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Biomedical subjects

N L Brown

Publications and source records attributed to N L Brown.

At least 19 recordsLinked to original sources
PubMed1998

Detection of heavy metal ions at femtomolar levels using protein-based biosensors.

Sensors based on proteins (GST-SmtA and MerR) with distinct binding sites for heavy metal ions were developed and characterized. A capacitive signal transducer was used to measure the conformational change following binding. The proteins were overexpressed in Escherichia coli, purified, and immobilized in different ways to a self-assembled thiol layer on a gold electrode placed as the working electrode in a potentiostatic arrangement in a flow analysis system. The selectivity and the sensitivity of the two protein-based biosensors were measured and compared for copper, cadmium, mercury, and zinc ions. The GST-SmtA electrodes displayed a broader selectivity (sensing all four heavy metal ions) compared with the MerR-based ones, which showed an accentuated selectivity for mercury ions. Metal ions could be detected with both electrode types down to femtomolar concentration. The upper measuring limits, presumably due to near saturation of the proteins' binding sites, were around 10(-10) M. Control electrodes similarly constructed but based on bovine serum albumin or urease did not yield any signals. The electrodes could be regenerated with EDTA and used for more than 2 weeks with about 40% reduction in sensitivity.

Amino Acid Sequence↗
PubMed1998

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein A and rabbit IgG.

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbent assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.

Animals↗
PubMed1998

Interleukin-1beta and bacterial endotoxin change the metabolism of prostaglandins E2 and F2alpha in intact term fetal membranes.

There is strong evidence that prostaglandins E2 and F2alpha (PGE2 and PGF2alpha) are involved in the initiation and maintenance of human parturition and that their production can be stimulated by a number of cytokines and in infection-induced preterm labour by bacterial endotoxin. This study used an intact fetal membrane disk model to investigate the regulation of PGE2 and PGF2alpha metabolism by interleukin-1 beta (IL-1beta) and bacterial endotoxin [lipopolysaccharide (LPS)]. Fetal membrane explants were incubated with IL-1beta (0.1 or 1.0 ng/ml) or LPS (10 ng/ml) for 24 h. A mixture of 3H-prostaglandin (0.1 microCi) and unlabelled prostaglandin (1 microg) was then added at selected times after the addition of inflammatory mediators. The radiolabelled prostaglandins and their metabolites were then extracted from the culture medium and quantified by high-pressure liquid chromatography. Levels of prostaglandin metabolites were generally decreased following incubation with IL-1beta or LPS, which is consistent with a decrease in the activity of 15-hydroxyprostaglandin dehydrogenase (PGDH). It is concluded that IL-1beta and LPS moderately decrease the metabolism of prostaglandins, which may contribute to increasing the local levels of active prostaglandins induced by these stimuli.

Adult↗
PubMed1998

Horizontal spread of mer operons among gram-positive bacteria in natural environments.

Horizontal dissemination of the genes responsible for resistance to toxic pollutants may play a key role in the adaptation of bacterial populations to environmental contaminants. However, the frequency and extent of gene dissemination in natural environments is not known. A natural horizontal spread of two distinct mercury resistance (mer) operon variants, which occurred amongst diverse Bacillus and related species over wide geographical areas, is reported. One mer variant encodes a mercuric reductase with a single N-terminal domain, whilst the other encodes a reductase with a duplicated N-terminal domain. The strains containing the former mer operon types are sensitive to organomercurials, and are most common in the terrestrial mercury-resistant Bacillus populations studied in this work. The strains containing the latter operon types are resistant to organomercurials, and dominate in a Minamata Bay mercury-resistant Bacillus population, previously described in the literature. At least three distinct transposons (related to a class II vancomycin-resistance transposon, Tn1546, from a clinical Enterococcus strain) and conjugative plasmids are implicated as mediators of the spread of these mer operons.

Bacillus↗
PubMed1998

Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis.

