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G M Smith

Publications and source records attributed to G M Smith.

At least 19 recordsLinked to original sources
PubMed1995

Cloning and characterisation of a bovine P2Y receptor.

Using a chick P2Y1 receptor cDNA probe we have isolated a mammalian P2Y receptor clone from a bovine aortic endothelial cell library. The sequence has a high degree of similarity to the chick P2Y1 clone. When transfected into the Jurkat cell line, the cDNA conferred sensitivity to purinoceptor agonists. Using fura-2 loaded cells the potency order at the receptor was found to be 2-methylthioadenosine 5' triphosphate = adenosine 5' diphosphate > adenosine 5' triphosphate >> alpha,beta-methyleneadenosine 5' triphosphate and uridine 5' triphosphate. This corresponds to the agonist potency order expected for the bovine aortic endothelial cell P2Y receptor.

Adenosine Diphosphate↗
PubMed1995

Synchrony between single-unit activity and local field potentials in relation to periodicity coding in primary auditory cortex.

1. We recorded responses from 136 single units and the corresponding local field potentials (LFPs) from the same electrode at 44 positions in the primary auditory cortex of 25 juvenile, ketamine-anesthetized cats in response to periodic click trains with click repetition rates between 1 and 32 Hz; to Poisson-distributed click trains with an average click rate of 4 Hz; and under spontaneous conditions. The aim of the study is to evaluate the synchrony between LFPs and single-unit responses, to compare their coding of periodic stimuli, and to elucidate mechanisms that limit this periodicity coding in primary auditory cortex. 2. We obtained averaged LFPs either as click-triggered averages, the classical evoked potentials, or as spike-triggered averages. We quantified LFPs by initial negative peak-to-positive peak amplitude. In addition, we obtained trigger events from negativegoing level crossings (at approximately 2 SD below the mean) of the 100-Hz low-pass electrode signal. We analyzed these LFP triggers similarly to single-unit spikes. 3. The average ratio of the LFP amplitude in response to the second click in a train and the LFP amplitude to the first click as a function of click rate was low-pass with a slight resonance at approximately 10 Hz, and, above that frequency, decreasing with a slope of approximately 24 dB/octave. We found the 50% point at approximately 16 Hz. In contrast, the LFP amplitude averaged over entire click trains was low-pass with a similar resonance but a high-frequency slope of 12 dB/octave and a 50% point at approximately 12 Hz. 4. The LFP amplitude for click repetition rates between 5 and 11 Hz often showed augmentation, i.e., the amplitude increased in response to the first few clicks in the train and thereafter decreased. This augmentation was paralleled by an increase in the probability of firing in single units simultaneously recorded on the same electrode. 5. We calculated temporal modulation transfer functions (tMTFs) for single-unit spikes and for LFP triggers. They were typically bandpass with a best modulating frequency of 10 Hz and similar shape for both single-unit spikes and LFP triggers. The tMTF per click, obtained by dividing the tMTF by the number of clicks in the train, was low-pass with a 50% cutoff frequency at approximately Hz, similar to that for the average LFP amplitude. 6. the close similarity of the tMTFs for single-unit spikes and LFP triggers suggests that single-unit tMTFs can be predicted from LFP level crossings.(ABSTRACT TRUNCATED AT 400 WORDS)

Acoustic Stimulation↗
PubMed1993

DNA-methylation of the E-selectin promoter represses NF-kappa B transactivation.

E-selectin is an adhesion molecule transiently and specifically expressed on endothelial cells upon stimulation with cytokines. We wished to determine whether methylation could play a role in cell-type specific expression of this gene. We found that the E-selectin promoter in cultured endothelial cells is under-methylated in comparison with non-expressing HeLa cells. Plasmid constructs carrying a reporter driven by the E-selectin promoter and methylated in vitro are no longer transcribed in either an in vitro transcription system or in transiently transfected cells. We identified the NF-kappa B site in the promoter as the likely target for this methylation-mediated repression by testing a minimal promoter carrying only this and an associated element. We conclude that methylation is likely to play a role in blocking E-selectin expression in non-endothelial cells.

Base Sequence↗
PubMed1993

Cyclic AMP has a differentiative effect on an immortalized oligodendrocyte cell line.

