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Biomedical subjects

N L Brown

Publications and source records attributed to N L Brown.

At least 37 records · Page 2Linked to original sources
PubMed2001

Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a predicted prolipoprotein signal peptidase. Downstream of pbrC, the pbrD gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration. Pb(II)-dependent inducible transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins. This is the first report of a mechanism for specific lead resistance in any bacterial genus.

Adenosine Triphosphatases↗
PubMed2001

Math5 is required for retinal ganglion cell and optic nerve formation.

The vertebrate retina contains seven major neuronal and glial cell types in an interconnected network that collects, processes and sends visual signals through the optic nerve to the brain. Retinal neuron differentiation is thought to require both intrinsic and extrinsic factors, yet few intrinsic gene products have been identified that direct this process. Math5 (Atoh7) encodes a basic helix-loop-helix (bHLH) transcription factor that is specifically expressed by mouse retinal progenitors. Math5 is highly homologous to atonal, which is critically required for R8 neuron formation during Drosophila eye development. Like R8 cells in the fly eye, retinal ganglion cells (RGCs) are the first neurons in the vertebrate eye. Here we show that Math5 mutant mice are fully viable, yet lack RGCs and optic nerves. Thus, two evolutionarily diverse eye types require atonal gene family function for the earliest stages of retinal neuron formation. At the same time, the abundance of cone photoreceptors is significantly increased in Math5(-/-) retinae, suggesting a binary change in cell fate from RGCs to cones. A small number of nascent RGCs are detected during embryogenesis, but these fail to develop further, suggesting that committed RGCs may also require Math5 function.

Animals↗
PubMed2000

MerF is a mercury transport protein: different structures but a common mechanism for mercuric ion transporters?

Mercury resistance determinants are widespread in Gram-negative bacteria, but vary in the number and identity of genes present. We have shown that the merF gene from plasmid pMER327/419 encodes a 8.7 kDa mercury transport protein, by determining in vivo mercury volatilisation when MerF is expressed in the presence of mercuric reductase. We have confirmed that MerC of Tn21 is also a mercuric ion transporter. We have been able to detect interaction of the periplasmic protein MerP only with the MerT transporter, and not with MerF or MerC. Hydropathy analysis led to the prediction of models for MerT, MerC and MerF having three, four and two transmembrane regions respectively. In all three cases one pair of cysteine residues is predicted to be within the inner membrane with a second pair of cysteine residues on the cytoplasmic face, and the second helix contains a proline and at least one charged residue. The mechanisms of mercuric ion transport may be similar in these transporters even though their structures in the membrane differ.

Amino Acid Sequence↗
PubMed2000

Bacterial metal-resistance proteins and their use in biosensors for the detection of bioavailable heavy metals.

We have expressed and purified metal-resistance and metal regulatory proteins from the bacterial determinants of resistance to heavy metals and utilised these in the development of biosensors for heavy metals. Both the metallothionein from the cyanobacterium Synechococcus PCC 7942 and the MerR regulatory protein from transposon Tn501 allow the detection of non-specific metal binding down to 10(-15) M concentrations of Hg(II), Cu(II), Zn(II) and Cd(II) in pure solution. Differential effects of the metals can be detected at both low and high concentrations, and the shape of the capacitance curves may reflect biologically relevant responses of the proteins to metals. Further work is required to establish the relationship between the detected binding of metal and the biological response of the protein, or to provide biosensors of use in the natural environment.

Bacterial Proteins↗
PubMed2000

The regulation of prostaglandin output from term intact fetal membranes by anti-inflammatory cytokines.