We have identified Math5, a mouse basic helix-loop-helix (bHLH) gene that is closely related to Drosophila atonal and Xenopus Xath5 and is largely restricted to the developing eye. Math5 retinal expression precedes differentiation of the first neurons and persists within progenitor cells until after birth. To position Math5 in a hierarchy of retinal development, we compared Math5 and Hes1 expression in wild-type and Pax6-deficient (Sey) embryos. Math5 expression is downregulated in Sey/+ eyes and abolished in Sey/Sey eye rudiments, whereas the bHLH gene Hes1 is upregulated in a similar dose-dependent manner. These results link Pax6 to the process of retinal neurogenesis and provide the first molecular correlate for the dosage-sensitivity of the Pax6 phenotype. During retinogenesis, Math5 is expressed significantly before NeuroD, Ngn2 or Mash1. To test whether these bHLH genes influence the fates of distinct classes of retinal neurons, we ectopically expressed Math5 and Mash1 in Xenopus retinal progenitors. Unexpectedly, lipofection of either mouse gene into the frog retina caused an increase in differentiated bipolar cells. Directed expression of Math5, but not Xath5, in Xenopus blastomeres produced an expanded retinal phenotype. We propose that Math5 acts as a proneural gene, but has properties different from its most closely related vertebrate family member, Xath5.

Amino Acid Sequence↗
PubMed1998

A spontaneous induction of fetal membrane prostaglandin production precedes clinical labour.

Fetal membranes from term human pregnancies produce prostaglandins, and may respond to bacterial endotoxin or interleukin-1 beta (IL-1 beta) with increased prostaglandin E2 (PGE2) production. The effects of endotoxin persisted for up to 24 h, whereas those of IL-1 beta were maximal 4-8 h after addition. The maximum levels of PGE2 (200-350 pg/ml) were similar in all experiments, and were independent of the stimulus used. Not all tissues responded to these stimuli; those which did not had basal levels of PGE2 production of 200-350 pg/ml, which was not further increased by endotoxin or IL-1 beta. The basal production from these tissues was therefore similar to the maximal production from those tissues which responded to endotoxin or IL-1 beta. The high basal production of PGE2 was attributed to prior in vivo activation of the membranes such that PGE2 synthesis could not be further stimulated in vitro. Overnight pretreatment with aspirin decreased basal PGE2 production from these activated membranes to < 100 pg/ml/4 h during subsequent culture in aspirin-free medium. Both endotoxin and IL-1 beta increased PGE2 production from the activated aspirin-pretreated membranes during this culture time, but this was transient as after 12 h of culture basal PGE2 production rose to over 200 pg/ml despite aspirin pretreatment.

Analysis of Variance↗
PubMed1998

Regulation of prostaglandin production in intact fetal membranes by interleukin-1 and its receptor antagonist.

There is strong evidence for the involvement of inflammatory mediators such as interleukin (IL)-1 in the biochemical mechanisms of parturition. Therefore the effects of the IL-1 family (IL-1alpha (1 ng/ml), IL-1beta (1 ng/ml) and the IL-1 receptor antagonist (IL-1ra) (10 ng/ml)) on the regulation of prostaglandin synthesis in term human fetal membranes were investigated. It was found that, after 4 h of culture, IL-1beta increased prostaglandin E2 (PGE2) output approximately twofold. This was associated with both a significant increase in cyclo-oxygenase-2 (COX-2) mRNA levels (approximately fourfold compared with control) and translocation of cytoplasmic phospholipase A2 (cPLA2) from the cytosol to the membrane fraction. IL-1alpha was less effective than IL-1beta at stimulating PGE2 production through similar mechanisms. IL-1ra had no effect on PGE2 output. However, in combination treatments, IL-1ra did not inhibit IL-1alpha- or IL-1beta-stimulated PGE2 output, and increased PGE2 production further compared with IL-1beta alone. IL-1ra decreased IL-1beta-induced COX-2 mRNA expression by about half and significantly increased cPLA2 protein levels, as detected by immunoblotting, when used alone and together with IL-1beta. These results suggest that IL-1ra has partial agonist properties when used together with IL-1alpha and IL-1beta in fetal membranes by increasing cPLA2 protein levels, which leads to an increase in the production of prostaglandins.

Analysis of Variance↗
PubMed1998

[Study on the induction of pco promoters from Escherichia coli with copper and other metal ions].

