extent to which adult children who have returned to the parental home ("boomerang kids") exchange several types of instrumental and affective support with their parents, and 2) whether there is symmetry or incongruence in perceptions of support among these family dyads. The data used for this study are drawn from interviews with one child and one parent from 218 families in which the child has recently returned home. Findings indicate that children receive more frequent instrumental and emotional (affective) support than parents receive, and that parents perceive that they receive considerably more emotional support than boomerang children acknowledge donating. Implications for family relationships over the life course and household living arrangements are considered.

Adult↗
PubMed1996

Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity.

The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.

Amino Acid Sequence↗
PubMed1996

Low-molecular-weight inhibitor of in vitro fibroblast colony formation from human urine.

The role of urinary toxins in interstitial cystitis (IC) has been suggested. This report describes the partial purification of a substance from human urine that inhibited in vitro colony formation by mouse fibroblasts. Urine samples from 15 women with IC and 17 healthy women serving as volunteers were fractioned by ultrafiltration and chromatography methods and tested by the inhibition of Swiss 3T3 fibroblast colony formation. The fibroblasts were cultured at low density with varying concentrations of whole or fractioned urine. Colonies were counted at 10 days. Colony formation was reduced by incubation with whole urine, ultrafiltrate, and nonadsorbed C18 fractions. Inhibition of colony formation by urine from healthy volunteers or women with IC was not significantly different. In vitro colony formation by Swiss 3T3 cells was inhibited by a component of human urine. The toxicity of urine from IC patients was not different from that of urine from healthy controls.

Adult↗
PubMed1995

Semiautomated quantitation of urinary protein and creatinine.

The application of personal computers to routine tasks such as the recording and manipulation of data can greatly increase the speed and accuracy of the analyses. This report describes the use of a Macintosh computer and a BioTek microplate reader for the analysis of urinary protein and creatinine. Previously published assay methods for protein and creatinine were adapted for analysis using 96-well microplates. The adapted assays had excellent linearity (r2 = 0.99), with reduced variability compared to the previous methods. This resulted in increased sensitivity as demonstrated by 95% confidence limits. In addition, reductions of both assay materials and sample sizes were realized. The direct connection of the computer and microplate reader reduced the time needed to record absorbance measurements and perform analyses when compared to the more common protocols and methods of analysis. The assay protocols, equipment and interface, data file management, and methods of analysis are described.

Albuminuria↗
PubMed1995

Patient satisfaction and the LoFric catheter for clean intermittent catheterization.

A pilot study was initiated to determine patient satisfaction with the use of a newly available pre-lubricated, hydrophilic, disposable LoFric* catheter for clean intermittent catheterization. The study population consisted of 16 new patients and 25 experienced with clean intermittent catheterization. Patients were asked questions regarding convenience, ease of handling, comfort and general opinion of the catheters. Four new and 8 experienced patients dropped out of the study. Of the new patients who completed the study 75% found clean intermittent catheterization less troublesome than expected and all wish to continue using the catheter. Of the experienced patients 81% had a more favorable general opinion of the disposable than of the previous catheter, 81% found the disposable catheter to be more convenient and 88% thought it was easier to handle. It appears that most people will be satisfied with the disposable catheter and will prefer it as an alternative to a plastic catheter with lubrication applied by the patient.

Adult↗
PubMed1994

Complement C3, eosinophil cationic protein and symptom evaluation in interstitial cystitis.

Interstitial cystitis is a painful, irritative voiding dysfunction of unknown etiology. In this study 19 women undergoing treatment for interstitial cystitis and 6 healthy women of similar age provided 2 urine and serum specimens with at least a 3-month interval between collections. Complement C3 and eosinophil cationic protein were determined by immunoassay methods, and symptom severity was quantitated with a visual analog scale questionnaire. Concentrations of complement C3 and eosinophil cationic protein from either serum or urine were not significantly different between interstitial cystitis patients and controls at either determination, although substantial differences were noted even between individual initial and followup determinations. Normalization of urine osmolality did not alter these results. Symptom severity scores were significantly greater in interstitial cystitis patients compared to controls but failed to correlate with the concentrations of complement C3 or eosinophil cationic protein. Therefore, the hypothesis that complement C3 and eosinophil cationic protein may be etiological factors of interstitial cystitis is not supported.

Adult↗
PubMed1993

Uptake and transport of fluorescent derivatives of dolichol in human fibroblasts.

