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M Wilson

Publications and source records attributed to M Wilson.

At least 19 recordsLinked to original sources
PubMed1996

Age-dependent silencing of globin transgenes in the mouse.

Variegation of transgene expression, a heterocellular or mosaic pattern of expression seen in all mice in a given transgenic line, is a frequently observed but unexplained phenomenon. We have encountered variegation with globin transgenes; when lacZ expression is driven by globin control elements a proportion of erythrocytes express beta-galactosidase (beta-gal), while the remaining erythrocytes express none. The percentage of expressing cells is constant within each line (at any particular developmental stage), but varies between lines. Such variation may account for much of the line-to-line variability which has been reported in the expression of a transgene construct. We have now extended these observations by studying expression of several globin/lacZ transgenes with increasing age. Expression of beta-gal is variegated in all lines in adult mice, including those made with a beta-globin promoter and locus control region driving lacZ. The extent of variegation differs widely between lines, but in all lines there is a marked decline in the number of erythrocytes expressing beta-gal with increasing age. Progression of silencing continues long past the point at which globin switching is complete, suggesting that it is not related to this process. We observe that age-dependent silencing is most severe in high copy number animals. Increasing variegation of transgene expression with ageing of mice is likely to complicate interpretation of the developmental regulation of transgenes. We speculate that it reflects a general mechanism of epigenetic regulation.

Aging↗
PubMed1996

Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors.

In order to study the regulation of the ribosomal protein S6 kinase, p70s6k, by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the alpha2 adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human alpha2C10 receptor, with the alpha2 agonist UK 14304 led to a transient increase in p70s6k activity. UK 14304 also activated p70s6k in a clone expressing the porcine alpha2A receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70s6k in alpha2 receptor-transfected and in nontransfected cells. Activation of p70s6k by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70s6k by both agents. Activation of p70s6k by UK 14304 and by LPA, but not by platelet-derived growth factor (PDGF), was blocked by preincubation of cells with pertussis toxin. Wortmannin, a selective inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of p70s6k by UK 14304, LPA, and PDGF. These data indicate that p70s6k is regulatable by Gi-coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70s6k by such agents appears to involve an isoform of PI 3-kinase.

Adrenergic alpha-Agonists↗
PubMed1996

Membrane exon sequences of the three Xenopus Ig classes explain the evolutionary origin of mammalian isotypes.

We have cloned and sequenced the genes corresponding to the membrane exons of the three immunoglobulin (Ig) heavy chain isotypes (mu, upsilon and chi) of Xenopus. Among membrane Ig (mIg) polypeptides, the transmembrane domain are the most highly conserved. The transmembrane and cytoplasmic domains of Xenopus mIgM are similar to the corresponding domains of all known vertebrate mIgM molecules, supporting the idea that amphibian mu gene is homologous, not just analogous, to the mu gene of higher vertebrates. The membrane forms of the two other Ig isotypes mIgX and mIgY exhibit the specific structure found in all Ig membrane exons, but are not homologous with any specific mammalian non-mu Ig isotype; they are most similar to Xenopus mIgM. Based on the conserved transmembrane domains of Xenopus mIgX, mIgY, we suggest that first the upsilon and later the chi genes arose by duplication from the original mu gene. The transmembrane and the 37-amino-acid-long cytoplasmic domains of Xenopus mIgY have conserved residues found in avian mIgY and mammalian mIgG and mIgE, suggesting that the modern isotypes might share a common ancestor with amphibian mIgY. However, while the sequence similarity between the membrane exons of avian mIgY and mammalian mIgG and IgE is striking, the overall similarity with Xenopus mIgY is very low. Thus, the genes giving rise to Xenopus mIgY and those eventually leading to avian mIgY and mammalian mIgG and mIgE must have diverged early in evolution, probably at the level of the primitive amphibians or before.

Amino Acid Sequence↗
PubMed1996

The killing of Helicobacter pylori by low-power laser light in the presence of a photosensitiser.