Two copper inducible promoters in the pco determinant of Escherichia coli were studied by determining the luciferase activity of report vector pUCD615. The results showed that in the absence of pBIN19pco, providing copper resistance genes in trans, the maximum induction for both PpcoA lux fusions was observed at 5 mmol/L CuSO4 and PpcoA box was a stronger promoter than PpcoA long. There were two peaks in the bioluminescences of both PpcoE-lux fusions induced with increasing copper concentration, the first peak was observed at about 0.5 mmol/L CuSO4, the second peak, also the maximum induction, was observed at about 5 mmol/L CuSO4, and PpcoE long was a stronger promoter than PpcoE box. The results indicated that the PpcoE promoter was a much stronger promoter than PpcoA promoter. The results also showed that the copper box was very important and essential to pco promoters, since both of the Ppco short-lux fusions failed to show any luciferase activity when they were induced with copper. In the presence of pBIN19pco, the maximum inductions of all of the Ppco-lux fragments were observed at 6 mmol/L CuSO4 and they were much higher than those observed in the absence of pBIN19pco, and the results also indicated that the cells were able to resistant to much higher copper concentration in the presence of pBIN19pco than in the absence of that. Zn2+ and Ni2+ could be inducers for all of the fragments and Zn2+ was a better inducer than Ni2+, and Cd2+ and Ag+ did not induce the pco system.

Base Sequence↗
PubMed1997

The influence of deck storage and initial processing on patulin levels in apple juice.

Patulin, a secondary metabolite produced by Penicillium expansum and some other fungal species, is a common contaminant of ripened apples used for the production of apple juice concentrates. The limited availability of suitable storage facilities may result in fruit being subjected to storage in the open ('deck storage') for extended periods of time, prior to processing. A study was conducted to determine the influence that deck storage and subsequent initial processing practices had on patulin levels in freshly pressed juice. Over the study period, triplicate samples were collected at four strategic processing points from individual consignments of Granny Smith apples deck-stored for 7, 15 and 33 days, respectively. Over the study period, mean patulin levels in non-processed fruit increased from 90 to 2445 ng/g, respectively, but decreased to between 75 and 695 ng/g, respectively, following a water wash step. Subsequent removal of rotten/damaged fruit decreased patulin levels further (to between 55 and 405 ng/g, respectively), although the numerical decreases between sampling points were not shown to be statistically significant (P > 0.05). However, patulin levels were significantly higher (P < 0.05) in the rejected rotten/damaged fruit (mean levels ranged from 1120 to 6235 ng/g, respectively). P. expansum was the major patulin-producing fungus isolated from the juice samples. The mycological analyses tended to support the chemical data, in that removal of the rotten/damaged fractions significantly reduced total fungal counts in the juice samples.

Beverages↗
PubMed1997

Development of a managed-care team in a traditional home healthcare agency.

Increasing numbers of home health clients have managed-care insurance plans. Issues related to the care of these clients in the home are different from those that relate to the care of Medicare clients. The Visiting Nurse Association (VNA) of Northern Virginia developed a managed-care team whose staff is trained to work effectively and efficiently with managed-care companies while maintaining a goal of quality care. The evolution of the managed-care team concept is described and offered as a model for other traditional home health agencies. Critical success factors for implementing a dedicated managed-care team are defined. Speculation about the future of the VNA's managed-care team and the implications for nursing and home healthcare of the managed-care team concept are discussed.

Community Health Nursing↗
PubMed1997

Copper-inducible transcriptional regulation at two promoters in the Escherichia coli copper resistance determinant pco.

The pco determinant of Escherichia coli plasmid pRI1004 encodes inducible resistance to the trace element copper. The identification of two copper-dependent transcriptional initiation regions within pco that each contain a similar upstream hyphenated dyad motif is described. Deletion constructs showed that this 'copper box' motif was essential for copper-inducible activity at both pco promoters, PpcoA and PpcoE. The placement of the motif differs in the two promoters, and PpcoA contains an extended -10 nonamer typical of promoters for which RNA polymerase does not bind specifically to -35 sequences. PpcoE does not contain this motif and is the more strongly expressed promoter. The transcript from PpcoA contains the pcoABCDRS genes, while PpcoE expresses only pcoE. The induction profiles for PpcoA- and PpcoE-IacZ fusions were flattened sigmoidal curves with a gradual response to increasing copper concentration. On high-copy-number plasmids, zinc was found also to induce transcription from both promoters in vivo. Both promoters showed inducible activity in the absence of pcoRS, the plasmid-borne two-component regulatory system, indicating that a second trans-acting regulatory system is present on the chromosome. The pcoR product showed repressor action in the absence of pcoS, while still allowing induction, suggesting the chromosome encoded a similar two-component system to pco. TnphoA insertion mutagenesis identified chromosomal genes which affected promoter expression, including ptsH, ptsI (sugar phosphotransferase system) and cya (adenylate cyclase). The results support that idea that pco-encoded copper resistance is an auxiliary mechanism for handling copper, the regulation of which is integrated with the chromosomal regulation of cellular copper metabolism.