Prostaglandins are some of the main mediators which control parturition, and their production by intrauterine tissues can be up-regulated by pro-inflammatory cytokines. Anti-inflammatory cytokines may oppose these effects, and in this study we have investigated how two such cytokines affected fetal membrane function. Interleukin-10 (IL-10) inhibited the output of prostaglandin E2 (PGE2) from intact fetal membranes under basal and lipopolysaccharide (LPS)-stimulated conditions, and there was a parallel decrease in the expression of mRNA for COX-2. IL-10 also inhibited the production of interleukin-1beta (IL-1beta) and the expression of mRNA for IL-1beta, indicating that this cytokine has a broad anti-inflammatory effect. Transforming growth factor-beta1 (TGF-beta1), which is generally considered to be anti-inflammatory had opposite effects on PGE2 production, in that it increased the output of PGE2 for up to 8 hr. TGF-beta1 increased levels of type-2 cyclo-oxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) protein, and also activated the cPLA2 enzyme present; the profile of effects is similar to that of the pro-inflammatory cytokine IL-1beta, and was not expected. Combinations of TGF-beta1 with IL-1beta also increased PGE2 output and caused appropriate changes in prostaglandin pathway enzymes, whereas TGF-beta1 and IL-1alpha had more limited effects. Further studies are needed to establish the physiological significance of these findings, but TGF-beta1 does not seem to act as an inhibitory cytokine in intact fetal membranes at term.

Cell Membrane↗
PubMed1999

Type II restriction endonucleases from Helicobacter pylori include an enzyme with a novel recognition sequence.

Type II restriction endonuclease activities of Helicobacter pylori strain Roberts and of the type strain H. pylori NCTC 11637 were detected and analysed by conventional techniques. The endonucleases were partially purified, their optima for activity and their recognition and cleavage sites were determined. H. pylori (Roberts) contained at least two enzymes: HpyBI was an isoschizomer of RsaI (GT/AC) and HpyBII was of a novel specificity (GTN/NAC). H. pylori NCTC 11637 was found to contain an isoschizomer of EcoRV (HpyCI: GAT/ATC) and at least one other enzyme which was too unstable to characterise.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1999

Detection of Alloiococcus otitis in mixed bacterial populations from middle-ear effusions of patients with otitis media.

BACKGROUND: Otitis media is a potentially serious disorder, since there is a risk of permanent hearing loss. Culture methods are not useful in characterisation of populations of bacteria in the middle ear. We have used a PCR-based method that does not depend on prior knowledge of the bacteria identified by culture. METHODS: Middle-ear effusion fluid was obtained from 12 patients with chronic otitis media with effusion. Total DNA was extracted from the samples, and the hypervariable regions of bacterial 16S rRNA genes were amplified by means of broad-range PCR primers. Individual PCR products were segregated by cloning to allow analysis of mixed bacterial populations. FINDINGS: Many bacterial species were detected by PCR, whereas with culture-based approaches, no bacterial growth was detected for ten of the 12 patients. The gram-positive bacterium Alloiococcus otitis (A. otitidis) was detected by 16S rDNA amplification in six of the twelve samples, but not by culture techniques. Interpretation The method may have general usefulness in characterising bacterial populations at the site of infection and may indicate, from small sample numbers, organisms that are candidates for further investigation.

Adult↗
PubMed1999

Iterative selection from a Salmonella typhimurium cosmid library can lead to the isolation of an atypically small plasmid.

We have constructed a DNA library from a virulent Salmonella typhimurium strain, in an avirulent strain. The process of selecting the components of interest from the library involved iterative growth in liquid culture. This resulted, after four cycles, in the culture becoming homogeneous for a single plasmid, which was much smaller than the average size for the library. We have identified the larger precursor of this plasmid which has two regions of sufficient homology to allow recombination resulting in the formation of the small plasmid. S. typhimurium carrying the small plasmid have a smaller genetic burden than other members of the library and survive better in spent culture medium, facilitating selection on repeated subculture. Such rapid adventitious selection has important implications for isolation of clones of interest from genomic libraries.

Cosmids↗
PubMed1999

Sphinganine/sphingosine ratio in plasma and urine as a possible biomarker for fumonisin exposure in humans in rural areas of Africa.