We investigated the effects of increasing the concentration of intracellular cyclic adenosine monophosphate (cAMP) on genes associated with oligodendrocyte differentiation in an immortalized glial cell line, 6E12, derived from the spinal cord of an MBP-SV40 large T-antigen transgenic mouse. Raising intracellular levels of cAMP induced expression of oligodendrocyte differentiation antigens recognized by O4 and anti-galactocerebroside antibodies, up-regulated expression of the proteolipid protein (PLP) gene, and down-regulated glial fibrillary acidic protein (GFAP) expression. There was no treatment effect on myelin-associated glycoprotein (MAG) expression. These phenotypic changes are consistent with oligodendrocyte differentiation. Treatment of 6E12 cells with dibutyryl cyclic AMP (DBC) down-regulated myelin basic protein (MBP) gene expression, perhaps, because it also up-regulated expression of a putative MBP repressor SCIP/Tst-1. Moreover, the 6E12 cells expressed high levels of MBP mRNA but no MBP translation products were detected in the presence or absence of DBC. This immortalized glial cell line is proposed as a CNS model for cAMP-modulated myelin gene expression and for post-transcriptional regulation of MBP.

Animals↗
PubMed1993

Distinct hypomyelinated phenotypes in MBP-SV40 large T transgenic mice.

To study the effect of SV40 large T-antigen expression in myelin-forming cells of both the central and peripheral nervous system, a series of transgenic mice were generated expressing the SV40 large T-antigen under control of the myelin basic protein (MBP) promoter. Two neurologic phenotypes, designated A and B, appeared among individual transgenic founders and their progeny. The A mice developed a severe action tremor at about 10 days of age that progressed into periods of convulsions and early death by three to four weeks of age. In contrast, the B mice exhibited a progressive hindlimb ataxia and had a more normal lifespan. The A mice displayed hypomyelinating lesions in the central nervous system (CNS), whereas the B mice had lesions in either the peripheral nervous system (PNS) alone or in both the PNS and CNS. Immunohistochemical staining of spinal cord sections of a type A mouse showed a substantial depletion in MBP. Moreover, T-antigen-positive cells appeared predominantly in white matter tracts as randomly distributed single cells. Double labeling immunocytochemistry demonstrated that some of these T-antigen-positive cells were positive for oligodendrocyte differentiation markers MBP and O4. Thus, T-antigen expression appeared to coincide with a terminal stage of oligodendrocyte differentiation.

Animals↗
PubMed1993

Modulation of adhesion molecule expression on rat cortical astrocytes during maturation.

During development of the vertebrate CNS the functional properties of astrocytes change significantly. Many of these functional changes result from modifications in the expression of cell surface adhesion molecules on astrocytes that mediate the interactions of astrocytes with other astrocytes, neurons, and growing axons. In this study we have compared the cell surface expression of HNK-1, NCAM, and laminin on rat cortical type-I-like astrocytes during maturation in vitro and in vivo. Both the proportion of immunoreactive cells and the relative levels of expression of these antigens on different aged astrocyte populations were assayed by flow cytometry. At birth, most cortical type-I astrocytes express high levels of HNK-1 and NCAM, while approximately 50% of the cells express laminin. During maturation in vitro, the proportion of cortical astrocytes that expressed these surface molecules decreased over a period of 28 days, even though cell size and glial fibrillary acidic protein content increased. During maturation in vivo, a qualitatively and temporally similar decrease in antigen expression on astrocytes was observed. This reduction in the expression of specific cell surface molecules on maturing astrocytes results from maturation of a single population of astrocytes and not differential proliferation of a nonexpressing subpopulation of astrocytes, as shown by cell cycle analysis of both immunoreactive and nonimmunoreactive cell populations. These data indicate that during maturation of rat cortical type-I-like astrocytes, the expression of cell surface adhesion molecules is regulated. Furthermore, this regulation appears to be cell autonomous and not dependent on environmental factors. Such regulation of adhesion molecule expression may have profound consequences for the functional properties of astrocytes during CNS maturation.

Aging↗
PubMed1992

Peripheral nerve injury down-regulates CNTF expression in adult rat sciatic nerves.