Chromosomes, Bacterial↗
PubMed1997

A process evaluation of condom availability in the Seattle, Washington public schools.

In 1993, Seattle began making condoms available in all of its 15 high schools. As part of an evaluation of this program, 16 focus groups with students from 13 high schools were conducted to explore students' perceptions of the school environment, program effectiveness, and suggestions for program improvement. The study also included data from a student survey conducted in 10 high schools. The focus group results revealed that students support school condom availability but have concerns about privacy when obtaining condoms. Students prefer obtaining condoms from baskets in private areas of school-based clinics to obtaining them from vending machines. Students do not believe that having condoms available has affected sexual activity or condom use among students. Recommendations for improving school condom availability include access to free condoms in private locations.

Adolescent↗
PubMed1997

[Conditions for the overexpressions of copper--resistant proteins from Escherichia coli].

There were five copper--resistant proteins encoded in the copper resistant plasmid pRJ1004 from Escherichia coli. The conditions for the overexpressions of two of the five, PcoC and PcoE, were reported in this paper. For the overexpression of PcoC using IPTG (Isopropyl-beta-D-thiogalactoside) as inducer, the best conditions were: medium, LB; IPTG concentration, 0.1 mmol/L; inducing time, one and half hours. For the overexpression of PcoE using CuSO4 as inducer, the best conditions were: medium, LB; CuSO4 concentration, 2.0 mmol/L; inducing time, one and halfhours.

Bacterial Proteins↗
PubMed1996

daughterless is required for Drosophila photoreceptor cell determination, eye morphogenesis, and cell cycle progression.

Initiation of Drosophila peripheral nervous system (PNS) development requires the achaete-scute complex (AS-C) and the atonal (ato) genes. The AS-C and ato encode basic helix-loop-helix (bHLH) transcription factors that dimerize in vitro with another bHLH protein, daughterless (da). da has many functions during Drosophila embryonic development, as it is required for proper sex determination, oogenesis, and neurogenesis. Here, we examine the expression and function of da within the developing Drosophila eye. The use of a monoclonal antibody to the Da protein revealed that Da levels are modulated across the developing eye disc. Within the morphogenetic furrow (MF) and photoreceptor cell R8, there is a cell-by-cell correspondence between high levels of Da protein expression and Ato protein expression. Mosaic analysis of adult tissue demonstrates that da function is cell autonomous and required within R2, R3, R4, R5, and R8. Examination of gene expression in da- imaginal disc clones reveals that da regulates Ato expression in the MF, affects the progression of the MF, and is necessary for the reestablishment of the G2 and M phases of the synchronized cell cycle posterior to the MF.

Animals↗
PubMed1996

Experimental venous thrombosis induced by homologous serum in the rat.

To investigate the effects of new drugs on the prevention of venous thrombosis, the Wessler stasis model is extensively employed usually in the rabbit. In this model, a hypercoagulable state is achieved by the administration of either heterologous serum (often human serum) or an activated coagulation factor followed by total stasis of the vein in which the thrombus is to be formed. Although classically the stasis model is performed in rabbits, it is possible to induce experimental venous thrombosis in a number of animal species, including rats. However, the use of homologous serum as the activating or thrombogenic agent has been previously described only in the dog and in the rabbit. The purpose of the present study was to compare the activities of heterologous and homologous serum as hypercoagulating agents in a rat stasis model of venous thrombosis using Wessler's technique. Heparin was employed as a reference antithrombotic compound in order to compare the two thrombogenic challenges.

Animals↗
PubMed1996

Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations.

The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system. Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity. Several mutant merT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Gly14Arg, Gly15Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter.

Amino Acid Sequence↗