Biomedical subjects

N L Brown

Publications and source records attributed to N L Brown.

At least 73 records · Page 4Linked to original sources

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↗

PubMed1996

The antithrombotic potential of dalteparin in man assessed indirectly by Wessler's technique.

The aim of this study was to demonstrate the effectiveness of a new experimental approach for assessing the antithrombotic potential of low molecular weight heparins (LMWH) such as dalteparin. In this approach, sera obtained from healthy volunteers treated with various i.v. doses of dalteparin (30, 60 or 120 anti-Xa IU/kg) or placebo (physiological saline) were used as the thrombogenic challenge in a Wessler's stasis model of experimental venous thrombosis in the rat. Sera from placebo-treated volunteers showed a stable thrombogenic activity (0.25 ml/kg of serum producing thrombi of about 50 mg wet weight). Sera from healthy volunteers having previously received dalteparin however demonstrated dose- and time-related reductions in their thrombogenic activity. Half-lives of these effects were 300. 444 and > 480 min for 30, 60 and 120 anti-Xa IU/kg dalteparin respectively. These values were significantly higher than the corresponding anti-Xa and anti-IIa half-lives.

Animals↗

PubMed1995

Hairy and emc negatively regulate morphogenetic furrow progression in the Drosophila eye.

The initial steps of pattern formation in the developing Drosophila eye involve the coordination of cell cycles, changes in cell shape, and the specification of the R8 photoreceptor cell. These events begin several cell rows ahead of the morphogenetic furrow and are positively regulated by secreted signaling proteins and the proneural HLH transcription factor atonal (ato). Two HLH regulatory proteins that function to suppress neuronal development in other tissues, extra macrochaetae (emc) and hairy (h), are expressed ahead of the morphogenetic furrow. While neither h nor emc is required for photoreceptor cell determination, in emc-h-clones the morphogenetic furrow and differentiated eye field advance up to eight ommatidial rows ahead of adjacent wild-type tissue. This indicates that morphogenetic furrow progression and neuronal differentiation are negatively regulated by a combination of anteriorly expressed HLH regulatory proteins.

Animals↗

PubMed1995

The effect of the beta-D-xyloside naroparcil on circulating plasma glycosaminoglycans. An explanation for its known antithrombotic activity in the rabbit.

Beta-D-Xylosides are known to initiate or prime free glycosaminoglycan (GAG) chain synthesis in cell and tissue culture. As such, the effect of the venous antithrombotic beta-D-xyloside, naroparcil, was investigated on the plasma GAG profile in the rabbit after oral administration. Using dose-response experiments, we showed that antithrombin activity via antithrombin III and heparin cofactor II was increased in parallel with GAG plasma levels compared to control. A more detailed qualitative examination of plasma GAGs by cellulose acetate electrophoresis and ion-exchange chromatography, following oral administration of naroparcil at 400 mg/kg, revealed the presence of higher density charged molecules compared to control. The extracted GAGs were found to activate inhibition of thrombin by heparin cofactor II and contained approximately 25% of a dermatan sulfate-like compound (undetectable in control), which could be responsible for the antithrombotic effect. Using radiolabeled naroparcil, we found radiolabeled GAG fractions and the fact that naroparcil was a substrate for galactosyltransferase I, the second enzyme responsible for GAG chain polymerization, suggested that the compound could initiate in vivo the biosynthesis of antithrombotic free GAG chains. This is, to our knowledge, the first description of the in vivo effect of a beta-D-xyloside on GAG biosynthesis; furthermore, this is correlated with an antithrombotic action.

Animals↗

PubMed1995

The role of cysteine residues in the transport of mercuric ions by the Tn501 MerT and MerP mercury-resistance proteins.

Each cysteine residue in the MerT and MerP polypeptides of bacterial transposon Tn501 was replaced by serine, and the mercury-resistance phenotypes of the mutants were determined in Escherichia coli. Cys-24 and Cys-25 in the first transmembrane region of MerT were essential for transport of mercuric ions through the cytoplasmic membrane, and mutations Cys-76-Ser, Cys-82-Ser or Gly-38-Asp in MerT or Cys-36-Ser in MerP all reduced transport and resistance. Deletion of the merP gene slightly reduced mercuric ion resistance and transport, whereas a Cys-33-Ser mutation in MerP appears to block transport of mercuric ions by MerT. The effects of deleting merP on mutations in merT were tested. The 116-amino-acid MerT protein is sufficient for mercuric ion transport across the cytoplasmic membrane.