This study was conducted in the Transkei region of the Eastern Cape and KwaZulu-Natal province, South Africa and in the Bomet district, western Kenya. The sphinganine (Sa)/sphingosine (So) ratios in the plasma and urine of male and female volunteers consuming a staple diet of home-grown maize in Transkei, were 0.34 +/- 0.36 (mean +/- standard deviation) (n = 154) and 0.41 +/- 0.72 (n = 153), respectively and in plasma samples from KwaZulu-Natal it was 0.44 +/- 0.23 (n = 26). In Kenya, the ratios in plasma and urine were 0.28 +/- 0.07 (n = 29) and 0.34 +/- 0.20 (n = 27), respectively. Mean total fumonisin level in home-grown maize, randomly collected in Transkei from the same region where the human volunteers lived, was 580 ng/g (n = 40), as compared to the KwaZulu-Natal province, where no fumonisin (n = 17) were detected (< 10 ng/g) in the home-grown maize. In Kenya, only one of seven samples was contaminated with 60 ng/g fumonisins. No significant differences were found in the Sa/So ratios between males and females within the regions nor between the different regions (P > 0.05). It is possible that the ratio is not sensitive enough to act as a biomarker for fumonisin exposure in humans at these levels of contamination in maize. This is the first report on Sa/So ratios determined in rural populations in Africa consuming home-grown maize as their staple diet.

Africa↗
PubMed1999

The production of interleukin-1beta from human fetal membranes is not obligatory for increased prostaglandin output.

Bacterial endotoxin increased the expression of mRNA (maximal after 4 hr) for interleukin-1beta (IL-1beta) and the release of mature protein from intact human fetal membranes. In contrast, the change in expression of mRNA for type 2 cyclo-oxygenase (COX-2) was biphasic, with peaks after 0.5-1 hr and after 8 hr of culture. An antibody to IL-1beta was without effect after 4 hr of culture, inhibited endotoxin-stimulated prostaglandin E2 (PGE2) production after 8 hr of culture, and caused a parallel decrease in the expression of mRNA for COX-2. We conclude that endotoxin induced the expression of COX-2 through IL-1beta-independent and IL-1beta-dependent mechanisms, and these differences are time dependent. Corticotrophin-releasing hormone (CRH) or platelet-activating factor (PAF) also increased the expression of mRNA for IL-1beta and the release of IL-1beta from some, but not all, fetal membranes. The antibody to IL-1beta did not affect CRH-stimulated or PAF-stimulated PGE2 production or COX-2 expression. We conclude that CRH and PAF can induce the expression of IL-1beta, but this is not obligatory for increased PGE2 release, and the effect of these stimuli on COX-2 expression is a direct, IL-1beta-independent effect.

Corticotropin-Releasing Hormone↗
PubMed1999

ZntR is a Zn(II)-responsive MerR-like transcriptional regulator of zntA in Escherichia coli.

We have identified the promoter/operator region of the zntA gene of Escherichia coli and shown that Zn(II) is the primary inducer of expression of this Zn(II)/Cd(II) export gene. The promoter PzntA shows sequence similarities to the promoters of mercury resistance (mer) operons, including a long spacer region containing an inverted repeat sequence. The gene encoding the transcriptional regulator of PzntA, designated zntR, has been identified from genome sequence data, by expression of the gene product and by insertional inactivation/complementation. The ZntR product is a member of the MerR family of transcriptional regulators and appears to act as a hypersensitive transcriptional switch. A hybrid MerR/ZntR protein has been constructed and indicates that the C-terminal region of ZntR recognizes Zn(II).

Adenosine Triphosphatases↗
PubMed1999

Corticotrophin-releasing hormone and platelet-activating factor induce transcription of the type-2 cyclo-oxygenase gene in human fetal membranes.

Corticotrophin-releasing hormone (CRH) and platelet-activating factor (PAF) are considered to be involved in the physiological processes of human labour. Both may have dual effects, directly regulating myometrial contractility and fetal membrane prostaglandin production. During this study, we investigated the mechanisms through which CRH and PAF exert their latter effect. CRH and PAF increased prostaglandin production from intact fetal membrane discs, with a maximum stimulation after 8 h of culture. Reverse transcription-polymerase chain reaction (RT-PCR) analyses using primers specific for type-2 cyclo-oxygenase (COX-2) showed that CRH and PAF increased the transcription of COX-2 mRNA two-fold after 8 h culture. These data indicate that the increased fetal membrane prostaglandin production in response to CRH or PAF may involve the induction of COX-2.