Ciliary neurotrophic factor (CNTF) is a 200-amino acid protein expressed in high concentrations by peripheral nerves and is thought to be important for the survival and regeneration of injured motoneurons (Lin et al., J Biol Chem 265:8942-8947, 1990). To better understand CNTF's role in nerve injury we have characterized the effects of crush injury on the expression of CNTF in adult rat sciatic nerves using specific antibody and RNA probes. Following a crush injury, both the protein and mRNA levels undergo pronounced decreases distal to the crush. These changes in CNTF expression were qualitatively distinct from changes in the expression of the low-affinity NGF receptor (p75NGFR), which increases following crush. Thus, the changes in CNTF levels do not reflect an overall down-regulation of mRNA during degeneration, and are inconsistent with the proposed role of CNTF in neuronal injury, since its levels are decreasing at the same time as the requirement for neurotrophic support is increasing.

Animals↗
PubMed1992

Effect of impression tray design upon the accuracy of stone casts produced from a single-phase medium-bodied polyvinyl siloxane impression material.

The study examined the accuracy of stone casts produced from impressions taken in stock polycarbonate trays, some of which had been strengthened with autopolymerizing polymethyl methacrylate resin and four designs of custom-made trays. Impressions of an acrylic master model of the mandibular arch on which two extracoronal crown preparations and one intracoronal inlay preparation had been carried out were taken using a medium-bodied polyvinyl siloxane material. Each preparation had been intended with a reference point for later measurement. Five impressions were taken for each type of tray and these were cast in die-stone after 24 h. The distances between the points were measured with a reflex microscope and the means determined for each design of tray. The mean difference between casts produced from the various tray designs and the acrylic master model were determined for each of the distances between the three measuring points. Statistical analysis showed that, with the stock trays, there were no significant differences between any of the modifications, or between them and the acrylic model, for any of the three distances (P greater than 0.1). When the results from the custom-made trays were analysed there were statistical differences between the acrylic model and the casts from the unperforated tray with no stops (P = 0.02), and between the unperforated tray with no stops and the perforated with stops for the distance A-B (P less than 0.01). There were no other significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Dental Impression Materials↗
PubMed1992

Immunofluorescent labeling of tight junctions in the rat brain and spinal cord.

Tight junctions may play an important role in maintaining the integrity of the blood-brain barrier. These junctions can be individually visualized using electron microscopy but no current technique is able to provide a more global picture of the presence and density of tight junctions in central nervous system tissue. We used an antibody that recognizes a high molecular weight protein (ZO-1) associated with tight junctions, to identify these specialized junctions within the rat brain and spinal cord. Immunofluorescent labeling showed a network of tight junctions between cells in the brain vasculature, leptomeninges and choroid plexus, and between tanycytes lining the floor of the third ventricle and the central canal of the spinal cord. Anti-ZO-1 labeled the majority of cells associated with the blood-brain barrier and may prove a useful marker, possibly in conjunction with functional dye studies, in evaluating the anatomical and functional integrity of the blood-brain barrier.

Animals↗
PubMed1992

Activity of amoxycillin-clavulanic acid against Legionella pneumophila in vitro and in an experimental respiratory infection model.

Amoxycillin and clavulanic acid show good activity against Legionella pneumophila in vitro, and synergy has been observed between the two agents. However, in tissue culture studies, amoxycillin was inactive against intracellular legionellae, whereas clavulanic acid and amoxycillin plus clavulanic acid were as effective as erythromycin in preventing bacterial growth. These latter findings were reflected in the results of therapy of a L. pneumophila pneumonia in the neutropenic rat. Amoxycillin was ineffective in reducing bacterial counts in the lungs of infected animals, but clavulanic acid and amoxycillin-clavulanic acid produced bactericidal effects similar to those of erythromycin. The data illustrate the bactericidal activity of amoxycillin-clavulanic acid and clavulanic acid against intracellular L. pneumophila in contrast to the lack of activity of amoxycillin.

Amoxicillin↗
PubMed1992

Bactericidal effects of co-amoxiclav (amoxycillin clavulanic acid) against a Legionella pneumophila pneumonia in the immunocompromised weanling rat.

