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Biomedical subjects

P Williams

Publications and source records attributed to P Williams.

At least 73 records · Page 4Linked to original sources
PubMed2001

Efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates.

We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of foreign gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 +/- 187 ng/g for the arm and 1692 +/- 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of beta-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable gene expression in human muscle without substantial toxicity.

Animals↗
PubMed2001

Impact desolvation of electrosprayed microdroplets--a new ionization method for mass spectrometry of large biomolecules.

Impact desolvation of electrosprayed microdroplets (IDEM) is a new method for producing gas-phase ions of large biomolecules. Analytes are dissolved in an electrolyte solution which is electrosprayed in vacuum, producing highly charged micron and sub-micron sized droplets (microdroplets). These microdroplets are accelerated through potential differences approximately 5 - 10 kV to velocities of several km/s and allowed to impact a target surface. The energetic impacts vaporize the droplets and release desolvated gas-phase ions of the analyte molecules. Oligonucleotides (2- to 12-mer) and peptides (bradykinin, neurotensin) yield singly and doubly charged molecular ions with no detectable fragmentation. Because the extent of multiple charging is significantly less than in atmospheric pressure electrospray ionization, and the method produces ions largely free of adducts from solutions of high ionic strength, IDEM has some promise as a method for coupling to liquid chromatographic techniques and for mixture analysis. Ions are produced in vacuum at a flat equipotential surface, potentially allowing efficient ion extraction.

Indicators and Reagents↗
PubMed2001

Quorum sensing as a population-density-dependent determinant of bacterial physiology.

The discovery that bacterial cells can communicate with each other has led to the realization that bacteria are capable of exhibiting much more complex patterns of co-operative behaviour than would be expected for simple unicellular microorganisms. Now generically termed 'quorum sensing', bacterial cell-to-cell communication enables a bacterial population to mount a unified response that is advantageous to its survival by improving access to complex nutrients or environmental niches, collective defence against other competitive microorganisms or eukaryotic host defence mechanisms and optimization of population survival by differentiation into morphological forms better adapted to combating environmental threats. The principle of quorum sensing encompasses the production and release of signal molecules by bacterial cells within a population. Such molecules are released into the environment and, as cell numbers increase, so does the extracellular level of signal molecule, until the bacteria sense that a threshold has been reached and gene activation, or in some cases depression or repression, occurs via the activity of sensor-regulator systems. In this review, we will describe the biochemistry and molecular biology of a number of well-characterized N-acylhomoserine lactone quorum sensing systems to illustrate how bacteria employ cell-to-cell signalling to adjust their physiology in accordance with the prevailing high-population-density environment.

4-Butyrolactone↗
PubMed2001

The influence of gender and the estrous cycle on learned helplessness in the rat.

Although the etiology of clinical depression is unknown, women are more likely to suffer from major depressive disorder than men. In addition, in some women, there is a clear association between depression and specific phases of the menstrual cycle. Surprisingly little research has examined gender differences and the influences of the estrous cycle in this and other animal behavioral models of clinical depression. Learned helplessness is a valid animal model of stress-induced behavioral depression in which prior exposure to inescapable stress produces deficits in escape testing. Learned helplessness was studied in rats using an inescapable tail shock stress followed by a shuttle box test to determine escape latencies. Animals with mean escape latencies of >or=20 s after shuttle-box testing are defined as learned helpless. Males and normal cycling female rats in the estrus and diestrus II phases were studied. Female rats in the diestrus II phase had significantly higher escape latencies and exhibited a more helpless behavior than female rats in the estrus phase. Male rat escape latencies were intermediate between the two female phases. These results suggest a role for gonadal hormones in the development of stress-induced behavioral depression or 'learned helplessness.'

Animals↗
PubMed2001

Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria.

Bacterial cell-to-cell communication (quorum sensing) relies upon the interaction of a small diffusible signal molecule with a sensor or transcriptional activator to couple gene expression with cell population density. In Gram-negative bacteria, it is now clear that N-acylhomoserine lactones bind directly to LuxR homologues and can be synthesized via one of three unrelated bacterial protein families and by transgenic plants. New chemical classes of signal molecules have been identified, some of which exhibit crosstalk with N-acylhomoserine-lactone-mediated quorum sensing. As the determinant of cell population density, quorum sensing is emerging as an integral component of bacterial global gene regulatory networks responsible for facilitating bacterial adaptation to environmental stress. N-acylhomoserine lactones are produced during experimental animal and human infections, and a function beyond quorum sensing has been suggested by their intrinsic immunomodulatory and pharmacological activities.