Amino Acid Sequence↗

PubMed1995

Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004.

The copper-resistance determinant (pco) of Escherichia coli plasmid pRJ1004 was cloned and sequenced. Tn1000 transposon mutagenesis identified four complementation groups, mutations in any of which eliminated copper resistance. DNA sequence analysis showed that the four complementation groups contained six open reading frames, designated pco-ABCDRS. The protein product sequences derived from the nucleotide sequence show close homology between this copper-resistance system and the cop system of a plasmid pPT23D of Pseudomonas syringae pv. tomato. The PcoR and PcoS protein sequences show homology to the family of two-component sensor/responder phosphokinase regulatory systems. A seventh reading frame (pcoE) was identified from DNA sequence data, and lies downstream of a copper-regulated promoter. Transport assays with 64Cu(II) showed that the resistant cells containing the plasmid had reduced copper accumulation during the log phase of growth, while increased accumulation had previously been observed during stationary phase. Chromosomal mutants defective in cellular copper management were obtained and characterized. In two of these mutants pco resistance was rendered totally inactive, whilst in another two mutants pco complemented the defective genes. These data indicate that plasmid-borne copper resistance in E. coli is linked with chromosomal systems for copper management.

Amino Acid Sequence↗

PubMed1995

Biological and genetic characterization of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis.

TnphoA transposon insertion mutants of phoN-negative derivatives of Salmonella typhimurium TML (of human gastroenteritic origin) were selected by growing mutagenized recipient bacteria under a variety of growth conditions. Ninety-seven individual mutants, which expressed alkaline phosphatase, were collected and tested for their ability to invade HEp-2 cells. Seven smooth mutants had a reduced ability to invade HEp-2 cells, and three smooth mutants were consistently more invasive than their corresponding parental strains. One rough mutant was of similar invasiveness and two were of reduced invasiveness when compared with that of parental strains. The seven smooth hypoinvasive mutants, the three smooth hyperinvasive mutants, and the three rough mutant strains were tested for their abilities to invade ileal enterocytes by the rabbit ileal invasion assay described previously (3). All smooth mutants exhibited parental levels of invasiveness. The rough mutants were hypoinvasive in the rabbit ileal invasion assay. The HEp-2 system is therefore not a good predictor of behavior in gut tissue in this model. DNA sequences flanking the transposon were determined for five mutants which were hypoinvasive in the HEp-2 cell assay. The mutations were found to be insertions in two previously identified invasion genes, invG and invH, and in a gene not normally associated with invasion, pagC. These observations lead one to be cautious in the interpretation of the biological significance of data obtained from invasion of tissue culture monolayers when extrapolated to gut tissue.

Alkaline Phosphatase↗

PubMed1995

Stimulus-response compatibility and videogame performance.

We investigated the role of stimulus-response compatibility in influencing manual reactions to a moving visual target in a videogame (pong). 40 right-handed university men were assigned randomly to one of two experimental conditions, the normal game condition or a reverse control condition in which the response device on the right controls the left game paddle and the device on the left controls the right paddle. Subjects in the normal condition performed marginally better playing pong when seated on the right when seated on the left, consistent with earlier findings. However, subjects in the reverse control condition showed the reverse effect, a left-side advantage. These findings suggest that compatibility between location of the moving target (the ball) and either handedness or hand used to respond may have been responsible for the right-side advantage observed in the normal-game condition.

Adult↗

PubMed1994

Accumulation and intracellular fate of tellurite in tellurite-resistant Escherichia coli: a model for the mechanism of resistance.

The tellurite accumulation properties of three Escherichia coli strains containing different tellurium-resistance determinants of Gram-negative origin, from plasmids pMER610, pHH1508a and RK2, were compared. In all three cases membrane-associated tellurium crystallization was observed, and neither reduced uptake nor increased export contributed to the resistance. Specific membrane-proximal reduction is proposed as the mechanism of resistance to tellurite coded by all three determinants, despite their lack of sequence homology.

Crystallization↗

PubMed1994

The venous antithrombotic profile of naroparcil in the rabbit.