To evaluate the activity of co-amoxiclav (amoxycillin/clavulanic acid) against Legionella pneumophila in vivo, a model of L. pneumophila pneumonia was developed in weanling rats rendered leukopenic by pre-administration of cyclophosphamide. Assessment of therapy was by lung bacterial counts and histological examination. Amoxycillin was ineffective in reducing bacterial counts in the lungs of infected rats, whereas erythromycin, the standard agent, was significantly more effective (P < 0.01). Co-amoxiclav and erythromycin, administered parenterally, produced significant bactericidal effects (P < 0.01), reducing the counts of L. pneumophila strain 1624 at 96 h to 1.2 log10 cfu/lungs compared with counts of 6 log10 cfu/lungs in the untreated animals. Clavulanic acid was also highly effective in preventing development of the infection, and was as efficacious as co-amoxiclav. Because of the significant reduction in bacterial numbers, a marked reduction in inflammation and consolidation of lung tissue was seen in rats treated with erythromycin, clavulanic acid or co-amoxiclav. The activity of co-amoxiclav was no greater than clavulanic acid alone, and no synergy was noted between the two components. When therapy was delayed until 48 h after infection, co-amoxiclav was as effective as erythromycin, with both treatments reducing bacterial numbers to 3.3 and 3.6 log10 cfu/lungs by 96 h, after only two days of therapy, in comparison with non-treated rats (5.6 log10 cfu/lungs). In a prolonged infection, produced by extending the period of leucopenia, co-amoxiclav and erythromycin were equally effective in preventing growth of the organism, with 1.5 and 1.6 log10 cfu/lungs, respectively, present at 96 h, in contrast to the non-treated rats with 5.7 log10 cfu/lungs (P < 0.01). After cessation of therapy, regrowth of L. pneumophila occurred in the erythromycin-treated group to such a degree that by 168 h, lung viable counts from these rats were significantly higher (4.8 log10 cfu/lungs) than in co-amoxiclav-treated rats (2.1 log10 cfu/lungs) (P < 0.05). Oral therapy of this infection with erythromycin or clavulanic acid, either alone or in combination with amoxycillin, resulted in counts of 3.3, 3.6 and 3.5 log10 cfu/lungs at 96 h, respectively. Although oral therapy was significantly less effective than parenteral therapy (P < 0.05), the bacterial counts in the treated groups were significantly lower than in the non-treated animals. The data show that co-amoxiclav displayed bactericidal activity consistently against intracellular L. pneumophila in vivo.

Administration, Oral↗
PubMed1992

Measurement of human phospholipase A2 in arthritis plasma using a newly developed sandwich ELISA.

A sandwich enzyme-linked immunosorbent assay (ELISA) for human non-pancreatic phospholipase A2 (PLA2) was developed using monoclonal antibodies raised against purified recombinant PLA2. This assay was shown to be specific for human non-pancreatic PLA2, showing no cross-reactivity with human pancreatic PLA2 or with snake venom PLA2 (Crotalus durissus). The immunoassay showed no cross-reactivity with plasma components, and was reproducible and quantitative between 39 pmol and 2.7 nmol PLA2/1. The levels of non-pancreatic PLA2 in the plasma of patients with arthritis was measured using this immunoassay. There were significantly higher levels of PLA2 in patients with rheumatoid arthritis than in those with osteoarthritis or healthy controls. Plasma PLA2 was highest in those patients with active rheumatoid arthritis.

Aged↗
PubMed1992

Elevated maternal plasma immunoreactive phospholipase A2 in human preterm and term labour.

Peripheral plasma concentrations of immunoreactive phospholipase A2 (irPLA2) (Type II, non-pancreatic) were determined in 110 women during pregnancy. The concentration of irPLA2 did not significantly change during pregnancy (5.7 +/- 0.6 ng/ml, n = 72) until the onset of labour. When compared with non-labouring women, irPLA2 concentrations were significantly elevated in association with both preterm labour (13.3 +/- 2.4 ng/ml, n = 15, p less than 0.02) and labour at term (10.4 +/- 1.7, n = 23, p less than 0.02). These data suggest that maternal plasma irPLA2 may be reflective of the mechanism(s) underlying the labour-associated increase in human gestational tissue eicosanoid formation.

Female↗
PubMed1991

High-resolution 1H- and 15N-NMR studies of Rhodospirillum rubrum cytochrome c2.

Rhodospirillum rubrum cytochrome c2 was uniformly enriched in 15N and studied by 1H- and 15N-NMR spectroscopy. Relaxation and NOE data allowed determination of the rotational correlation time and indicated more rapid side-chain motion in the native protein and increased segmental motion in the base-denatured protein. The pi nitrogen of the ligand histidine and the indolic nitrogen of the invariant tryptophan both remain protonated and act as proton-donors in hydrogen bonds over a wide pH range and therefore do not contribute to pH-related changes in the midpoint potential. pK values identified by numerous methods in the ferrocytochrome at pH 6.9 and in the ferricytochrome at pH 6.2 arise from His-42. At pH values below the pK, the imidazolium group participates in a salt bridge or in a hydrogen bond with the carboxylate group of the inner propionate of the heme. Loss of the proton causes a local conformational change which alters the midpoint potential. The pK values of the amino terminus and lysines were also determined from pH titrations monitored by 15N-NMR. Similar titrations of the ferricytochrome monitored by 1H-NMR showed structural heterogeneity in that the resonance of heme ring methyl 8 split into a doublet as the pH was raised.

