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Biomedical subjects

L J Chandler

Publications and source records attributed to L J Chandler.

At least 19 recordsLinked to original sources
PubMed2005

Alterations in glutamatergic and gabaergic ion channel activity in hippocampal neurons following exposure to the abused inhalant toluene.

Toluene, a representative member of the large class of abused inhalants, decreases neuronal activity and depresses behavior in both animals and humans. The sites of action of toluene are not completely known but recent studies suggest that ion channels that regulate neuronal excitability may be particularly sensitive. Previous studies with recombinant receptors showed that toluene decreases currents carried by N-methyl-D-aspartate (NMDA)-glutamate receptors without affecting those gated by non-NMDA receptors. In addition, toluene increases currents generated by GABA and glycine receptors. In the present study, primary cultures of rat hippocampal neurons were used to investigate the effects of acute and chronic toluene exposure on native excitatory and inhibitory ligand-gated ion channels. Toluene dose-dependently inhibited NMDA-mediated currents (IC50 1.5 mM) but had no effect on responses evoked by the non-NMDA agonist kainic acid. Prolonged treatment of neurons with toluene (1 mM; 4 days) increased whole-cell responses to exogenously applied NMDA, reduced those evoked by GABA but did not alter responses generated by kainic acid. Immunoblot analysis revealed that prolonged toluene exposure increased levels of NR2A and NR2B NMDA receptor subunits with no change in NR1. Immunohistochemical analysis with confocal imaging showed that toluene-treated neurons had significant increases in the density of NR1 subunits as compared with control neurons. Toluene exposure increased the amplitude of synaptic NMDA currents and decreased those activated by GABA. The results from this study suggest that toluene induces compensatory responses in the functional expression of ion channels that regulate neuronal excitability.

Amino Acid Transport System X-AG↗
PubMed2002

Clinical evaluation of the Vitek automated system with cards GNS 122 and 127 and VTK-R07.01 software for antimicrobial susceptibility testing of Pseudomonas aeruginosa.

The performance of the Vitek Automated Susceptibility Testing System software version VTKR07.01 (bioMerieux Vitek, Hazelwood, MO), for testing Pseudomonas aeruginosa was evaluated by comparing results for 200 clinical isolates with those of disk diffusion and manual broth microtiter dilution testing. For cefepime, the restricted major error rate was 0.53% and the minor error rate was 12.5%. For piperacillin, the restricted major error rate was 2.15%. For ticarcillin/clavulanic acid, restricted very major and major error rates of 6.5% and 3.2%, respectively, occurred. The results of our study indicate that the Vitek system performs within acceptable limits when testing piperacillin, but remains problematic for testing cefepime and ticarcillin-clavulanic acid.

Automation↗
PubMed2001

Development of rolipram-sensitive, cyclic AMP phosphodiesterase (PDE4) in rat primary neuronal cultures.

The development of PDE4 was examined in primary neuronal cultures of rat cerebral cortex. Three days after culturing, neurons exhibited relatively low PDE4 activity (i.e., rolipram-sensitive PDE activity). It gradually increased over time, approximately doubling by day 12. The increase in activity was accompanied by an increase in the expression of the PDE4A variants, PDE4A5 and PDE4A1, as well as of the synaptic marker protein synapsin I. There was a strong correlation between the expression of the PDE4A variants with that of synapsin I, which suggests that as neurons develop and signal transduction increases there is a regulated increase in PDE4 expression and activity. Consistent with this interpretation, it was found that treatment with the sodium channel blocker tetrodotoxin, which inhibits depolarization-induced neurotransmitter release, reduced the expression of the PDE4A variants. These data demonstrate the developmental regulation of PDE4 in neurons and offer a manner by which the association of PDE4 variants with particular signal transduction pathways may be studied in vitro.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Phylogenetic analysis of the envelope gene of St. Louis encephalitis virus.