Helicobacter pylori is associated with various gastrointestinal disorders. Lethal photosensitisation was investigated as a possible technique for killing H. pylori which might offer a better alternative to antibiotics. The susceptibility of H. pylori to lethal photosensitisation was determined by mixing suspensions of H. pylori with various photosensitisers and plating out on blood agar before irradiation with low-power laser light. Five sensitisers were studied further by mixing them with H. pylori in a tissue-culture plate and counting survivors after irradiation as a function of laser exposure time, dye concentration and pre-irradiation time. Crystal violet and thionine were ineffective as sensitisers, but zones of inhibition appeared with methylene blue (MB), protoporphyrin IX (PPIX), haematoporphyrin derivative (HPD), toluidine blue O (TBO) and disulphonated aluminium phthalocyanine (S2). Laser light or sensitiser alone did not affect bacterial viability. S2 (100 microg/ml) with a laser light energy density of 16 J/cm2, HPD (10O microg/ml) with 160 J/cm2, MB (100 microg/ml) with 21 J/cm2, PPIX (150 microg/ml) with 320 J/cm2 and TBO (50 microg/ml) with 160 J/cm2 all reduced bacterial viability by >99%. The killing of sensitised H. pylori by laser light offers a new approach to the treatment of localised infections when all colonised areas are accessible to light.

Coloring Agents↗
PubMed1996

Infection control and urine drainage bag design.

Catheterisation carries a significant risk of infection. Catheter and drainage system design should reduce this risk. No one system will suit all users; new designs need extensive trials in use.

Cross Infection↗
PubMed1995

The kinetics of quantal transmitter release from retinal amacrine cells.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Animals↗
PubMed1995

Distinct skeletal abnormalities in four girls with Shprintzen-Goldberg syndrome.

We describe 4 girls with Shprintzen-Goldberg syndrome. Skeletal abnormalities common to 3 of them include bowing of long bones (with a variable degree of progression over time), flare of the metaphyses, a large anterior fontanel with persistent patency into the second to fourth years of life, 13 pairs of ribs, distinct vertebral abnormalities which were absent neonatally but evolved by the second year of life, and progressive osteopenia. These abnormalities were generalized and, in one case, progressive over the first few years of life. Communicating hydrocephalus was present in all 4 cases. The eldest, an 11-year-old girl, had additional anomalies not reported previously in this syndrome, including intestinal malrotation, an anteriorly placed anus, and mild cerebral atrophy. This is the first detailed report of skeletal manifestations in this rare disorder of unknown cause. These cases, in conjunction with a review of the literature, suggest that skeletal abnormalities are common in Shprintzen-Goldberg syndrome.

Abnormalities, Multiple↗
PubMed1995

Dexamethasone up-regulates A3 adenosine receptors in rat basophilic leukemia (RBL-2H3) cells.

The cross-linking of surface IgE receptors by multi-functional Ags promotes the degranulation of mast cells. Previous studies have indicated that the nucleoside adenosine potentiates this response by activating putative A3 adenosine receptors (AR) coupled to phospholipase C in mast cells or their cultured analogues, rat basophilic leukemia (RBL-2H3) cells. Moreover, it has been shown that exposure of RBL-2H3 cells to dexamethasone attenuated antigen-mediated mast cell degranulation, but potentiated the response elicited by adenosine. To determine whether the A3AR is a potential site of action of dexamethasone, we have assessed the status of these receptors in RBL-2H3 cells treated with and without dexamethasone. Treatment with dexamethasone (100 nM) for 24 h resulted in an increase in the number of A3AR to 217 +/- 50% of control. The increased receptor expression was both time- and concentration-dependent, with optimal increases observed following 16 h of treatment and using 100 nM of dexamethasone. No increase in the level of the A2aAR was detectable following dexamethasone treatment. Northern blotting studies indicated a 2.7 +/- 0.3-fold increase in A3AR mRNA in RBL-2H3 cells treated with dexamethasone for 24 h. Dexamethasone also increased the expression of G protein alpha i2, alpha i3, alpha s, and beta subunits by two- to threefold. Activation of the A3AR by aminophenylethyladenosine (APNEA) following dexamethasone treatment enhanced the production of inositol phosphates and the mobilization of intracellular Ca2+. From these data, it is concluded that dexamethasone increases the expression of both A3AR and G proteins in RBL-2H3 cells which contributes to the enhanced response to adenosine.