Gene Expression Regulation, Bacterial↗
PubMed2001

Natural levels of dimethyl sulfide in rough rice and its products.

Natural levels of dimethyl sulfide (DMS) in rough rice and its products (polished rice, brown rice, and broken rice) were determined by a gas chromatograph equipped with a flame photometric detector and sulfur mode, after extraction with 25% KBr solution in a sealed system. DMS was found to occur naturally in nine newly harvested and stored Australian varieties of rough rice and its products and decreased during storage after harvesting. Natural levels of DMS in rough rice and its products varied with variety, fraction, and period of storage. The order of levels of DMS was rough rice = brown rice > polished rice = broken rice. The range of values was 0.002-30 mg kg(-1) (ppm, w/w).

Australia↗
PubMed2001

Haemodynamic effects of the bacterial quorum sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, in conscious, normal and endotoxaemic rats.

N-acylhomoserine lactones (AHLs) are small, diffusible signalling molecules, employed by Gram-negative bacteria to coordinate gene expression with cell population density. Recent in vitro findings indicate that AHLs may function as virulence determinants per se, through modification of cytokine production by eukaryotic cells, and by stimulating the relaxation of blood vessels. In the present study, we assessed the influence of AHLs on cardiovascular function in conscious rats, and draw attention to the ability of the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), a signal molecule produced by P. aeruginosa, to cause marked bradycardia. This bradycardic effect was blocked by atropine and atenolol, and did not occur in vitro. Furthermore, modification of the acyl side chain length resulted in the loss of activity, whereas removal of the homoserine lactone ring, did not. The bradycardic effect of 3-oxo-C12-HSL was also observed in endotoxaemic animals, albeit attenuated. In normal rats, 3-oxo-C12-HSL caused initial mesenteric and hindquarters vasoconstriction, but only slight, and delayed signs of vasodilatation in the renal and mesenteric vascular beds. Furthermore, administration of 3-oxo-C12-HSL (pre-treatment or 2 h post-treatment) together with LPS, did not modify the established regional haemodynamic effects of the LPS, 6 h after the onset of its infusion. Our observations do not provide any clear evidence for an ability of 3-oxo-C12-HSL to modify the haemodynamic responses to LPS infusion. However, they are not inconsistent with the hypothesis that some of the cardiovascular sequelae of bacterial infection may be modulated by an influence of bacterial quorum sensing signalling molecules on the host.

4-Butyrolactone↗
PubMed2001

Structure, activity and evolution of the group I thiolactone peptide quorum-sensing system of Staphylococcus aureus.

In Staphylococcus aureus, the agr locus is responsible for controlling virulence gene expression via quorum sensing. As the blockade of quorum sensing offers a novel strategy for attenuating infection, we sought to gain novel insights into the structure, activity and turnover of the secreted staphylococcal autoinducing peptide (AIP) signal molecules. A series of analogues (including the L-alanine and D-amino acid scanned peptides) was synthesized to determine the functionally critical residues within the S. aureus group I AIP. As a consequence, we established that (i) the group I AIP is inactivated in culture supernatants by the formation of the corresponding methionyl sulphoxide; and (ii) the group I AIP lactam analogue retains the capacity to activate agr, suggesting that covalent modification of the AgrC receptor is not a necessary prerequisite for agr activation. Although each of the D-amino acid scanned AIP analogues retained activity, replacement of the endocyclic amino acid residue (aspartate) located C-terminally to the central cysteine with alanine converted the group I AIP from an activator to a potent inhibitor. The screening of clinical S. aureus isolates for novel AIP groups revealed a variant that differed from the group I AIP by a single amino acid residue (aspartate to tyrosine) in the same position defined as critical by alanine scanning. Although this AIP inhibits group I S. aureus strains, the producer strains possess a functional agr locus dependent on the endogenous peptide and, as such, constitute a fourth S. aureus AIP pheromone group (group IV). The addition of exogenous synthetic AIPs to S. aureus inhibited the production of toxic shock syndrome toxin (TSST-1) and enterotoxin C3, confirming the potential of quorum-sensing blockade as a therapeutic strategy.