We are using fluorescent derivatives to visualize the endocytic transport of dolichol intermediates from the cell surface to the lysosome, and to estimate their rate of turnover within the lysosome. Anthroyl dolichol and anthroyl [1-14C]dolichol were synthesized and purified by chromatography on silica and C18 Sep-Paks followed by high-performance liquid chromatography on C18. The successful synthesis of anthroyl polyisoprenoid alcohols was confirmed by the use of uv-visible spectrometry and by fluorescence spectrometry. The purified esters were taken up into Ham's media containing 10-30% fetal calf serum or alternatively reconstituted into phospholipid liposomes for delivery to human fibroblasts in culture. The uptake of fluorescent dolichol esters into the cells and into lysosomes was demonstrated using fluorescence microscopy. The localization of anthroyl dolichol in lysosomes was further documented by simultaneously labeling fibroblasts with anthroyl dolichol and FITC-dextran a recognized lysosomal marker. Fibroblasts generally showed several groupings (domains) of lysosomes, some were dually labeled while others were labeled exclusively with either anthroyl dolichol or FITC-dextran. Labeling with anthroyl dolichol was very slow relative to labeling of the same fibroblasts with FITC-dextran suggesting that anthroyl dolichol acts as a labeling agent for intracellular membranes, particularly those of the lysosome while the dextran fluorescence is presumably of lysosolic origin. Several types of experiments were done with anthroyl [1-14C]dolichol to establish that the fluorescence seen in lysosomes represents anthroyl dolichol. Anthroyl dolichol appears to enter fibroblasts intact, since we were unable to recover any free [1-14C]dolichol from total lipid extracts of (i) media used for the uptake of anthroyl dolichol or (ii) the media removed from cells labelled for 42 h. In addition, attempts to hydrolyze anthroyl [1-14C]dolichol in vitro using whole fibroblast homogenates at pH 4.0 and 7.5 were unsuccessful, even though the fibroblasts expressed acid lipase activity using 4-methylumbelliferyl palmitate as substrate.

Biological Transport↗
PubMed1984

Prevalence of mastitis pathogens in herds participating in a mastitis control program.

Bacteriological data from herd surveys by the Massachusetts Mastitis Laboratory were analyzed. Comparisons of percent cows and quarters with major mastitis pathogens and types of bacteria isolated were between Streptococcus agalactiae-positive (1105 cows from 17 herds) and Streptococcus agalactiae-negative herds (1088 cows from 17 herds). Major mastitis pathogens were isolated from 58.5% of cows and 37.0% quarters in Streptococcus agalactiae-positive herds. The most frequently isolated bacteria were Streptococcus agalactiae and Staphylococcus aureus, and together these accounted for 87% of organisms isolated. In contrast, major mastitis pathogens were isolated from only 26.3% of cows and 10.2% of quarters in Streptococcus agalactiae negative herds. Streptococci other than Streptococcus agalactiae, Staphylococcus aureus, and coliforms were the predominant organisms isolated. Seventeen additional Streptococcus agalactiae negative herds were surveyed annually over 6 yr. An average of 25.8% of cows and 10.3% of quarters were positive for major mastitis pathogens during this time. The predominant bacteria isolated were streptococci other than Streptococcus agalactiae, Staphylococcus aureus, and coliforms. There was little variation between years.

Animals↗
PubMed1983

Susceptibility of bovine mammary gland to infections during the dry period.

Quarter foremilk samples (1,574) were collected for bacteriological analysis from 40 cows during late lactation, early involution, prior to parturition, parturition, and during early lactation. Six of 160 quarters were infected with major pathogens during late lactation. Twelve new infections occurred during early involution. Twenty-two quarters were infected at parturition. Of these infections, 12 occurred after early involution, and 10 infections that originated during early involution persisted throughout the dry period. During early lactation, 21 major pathogen infections were observed. Nine occurred after calving, and 12 persisted from parturition. Most major pathogen infections (93.8%) were caused by coliforms and streptococcal species other than Streptococcus agalactiae. Numbers of streptococcal infections were highest at parturition and early lactation. Numbers of quarters infected with coliforms were similar during early involution, parturition, and early lactation. Coagulase negative staphylococcal infections increased during the dry period, whereas Corynebacterium bovis infected quarters decreased. Udder infections were few in cows completing first or second dry periods. The incidence of udder infection was highest in cows completing third or later dry periods.

Animals↗
PubMed1983

Intramammary infections in primigravid heifers near parturition.

Mammary secretion from 32 primigravid heifers was obtained aseptically to determine frequency of bacterial isolation and incidence of intramammary infection near parturition. Quarter samples were collected 14 and 7 days prior to expected parturition, at parturition, and 7 and 14 days postparturition. Analysis of culture data indicated that 77.1% of samples were bacteriologically negative, and 15.7% contained coagulase negative staphylococci, 4.4% streptococci other than Streptococcus agalactiae, 3.8% coliforms, .8% coagulase positive staphylococci, and .1% Corynebacterium bovis. Frequency of bacterial isolation was highest in samples obtained prior to and at parturition. Thirty-five of 128 quarters were infected at parturition. Twenty-six of the 35 infections were caused by coagulase negative staphylococci, 4 by streptococci other than Streptococcus agalactiae, 4 by coliforms, and 1 by coagulase positive staphylococci. Twenty infections were observed during the early postpartum period. Coagulase negative staphylococcal infections decreased markedly. However, the number and type of primary pathogen infections during early lactation were similar to those at parturition.

Animals↗
PubMed1980

Variation of lactoferrin and mastitis and their heritabilities.

Analyses were of lactoferrin and mastitis of milk samples taken every other month from 830 Holstein cows in eight herds for 1 yr. Patterns of variation and amounts in lactation, colostrum, and dry period were similar to reports. In negative mastitis tests of milk samples, lactoferrin content was lower during lactation and lower as age of the cow advanced than for positive tests. However, heritability of mastitis was .14, whereas for lactoferrin was .44 in records of 289 cows. This latter is high enough to be useful but unreliable with large standard error .30. Several sire groups differed significantly.

Animals↗