The complete nucleotide sequences of the envelope gene of 62 geographic isolates of St. Louis encephalitis (SLE) virus were determined. Phylogenetic analyses of the sequences, conducted using both maximum parsimony and neighbor-joining methods, included four other members of the Japanese encephalitis serogroup. The results indicated that the SLE isolates formed a monophyletic group in which isolates generally clustered according to geographic origin. Isolates from Panama and South America predominantly formed two large groupings, while isolates from the U.S. formed two other major groups. Several South and Central American strains were more closely related to strains isolated in the U.S., e.g., one isolate from Mexico and Panama, each, were closely related to two Tampa Bay, Florida, isolates, and an isolate, from Brazil was closely related to three isolates from Texas. The U.S. isolates also were not strictly grouped according to geographic source, e.g., some California isolates were closely related to Texas or midwestern isolates, and a Florida isolate was closely related to three isolates from Maryland. The results of the phylogenetic analyses indicated that SLE virus is predominantly maintained locally, but has been transported occasionally between areas, both within and outside the U.S.

Americas↗
PubMed2000

N-methyl D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures.

N-Methyl D-aspartate (NMDA) receptor activation of extracellular-signal regulated kinase (ERK) was examined in primary cortical cultures. Tetrodotoxin, NMDA receptor antagonists, or reduced extracellular calcium (0.1 mm) greatly decreased basal levels of phospho-ERK2, indicating that activity-dependent activation of NMDA receptors maintained a high level of basal ERK2 activation. This activity-dependent activation of phospho-ERK2 was blocked by pertussis toxin and inhibition of calcium/calmodulin-dependent kinase II and phosphatidylinositol 3-kinase but not by inhibition of protein kinase C or cAMP-dependent protein kinase. Addition of a calcium ionophore or 100 microm NMDA decreased phospho-ERK2 in the presence of 1 mm extracellular calcium but enhanced phospho-ERK2 in 0.1 mm extracellular calcium. The reduction in basal phospho-ERK2 by 100 microm NMDA was also reflected as a decrease in phospho-cAMP response element-binding protein. Inhibition of tyrosine phosphatases and serine/threonine phosphatases protein phosphatase 1 (PP1), PP2A, and PP2B did not prevent the inhibitory effect of NMDA. In the presence of tetrodotoxin, NMDA produced a bell-shaped dose-response curve with stimulation of phospho-ERK2 at 10, 25, and 50 microm NMDA and reduced stimulation at 100 microm NMDA. NMDA (50 microm) stimulation of phospho-ERK2 was completely blocked by pertussis toxin and inhibitors of phosphatidylinositol 3-kinase and was partially blocked by a calcium/calmodulin-dependent kinase II inhibitor. These results suggests that NMDA receptors can bidirectionally control ERK signaling.

Animals↗
PubMed2000

Comparison of four methods for identifying Streptococcus pneumoniae.

Four methods (bile solubility, optochin, latex agglutination, and DNA probe) were compared for identification of clinical isolates of Streptococcus pneumoniae. Of 209 isolates tested, 151 (Group I) were selected based on typical colony morphology of S. pneumoniae, while 58 (Group II) were selected on the basis of alpha-hemolysis alone. Using the DNA probe as a reference method, 141 isolates from Group I and 10 from Group II were identified as S. pneumoniae. The optochin disk test and the latex agglutination test both exhibited a 100% sensitivity and specificity for Group I isolates; bile solubility identified all but 1 isolate in this group. For Group II isolates, the sensitivity and specificity of optochin testing was 100%, 80 and 94% for latex and 80 and 100% for bile. The results of this study indicate that all methods tested give reliable results for isolates with typical colony morphology of S. pneumoniae. Bile solubility and latex may not be as reliable when testing alpha-hemolytic colonies without colony morphology typical S. pneumoniae.

Bacterial Typing Techniques↗
PubMed2000

Inhibition of cyclic AMP phosphodiesterase (PDE4) reverses memory deficits associated with NMDA receptor antagonism.

Rolipram, a selective inhibitor of type 4 cyclic AMP phosphodiesterase (PDE4), completely reversed the amnesic effects of MK-801 on working and reference memory (F[4,64] = 11.10; p <.0001 and F[4,64] = 2.53; p <.05, respectively) at doses of 0.01-0.1 mg/kg in the radial-arm maze task. Similar antagonism by rolipram of the effects of MK-801 was observed on inhibitory avoidance behavior (F[3,35] = 190.8; p <.0001). In vitro evidence suggests that an increase in cAMP concentrations may mediate the observed behavioral effects of rolipram. In the absence of PDE4 inhibition, NMDA did not increase cAMP concentrations in primary cultures of rat cerebral cortical neurons. However, when PDE4 was inhibited with rolipram, NMDA markedly elevated cAMP. These observations suggest that PDE4 is an integral component of the NMDA receptor-mediated signal transduction pathway involved in memory processes. Inhibitors of PDE4 may act on this pathway to produce their effects on memory and may represent a new class of cognitive enhancers.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Primary and booster immune responses to SA14-14-2 Japanese encephalitis vaccine in Korean infants.