Animals↗
PubMed1995

Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum.

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Surface-associated proteins from Staphylococcus aureus demonstrate potent bone resorbing activity.

Staphylococcus aureus infections are associated with rapid bone destruction in conditions such as osteomyelitis, bacterial arthritis, and infected orthopedic implant failure. How this bacterium induces bone destruction has not been defined. In studies of the role of oral Gram-negative bacteria in periodontal pathology, we have established that cell surface-associated proteins (SAPs) are potent stimulators of bone resorption. The surface-associated components from S. aureus have now been isolated and demonstrated to be extremely potent stimulators of bone resorption in the murine calvarial bone resorption assay. Bone resorption appears to be due to proteins, is not the result of contamination with lipoteichoic acid or muramyl dipeptide, and is potently inhibited by indomethacin and can be completely blocked by high concentrations of interleukin-1 receptor antagonist or TN3-19.12, a neutralizing monoclonal antibody to murine TNF. The SAP fraction can stimulate fibroblasts or monocytes to release osteolytic cytokines, but only at high concentrations. Fractionation of the SAPs by high performance liquid chromatography demonstrated that a number of fractions were osteolytically active. The most active contained a heterodimeric protein of molecular weight 32-36 kD. The presence of this osteolytically active surface-associated fraction may account for the bone resorption associated with local infection with S. aureus.

Animals↗
PubMed1995

Lethal photosensitisation of Staphylococcus aureus in vitro: effect of growth phase, serum, and pre-irradiation time.

BACKGROUND AND OBJECTIVES: Staphylococcus aureus can be killed by low-power laser light in the presence of aluminium disulphonated phthalocyanine (AlPcS2). The purpose of this study was to determine the effect of pre-irradiation time (PIT), the presence of serum, and the physiological state of the organism on the kills achieved. STUDY DESIGN/MATERIALS AND METHODS: To determine the effect of PIT on killing, suspension of methicillin-resistant S. aureus (MRSA) were incubated in the dark with 12.5 micrograms/ml of AlPcS2 for 60 s or 300 s, and survivors were enumerated after exposure to 1.2 J of light from an 11-mW gallium aluminium arsenide laser. The susceptibility of MRSA in its various growth phases was determined in a similar manner using a PIT of 300 s. The effect of serum on killing was determined using stationary phase cells resuspended in horse serum. RESULTS: Using a PIT of either 60 s or 300 s, 10(6) cfu (99.9%) of MRSA were killed. There was little difference in the susceptibility of lag-, logarithmic-, or stationary-phase cells, the kills being 99.9%, 99.8%, and 99.9%, respectively. Although kills were reduced in the presence of serum, 99.6% of MRSA were killed using a light dose of 1.2 J. CONCLUSION: These results demonstrate that MRSA can be rapidly sensitised by AlPcS2 to killing by low-power laser light, that killing is not dependent on the organism's growth phase, and that substantial kills can be achieved in the presence of serum.

Animals↗
PubMed1995

Early prenatal diagnosis of cartilage-hair hypoplasia (CHH) with polymorphic DNA markers.

Cartilage-hair hypoplasia (CHH) is an autosomal recessive disorder resulting in short stature and hypoplasia of hair. Associated features include impaired T-cell-mediated immunity, deficient erythropoiesis, gastrointestinal dysfunction, and an increased risk of malignancies. As the condition may, in some cases, be severe or even fatal during childhood, families with a previous history of CHH may wish to have prenatal diagnosis. We have previously assigned the gene for CHH to the proximal 9p by linkage analysis using several polymorphic DNA markers. Here we report the prenatal testing for CHH in three Finnish and one Australian family using three DNA markers closely linked to the CHH gene. In three cases a fetus unaffected with CHH was predicted at the probability level of more than 94 per cent. In one case, an affected fetus was predicted. The results were in concordance with ultrasonography performed for all fetuses. The three children born to date were unaffected as predicted. The DNA marker-based analysis thus provides a useful method for early prenatal testing for CHH.

Base Sequence↗
PubMed1995

Interleukin 6 production by lipopolysaccharide-stimulated human fibroblasts is potently inhibited by naphthoquinone (vitamin K) compounds.