Corticotropin-Releasing Hormone↗
PubMed1999

Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human fetal membranes throughout pregnancy and at term.

Lipoxygenase metabolites may be involved in human parturition. 5-lipoxygenase (5-LOX) catalyses the first steps in the synthesis of leukotrienes from arachidonic acid, and its activity is dependent on 5-LOX activating protein (FLAP). The expression of 5-LOX and FLAP were investigated in fetal membranes to determine whether there are changes with gestational age or at term with the onset of labour. No significant differences were found in the expression of 5-LOX or FLAP mRNA in the amnion at different gestational ages or at term. In the chorion-decidua, 5-LOX mRNA expression was significantly higher in the first trimester of pregnancy than in the second and third trimesters. At term, there was a significant increase in both 5-LOX mRNA and protein expression in the chorion-decidua in the time after labour, compared with the time before labour. The expression of FLAP mRNA was also significantly higher in the chorion-decidua in the first trimester of pregnancy compared with the third trimester, and at term in the time after labour compared with the time before labour. Expression of FLAP protein was not studied, as an antibody is not currently available. These results are consistent with a role for 5-LOX and FLAP in the control of parturition at term, and also suggest an involvement earlier in pregnancy.

5-Lipoxygenase-Activating Proteins↗
PubMed1999

Transcriptional organization of the czc heavy-metal homeostasis determinant from Alcaligenes eutrophus.

The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. DNA sequencing of the region upstream of the czcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5' end of czcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of czc. Additional open reading frames upstream of czc had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide czcN message and a 400-nucleotide czcI message were found, and the presence of 6, 200-nucleotide czcCBA message (D. Van der Lelie et al., Mol. Microbiol. 23:493-503, 1997) was confirmed. Induction of czcN, czcI, czcCBA, and czcDRS followed a similar pattern: transcription was induced best by 300 microM zinc, less by 300 microM cobalt, and only slightly by 300 microM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from czcN to czcC and from czcD to czcS, but not between czcA and czcD nor between czcS and a 131-amino-acid open reading frame following czcS. The CzcR putative response regulator was purified and shown to bind in the 5' region of czcN. A reporter strain carrying a czcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were DeltaczcR and DeltaczcS mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn2+, (ii) induction of the beta-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of czcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.

Alcaligenes↗
PubMed1999

The impact of condom availability [correction of distribution] in Seattle schools on sexual behavior and condom use.

OBJECTIVES: Ten Seattle high schools made condoms available through vending machines, baskets in school clinics, or both. This study measured the number of condoms that students obtained and subsequent changes in sexual behavior and condom use. METHODS: Schoolwide surveys were administered in spring 1993 and in spring 1995, before and during the condom availability program. These data were compared with data from nationally representative surveys administered at the same time. RESULTS: Seattle students obtained an average of 4.6 condoms per year, the vast majority from baskets and very few from vending machines. Relative to the national samples, the percentage of Seattle students who had ever had sex remained stable after the program began; current sexual activity decreased significantly; and the percentage of sexually experienced students who used a condom the last time they had sex decreased significantly, particularly in the 5 schools with baskets of condoms in clinics. CONCLUSIONS: Making condoms available in Seattle schools enabled students to obtain relatively large numbers of condoms but did not lead to increases in either sexual activity or condom use.

Adolescent↗
PubMed1999

Generation of vector-insensitive dig-labeled probes from large cosmid library inserts using PCR.

We have devised a general method for producing vector-insensitive probes from clones in which insert DNA (ca. 40 kb) could not be amplified in one piece nor be excised from the vector sequence. The method involves PCR and vector-specific primers in combination with restriction digestion and ligation. It yields specific PCR products that could subsequently be labeled using DIG-11-dUTP in a single-cycle PCR. In colony blot hybridization, the probes were specific for the insert DNA from which the probe was derived and, importantly, did not detect vector sequences.

Blotting, Southern↗