Attenuated SA14-14-2 Japanese encephalitis (JE) vaccine has been administered safely and effectively to more than 100 million children in China since 1988 and recently, licensure of the vaccine in Korea has been sought. In the first clinical evaluation of the vaccine outside of China, we monitored side effects in 84 children and evaluated antibody responses to a single dose given as primary JE vaccination in 68 children, 1-3 years old (mean age 27 months). No significant adverse events were noted. Neutralizing antibodies (geometric mean titer [GMT] of 188) were produced in 96% of the 68 subjects. In 10 other children who previously had been immunized with two or three doses of inactivated JE vaccine, the booster administration of SA14-14-2 vaccine produced an anamnestic response in all, with a GMT of 3378. In a comparison group of 25 children previously immunized with two doses of inactivated vaccine, neutralizing antibody titers were detected in 16 (64%). Viral specific IgM was detected in nine primary vaccinees (13%) but in others, IgM may have declined to undetectable levels in the four week postimmunization sample. Live attenuated SA14-14-2 JE vaccine is a promising alternative to the only commercially available JE vaccine for national childhood immunization programs in Asia.

Antibodies, Viral↗
PubMed1999

Identification of genetic variation among St. Louis encephalitis virus isolates, using single-strand conformation polymorphism analysis.

A single-strand conformation polymorphism (SSCP) technique was developed for identification of genetic variation among 26 isolates of St. Louis encephalitis (SLE) virus. A 750-bp portion of the envelope gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the products analyzed by SSCP. SSCP reliably identified genetic variation among the isolates from the US, Central and South America. Closely related isolates from a smaller geographic area (Panama) were also distinguishable by SSCP. The sensitivity of this technique was demonstrated by sequencing each of the isolates used; SSCP was capable of discriminating between isolates that had as few as 1-6 nucleotide differences. These results indicate that SSCP has excellent potential as a tool to screen rapidly SLE virus isolates for genetic variation and could be incorporated into molecular epidemiology studies.

Animals↗
PubMed1999

Fyn tyrosine kinase reduces the ethanol inhibition of recombinant NR1/NR2A but not NR1/NR2B NMDA receptors expressed in HEK 293 cells.

NMDA receptors are potentiated by phosphorylation in a subunit- and kinase-specific manner. Both native and recombinant NMDA receptors are inhibited by behaviorally relevant concentrations of ethanol. Whether the phosphorylation state of individual subunits modulates the ethanol sensitivity of these receptors is not known. In this study, the effects of Fyn tyrosine kinase on the ethanol sensitivity of specific recombinant NMDA receptors expressed in HEK 293 cells were investigated. Whole-cell mode patch clamp and ratiometric calcium imaging demonstrated that the degree of ethanol inhibition of NR1/NR2B receptors was unaffected by Fyn tyrosine kinase. In contrast, the inhibition of NR1/NR2A receptors by ethanol (100 mM) was significantly reduced under conditions of enhanced Fyn-mediated tyrosine phosphorylation of the NR2A subunit. This effect was not observed at lower concentrations of ethanol (< or = 50 mM). These results suggest that tyrosine phosphorylation of specific NMDA receptors by Fyn tyrosine kinase may regulate the sensitivity of these receptors to the sedative/hypnotic concentrations of ethanol.

Cells, Cultured↗
PubMed1999

Mayaro virus disease: an emerging mosquito-borne zoonosis in tropical South America.