Naphthoquinone vitamins (vitamins K) are widely recognized for their role in the gamma-carboxylation of specific glutamyl residues in coagulation, anti-coagulation and extra-hepatic proteins. Recently, however, there have been reports that these compounds can exert actions other than those normally associated with protein gamma-carboxylation. These observations suggest that naphthoquinones may have effects on the production of inflammatory mediators including cytokines. Fibroblasts are now recognized as a rich source of cytokines and we have examined the effect of various naphthoquinones on the production of interleukin 6 (IL-6) by lipopolysaccharide-stimulated human gingival fibroblasts. Compounds examined in this study include: phylloquinone (K1), menaquinone-4 (K2), menadione (K3), 2,3-dimethoxy-1,4-naphthoquinone (DMK) and a synthetic product of vitamin K catabolism, 2-methyl, 3-(2'methyl)-hexanoic acid-1,4-naphthoquinone (KCAT). All of these compounds are capable of inhibiting IL-6 production with a rank order of potency: KCAT > K3 > DMK > K2 > K1. The most potent compound, KCAT, inhibited IL-6 production with an IC50 of 3 x 10(-7)M. The mechanism of action of these naphthoquinones on fibroblast IL-6 production is unknown. Given that K3 and KCAT are inactive in the gamma-carboxylation reaction, we suggest that this activity is not essential for the inhibition of IL-6 production and that activity may be related to the redox capacity of these naphthoquinones.

Cells, Cultured↗
PubMed1995

Relative cytokine-stimulating activities of surface components of the oral periodontopathogenic bacterium Actinobacillus actinomycetemcomitans.

The purpose of this study was to determine whether bacterial surface components other than lipopolysaccharide (LPS) could stimulate pro-inflammatory cytokine synthesis by mesenchymal and myelomonocytic cells in vitro. LPS, lipid A-associated proteins (LAP) and saline-extractable surface-associated material (SAM) were isolated from the periodontopathogenic bacterium Actinobacillus actinomycetemcomitans and added to cultures of human gingival fibroblasts (HGFs), human PBMCs and the human myelomonocytic MonoMac-6 cell line. Pro-inflammatory cytokine release into culture supernatants was determined by two-site ELISAs. Contrary to expectation, the highly purified LPS extracted from this bacterium was significantly less potent than the other surface extracts in stimulating release of IL-1 beta, IL-6 and TNF-alpha by all three cell types. The SAM was the most potent cytokine-stimulating agent showing equivalent activity to highly purified E. coli LPS in stimulating IL-6 release by PBMCs. LAP also had cytokine-stimulating activity although it was generally significantly less potent than the SAM. Thus in the case of this organism, which is involved in the pathology of chronic inflammatory diseases the LPS does not appear to be the major cytokine-stimulating component.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

Modulins: a new class of cytokine-inducing, pro-inflammatory bacterial virulence factor.

Despite the fact that the inflammatory and immune responses have evolved to combat microorganisms, the present generation of inflammation researchers has evinced relatively little interest, with the exception of septic shock, in microbially-induced inflammation. This in spite of the fact that the Gram-negative cell wall constituent, lipopolysaccharide, has been widely used as a tool in inflammation research. The reason for such lack of interest has been due to the therapeutic efficacy of antibiotics which are the treatment of choice for infections and their inflammatory sequelae. However, this is likely to change within the next decade or so, with the relentless increase in the incidence of antibiotic-resistant strains of bacteria. This will return therapy to the stage where clinicians will have to treat the inflammatory symptoms of infection. Many of these symptoms are due to the stimulation of cytokine synthesis. The capacity of bacteria to induce cytokine synthesis has, until the past few years, centred exclusively on lipopolysaccharide. However, it has been established during the past 5-10 years that a range of other molecules, mainly associated with the surface of bacteria, have the capacity to induce cytokine production. Some of these are exquisitely potent stimulators of pro-inflammatory cytokine synthesis. The nature and mechanism of action of these various cytokine-inducing molecules, for which we have devised the name modulins, is the subject of this review. It is clear that bacteria still have many surprises for us, as exemplified by the recent discovery of the role played by Helicobacter pylori in gastritis, gastric ulceration and gastric cancer.

Animals↗