Bacterial Proteins↗
PubMed2001

Health information: an evaluation of the use of touch screen kiosks in two hospitals.

An exit questionnaire survey of 150 hospital users of a touch screen health information kiosk was undertaken. It was compiled by Royal Cornwall Hospitals NHS to evaluate kiosk use and was reanalysed by researchers at City University to determine whether the information obtained went anyway to meeting patients' information needs. The study established two effective outcome variables on kiosk use: whether the information found answered the users' questions and if users intended, after using the kiosk, to find more information elsewhere. Two-thirds of respondents said that the information found had answered their question, whilst 16% said that after using the kiosk they still had further questions and intended to talk to a health professional to obtain this information. The variables found to have an impact on the information outcome included: gender, navigational ease, object of search, and ease of reading and understanding the information.

Adolescent↗
PubMed2001

Synthesis of multiple N-acylhomoserine lactones is wide-spread among the members of the Burkholderia cepacia complex.

Seventy strains of the Burkholderia cepacia complex, which currently comprises six genomic species, were tested for their ability to produce N-acylhomoserine lactone (AHL) signal molecules. Using thin layer chromatography in conjunction with a range of AHL biosensors, we show that most strains primarily produce two AHLs, namely N-octanoylhomoserine lactone (C8-HSL) and N-hexanoylhomoserine lactone (C6-HSL). Furthermore, some strains belonging to B. vietnamiensis (genomovar V) produce additional long chain AHL molecules with acyl chains ranging from C10 to C14. For B. vietnamiensis R-921 the structure of the most abundant long chain AHL was confirmed as N-decanoylhomoserine lactone (C10-HSL) by liquid chromatography-mass spectrometry (LC-MS) in combination with total chemical synthesis. Interestingly, a number of strains, most notably all representatives of B. multivorans (genomovar II), did not produce AHLs at least under the growth conditions used in this study. All strains were also screened for the production of extracellular lipase, chitinase, protease, and siderophores. However, no correlation between the AHL production and the synthesis of these exoproducts was apparent. Southern blot analysis showed that all the B. cepacia complex strains investigated, including the AHL-negative strains, possess genes homologous to the C8-HSL synthase cepI and to cepR, which encodes the cognate receptor protein. The nucleotide sequence of the cepI and cepR genes from one representative strain from each of the six genomovars was determined. Furthermore, the cepI genes from the different genomovars were expressed in Escherichia coli and it is demonstrated that all genes encode functional proteins that direct the synthesis of C8-HSL and C6-HSL. Given that cepI from the B. multivorans strain encodes a functional AHL synthase, yet detectable levels of AHLs were not produced by the wild-type, this suggests that additional regulatory functions may be present in members of this genomovar that negatively affect expression of cepI.

4-Butyrolactone↗
PubMed2001

Quorum sensing and the regulation of virulence gene expression in pathogenic bacteria.

For many pathogens, the outcome of the interaction between host and bacterium is strongly affected by the bacterial population size. Coupling the production of virulence factors with cell population density ensures that the mammalian host lacks sufficient time to mount an effective defence against consolidated attack. Such a strategy depends on the ability of an individual bacterial cell to sense other members of the same species and in response, differentially express specific sets of genes. Such cell-cell communication is called "quorum sensing" and involves the direct or indirect activation of a response regulator by a small diffusible signal molecule. A number of chemically distinct quorum-sensing signal molecules have been described including the N-acyl-L-homoserine lactones (AHLs) in Gram-negative bacteria and post-translationally modified peptides in Gram-positive bacteria. For example, the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus employ AHLs and peptides, respectively, to control the expression of multiple virulence genes in concert with cell population density. Apart from their role in signal transduction, certain quorum-sensing signal molecules, notably N-(3-oxododecanoyl)homoserine lactone, possess intrinsic pharmacological and immunomodulatory activities such that they may function as virulence determinants per se. While quorum-sensing signal molecules have been detected in tissues in experimental animal model and human infections, the mutation of genes involved in either quorum-sensing signal generation or signal transduction frequently results in the attenuation of virulence. Thus, interference with quorum sensing represents a promising strategy for the therapeutic or prophylactic control of infection.