The venous antithrombotic profile of naroparcil or (4-[4-cyanobenzoyl]-phenyl)-1.5-dithio-beta-D-xylopyranoside was investigated in the rabbit following single i.v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i.v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i.v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same dose of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human alpha-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

PubMed1993

Construction and characterization of a mercury-independent MerR activator (MerRAC): transcriptional activation in the absence of Hg(II) is accompanied by DNA distortion.

The MeR regulatory protein of transposon Tn501 controls the expression of the mercury resistance (mer) genes in response to the concentration of mercuric ions. MerR is unique among prokaryotic regulatory proteins so far described in that it acts as a repressor [-Hg(II)] and an activator [+Hg(II)] of transcription of the mer genes, but binds to a single site on the DNA in both cases. This transcriptional activation process has been postulated to involve a protein-induced conformational change in the DNA that allows RNA polymerase more readily to form an open complex at the promoter. It has been shown [Frantz and O'Halloran (1990) Biochemistry, 29, 4747-4751] that activation of transcription by MerR in the presence of mercury is accompanied by hypersensitivity of the operator to chemical nucleases that are sensitive to local distortion in DNA structure. Here we describe specific mutations in MerR that allow the protein to stimulate transcription in the absence of the allosteric activator Hg(II). We demonstrate that the degree of activation caused by these mutants directly correlates with the degree of DNA distortion as measured by the hypersensitivity of MerR-DNA complexes to the nuclease Cu-5-phenyl-o-phenanthroline. These results support the model described above.

Allosteric Regulation↗

PubMed1993

Copper-resistant enteric bacteria from United Kingdom and Australian piggeries.

Thirty-three enteric isolates from Australian (Escherichia coli only) and United Kingdom (U.K.) (Salmonella sp., Citrobacter spp., and E. coli) piggeries were characterized with respect to their copper resistance. The copper resistance phenotypes of four new Australian E. coli isolates were comparable with that of the previously studied E. coli K-12 strain ED8739(pRJ1004), in that the resistance level in rich media was high (up to 18 mM CuSO4) and resistance was inducible. Copper resistance was transferable by conjugation from the new Australian isolates to E. coli K-12 recipients. DNA similarity between the new Australian isolates and the pco copper resistance determinant located on plasmid pRJ1004 was strong as measured by DNA-DNA hybridization; however, the copper resistance plasmids were nonidentical as indicated by the presence of restriction fragment length polymorphisms between the plasmids. DNA-DNA hybridization and polymerase chain reaction analysis demonstrated DNA homology between the pco determinant and DNA from the U.K.E. coli, Salmonella sp., and Citrobacter freundii isolates. However, the copper resistance level and inducibility were variable among the U.K. strains. Of the U.K. E. coli isolates, 1 demonstrated a high level of copper resistance, 4 exhibited intermediate resistance, and 16 showed a low level of copper resistance; all of these resistances were expressed constitutively. A single U.K. C. freundii isolate, had a high level of copper resistance, inducible by subtoxic levels of copper. Transconjugants from one E. coli and one C. freundii donor, with E. coli K-12 strain UB1637 as a recipient, showed copper resistance levels and inducibility of resistance which differed from that expressed from plasmid pRJ1004.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Husbandry↗

PubMed1992

The venous antithrombotic effect of LF 1351 in the rat following oral administration.

The venous antithrombotic effects of a novel chemical entity, LF 1351, were investigated in rats following single oral administration, in comparison with i.v. administered heparin. LF 1351 demonstrated a dose-related antithrombotic effect in three models of venous thrombosis. The compound was approximately equipotent in two models involving complete stasis of the vena cava and administration of factor Xa or porcine serum, giving respective ED50 values of 48.7 mg/kg and 36.7 mg/kg. LF 1351 was less effective in a model involving partial stasis in the presence of an endothelial lesion. In this case, the antithrombotic effect did not exceed 60-65%, the ED50 being 150 mg/kg. Heparin (50-300 micrograms/kg; 7.5-45.0 U/kg) was effective in all three models. At the approximate ED80 value against factor Xa-induced thrombosis (75 mg/kg) the antithrombotic effect of LF 1351 persisted for 6 h. The antithrombotic effect of LF 1351 (300 mg/kg) occurred without significant changes in APTT or thrombin time.