Cytochrome c Group↗
PubMed1991

Immature type-1 astrocytes suppress glial scar formation, are motile and interact with blood vessels.

Previous studies have shown that immature but not mature astrocytes have the capacity to suppress glial scar formation and enhance axon outgrowth when transplanted into the adult mouse brain. We report here that glial scar formation is suppressed following transplantation of purified immature but not mature cultured type-1 rat cortical astrocytes into the adult rat brain. To examine the fate of transplanted cells, cultured astrocytes were labeled with either fluorescent beads or BSA-conjugated colloidal gold and traced after transplantation using both light and electron microscopy. While both immature and mature astrocytes survived transplantation, mature astrocytes appeared more susceptible to phagocytosis by cells of the immune system than immature astrocytes. Furthermore, while mature astrocytes were restricted to the region of the implant, immature astrocytes migrated into the surrounding CNS and became closely associated with host blood vessels. Such blood vessels were impermeable to the diffusion of systematically applied Evans blue dye. To determine whether immature astrocytes were intrinsically more motile than mature astrocytes, their rate of translocation was compared in vitro. Immature astrocytes translocated more than twice as fast as mature astrocytes. This ability of immature astrocytes to translocate throughout the host CNS and become associated with blood vessels may be a major factor in their ability to suppress glial scar formation in the adult animal.

Animals↗
PubMed1991

Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis.

Elevation of circulating phospholipase A2 (PLA2) activity is associated with sepsis and septic shock. Elevated levels of PLA2 activity also are seen in association with chronic inflammatory disorders such as rheumatoid arthritis. The relationship between these phospholipases is unclear. We have developed a highly specific enzyme-linked immunosorbent assay (ELISA) capable of measuring human synovial PLA2 in plasma, using monoclonal antibodies raised to recombinant synovial PLA2. This ELISA has been used to quantitate circulating PLA2 levels in patients clinically diagnosed with sepsis. These elevated levels positively correlated with the elevation seen in plasma PLA2 enzyme activity. The antibodies also have been used to purify immunoreactive PLA2 from plasma of patients with sepsis, thus enabling characterization of the purified protein by amino-terminal sequence analysis. We conclude from this study that the increase in PLA2 activity seen in association with sepsis and septic shock results from a dramatic elevation in levels of a circulating PLA2 enzyme. This inflammatory PLA2 is indistinguishable, both immunologically and chemically, from that associated with rheumatoid arthritis. Therapeutic agents directed towards inhibition of this inflammatory PLA2 enzyme may have utility in the treatment of both chronic and acute inflammatory disease.

Adolescent↗
PubMed1991

Effect of impression tray design and impression technique upon the accuracy of stone casts produced from a putty-wash polyvinyl siloxane impression material.

This study examined the accuracy of stone casts produced from impressions taken in stock polycarbonate trays, some of which had been strengthened with autopolymerizing polymethyl methacrylate resin. Three techniques were used to make the impression of an acrylic master model of the mandibular arch on which two extracoronal preparations for bridgework and one intracoronal inlay preparation had been carried out. Each preparation had been indented with a reference point for later measurement. The impression material was a putty-wash polyvinyl siloxane material. Five impressions were taken for each type of tray for each impression technique and these were cast in die-stone after 24 h. The distances between the points were measured with a reflex microscope and the means determined for each design of tray. The mean difference between casts produced from the various tray designs and the acrylic master model were determined for each of the distances between the three measuring points for the various impression techniques. Statistical analysis showed that, with the polycarbonate stock trays, there were significant differences between some of the modifications and between them and the acrylic model, for the three distances (P less than 0.05). These differences were limited to one measurement for one design of tray for each of the two-stage impression methods. With the one-stage technique the unreinforced tray and those reinforced with acrylic, over the heels and anteriorly, and the barred design were statistically significantly different from the acrylic model for measurement A-B.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesives↗