Gene Expression Regulation, Bacterial↗
PubMed2001

Influence of lysates of the salivary glands of Lutzomyia longipalpis on the development of a Leishmania-major-like parasite in the skin of the golden hamster.

Twelve years ago, some mice inoculated with Leishmania major were found to develop larger lesions, containing more amastigotes, if the inoculum used to infect them contained a lysate of salivary glands from Lutzomyia longipalpis than if no lysate was included. In the present study, outbred golden hamsters (Mesocricetus auratus) were each inoculated in a footpad with 10(4), 10(5), 10(6) or 10(7) stationary-phase promastigotes of a Leishmania-major-like parasite (MHOM/BR/71/BH49). Some of the inocula used each contained a lysate of the salivary glands from a laboratory-reared, female Lu. longipalpis. Only the hamsters inoculated with 10(7) promastigotes each developed macroscopic cutaneous lesions (all 10 co-inoculated with lysate but only two of the 10 co-inoculated with diluent). Each of the lesions developed into cutaneous nodule affecting the dermis and underlying subcutaneous tissue of the inoculated footpad, with, histologically, an intensive, diffuse and productive, inflammatory reaction. There were no apparent differences between the lesions of hamsters infected with inocula containing salivary-gland lysate and those seen in the animals infected with lysate-free inocula. Future studies will follow the histological changes at the sites of Lu. longipalpis bites.

Analysis of Variance↗
PubMed2001

agr expression precedes escape of internalized Staphylococcus aureus from the host endosome.

Staphylococcus aureus is a versatile pathogen capable of causing life-threatening infections. Many of its cell wall and exoproduct virulence determinants are controlled via the accessory gene regulator (agr). Although considered primarily as an extracellular pathogen, it is now recognized that S. aureus can be internalized by epithelial and endothelial cells. Traditional experimental approaches to investigate bacterial internalization are extremely time-consuming and notoriously irreproducible. We present here a new reporter gene method to assess intracellular growth of S. aureus in MAC-T cells that utilizes a gfp-luxABCDE reporter operon under the control of the Bacillus megaterium xylA promoter, which in S. aureus is expressed in a growth-dependent manner. This facilitates assessment of the growth of internalized bacteria in a nondestructive assay. The dual gfp-lux reporter cassette was also evaluated as a reporter of agr expression and used to monitor the temporal induction of agr during the MAC-T internalization process. The data obtained suggest that agr induction occurs prior to endosomal lysis and that agr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.

Acyltransferases↗
PubMed2001

The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone.

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).

4-Butyrolactone↗
PubMed2001

The global posttranscriptional regulator RsmA modulates production of virulence determinants and N-acylhomoserine lactones in Pseudomonas aeruginosa.

Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a Pseudomonas aeruginosa gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of rsmA resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of rsmA in P. aeruginosa had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on N-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL) produced by the rsmA mutant and the rsmA-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using lasI and rhlI translational fusions. These data also highlighted the temporal expression control of the lasI gene, which was induced much earlier and to a higher level during the exponential growth phase in an rsmA mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control, hcn translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (hcnABC). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the hcnA ribosome-binding site. This suggests that, in P. aeruginosa, RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.

4-Butyrolactone↗
PubMed2001

Muscle fibre damage and regeneration resulting from surgical limb distraction.

Using an animal model of limb distraction, the extent of muscle fibre damage and atrophy resulting from distraction at two different rates (1.3 or 3.0 mm day(-1)) was investigated. It was found that at the high rate of distraction there was a significantly greater loss of range of joint movement and more muscle fibre atrophy and fibre damage than at the low rate. Muscle fibre damage is usually followed by regeneration. This involves the expression of the neonatal form of myosin heavy chain, which can therefore be used as an indicator of regeneration. It was found that whilst many more fibres showed evidence of damage at the high compared to the low rate, the number of fibres expressing neonatal myosin was significantly reduced, indicating the presence of a population of fibres which was undergoing degeneration without subsequent regeneration. Thus it would appear that beyond a certain rate of distraction, regeneration may be insufficient to replace contractile material damaged by overstretching. It is suggested that these fibres are replaced with connective tissue. This process may contribute to the muscle weakness and loss of range of joint movement which sometimes accompanies limb distraction procedures.

Animals↗