Administration, Oral↗

PubMed1992

Copper resistance determinants in bacteria.

Copper is an essential trace element that is utilized in a number of oxygenases and electron transport proteins, but it is also a highly toxic heavy metal, against which all organisms must protect themselves. Known bacterial determinants of copper resistance are plasmid-encoded. The mechanisms which confer resistance must be integrated with the normal metabolism of copper. Different bacteria have adopted diverse strategies for copper resistance, and this review outlines what is known about bacterial copper resistance mechanisms and their genetic regulation.

Amino Acid Sequence↗

PubMed1991

A complex family of class-II restriction endonucleases, DsaI-VI, in Dactylococcopsis salina.

A series of class-II restriction endonucleases (ENases) was discovered in the halophilic, phototrophic, gas-vacuolated cyanobacterium Dactylococcopsis salina sp. nov. The six novel enzymes are characterized by the following recognition sequences and cut positions: 5'-C decreases CRYGG-3' (DsaI); 5'-GG decreases CC-3' (DsaII); 5'-R decreases GATCY-3' (DsaIII); 5'-G decreases GWCC-3' (DsaIV); 5'-decreases CCNGG-3' (DsaV); and 5'-GTMKAC-3' (DsaVI), where W = A or T, M = A or C, K = G or T, and N = A, G, C or T. In addition, traces of further possible activity were detected. DsaI has a novel sequence specificity and DsaV is an isoschizomer of ScrFI, but with a novel cut specificity. A purification procedure was established to separate all six ENases, resulting in their isolation free of contaminating nuclease activities. DsaI cleavage is influenced by N6-methyladenine residues [derived from the Escherichia coli-encoded DNA methyltransferase (MTase) M.Eco damI] within the overlapping sequence, 5'-CCRYMGGATC-3'; DsaV hydrolysis is inhibited by a C-5-methylcytosine residue in its recognition sequence (5'-CMCNGG-3'), generated in some DsaV sites by the E. coli-encoded MTase, M.Eco dcmI.

Base Sequence↗

PubMed1991

Bacterial resistances to mercury and copper.

Heavy metals are toxic to living organisms. Some have no known beneficial biological function, while others have essential roles in physiological reactions. Mechanisms which deal with heavy metal stress must protect against the deleterious effects of heavy metals, yet avoid depleting the cell of a heavy metal which is also an essential nutrient. We describe the mechanisms of resistance in Escherichia coli to two different heavy metals, mercury and copper. Resistance of E. coli to mercury is reasonably well understood and is known to occur by transport of mercuric ions into the cytoplasmic compartment of the bacterial cell and subsequent reductive detoxification of mercuric ions. Recent mutational analysis has started to uncover the mechanistic detail of the mercuric ion transport processes, and has shown the essential nature of cysteine residues in transport of Hg(II). Resistance to copper is much less well understood, but is known to involve the increased export of copper from the bacterial cell and modification of the copper; the details of the process are still being elucidated. Expression of both metal resistance determinants is regulated by the corresponding cation. In each case the response enables the maintenance of cellular homeostasis for the metal. The conclusions drawn allow us to make testable predictions about the regulation of expression of resistance to other heavy metals.

Amino Acid Sequence↗

PubMed1991

hairy gene function in the Drosophila eye: normal expression is dispensable but ectopic expression alters cell fates.

The regulatory gene hairy is expressed and required during early embryogenesis to control segmentation gene expression properly and during larval and pupal development to control the pattern of certain adult sensory structures. We have found the hairy protein to be expressed transiently during two stages of eye imaginal disc development, including all cells immediately anterior to the morphogenetic furrow that traverses the developing eye disc, and again in the presumptive R7 photoreceptor cells of the developing ommatidia. This pattern is conserved in a significantly diverged Drosophila species. We show that, surprisingly, ommatidia formed by homozygous hairy- mutant clones are apparently normal, indicating that hairy function in the eye is dispensable. However, we do find that ectopic expression of hairy causes numerous structural abnormalities and the alteration of cell fates. Thus, proper regulation of hairy is still essential for normal eye development. We suggest that the loss of hairy function may be compensated by other regulatory proteins, as has been observed previously for several structurally and functionally related genes involved in sensory organ development. The effects of ectopic hairy expression may result from interactions with proneural genes involved in the development of the eye and other sensory organs.

Animals↗