This report describes the clinical, laboratory, and epidemiological findings on 27 cases of Mayaro virus (MV) disease, an emerging mosquito-borne viral illness that is endemic in rural areas of tropical South America. MV disease is a nonfatal, dengue-like illness characterized by fever, chills, headache, eye pain, generalized myalgia, arthralgia, diarrhea, vomiting, and rash of 3-5 days' duration. Severe joint pain is a prominent feature of this illness; the arthralgia sometimes persists for months and can be quite incapacitating. Cases of two visitors from the United States, who developed MV disease during visits to eastern Peru, are reported. MV disease and dengue are difficult to differentiate clinically.

Adult↗
PubMed1999

Use of base excision sequence scanning for detection of genetic variations in St. Louis encephalitis virus isolates.

Twenty-two isolates of St. Louis encephalitis (SLE) virus of various geographical origins (Brazil, Argentina, Panama, Texas, Missouri, Maryland, California, and Florida) were examined for genetic variation by the base excision sequence scanning (BESS T-scan) method. A fragment was amplified in the envelope gene with the forward primer labeled in the PCR. The BESS T-scan method determined different clusters according to the profiles generated for the isolates and successfully grouped the isolates according to their geographical origins. Two major clusters, the North American cluster (cluster A) and the South and Central American cluster (cluster B), were defined. Two subgroups, the Texas-California subgroup (subgroup A1) and the Missouri-Maryland-Florida subgroup (subgroup A2), were distinguished within group A. Similarly, group B strains were subclustered to a South American subgroup (subgroup B1) and a Central American subgroup (subgroup B2). These results were consistent with those obtained by DNA sequencing analysis. The ability of the BESS T-scan method to discriminate between strains that present with high degrees of nucleotide sequence similarity indicated that this method provides reliable results and multiple applications for other virus families. The method has proven to be suitable for phylogenetic comparison and molecular epidemiology studies and may be an alternative to DNA sequencing.

Animals↗
PubMed1999

Cardiac impairment and nitric oxide synthase activity in the chronic portal vein-stenosed rat.

Decreased cardiac contractility and beta-adrenergic responses have been observed in the chronic portal vein-stenosed (PVS) rat. Because nitric oxide (NO) may be increased in PVS and has been recognized as a negative inotropic agent, we investigated the induction of NO synthase (NOS2) and/or changes in constitutive NOS (NOS3) as factors in the cardiac dysfunction of the PVS rat. Ten to twelve days after portal vein stenosis or sham operation, cardiac function was evaluated in paced left ventricular papillary muscles (LVPM) and right ventricular strips (RV). To determine if NO modulation of contractile function was altered in PVS, we examined the increase in developed tension produced by the effect of Nomega-nitro-L-arginine (L-NNA) on the myocardial force-frequency relationship. Cardiac tissue NOS2 and NOS3 activities were assayed, Western blot analyses of NOS2 and NOS3 expression were performed, and circulating nitrate-nitrite (NOX) levels (an indicator of in vivo NOS activity) were assayed. Basal LVPM and RV contractile indexes were significantly reduced in PVS (30-50%), without a change in the relaxation rate. No between-group differences in the cardiac NOS2 or NOS3 enzymatic activities of PVS and sham-operated (SO) rats were observed. Western blots revealed no cardiac NOS2 expression in either SO or PVS rats. In contrast, NOS3 was expressed in both SO and PVS rats, but there was no quantitative difference in expression between the two groups. Changes in the cardiac force-frequency relationship (staircase effect) after L-NNA were consistent with NOS3 modulation of contractile function in both SO and PVS rats, but there was no between-group difference in the modulation. Circulating NOX concentrations did not differ between SO and PVS rats. In conclusion, protein expression data, enzymatic assays, end-product assays, and functional data indicate that between-group differences in NOS2 and NOS3 activity are not responsible for the cardiac impairment that has been observed in the chronic PVS rat.

Animals↗
PubMed1999

Effects of c-Src tyrosine kinase on ethanol sensitivity of recombinant NMDA receptors expressed in HEK 293 cells.

N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that are important mediators of the actions of the excitatory amino acid neurotransmitter glutamate. Previous studies have shown that ethanol inhibits the function of both wild-type receptors found in neurons and recombinant NMDA receptors expressed in heterologous cells, such as oocytes and transfected mammalian cells. Although some studies have reported that certain subunit combinations display an enhanced sensitivity to ethanol, this effect is not observed in all experimental systems. This discrepancy may be due to varying levels of endogenous modulators, such as kinases, between different cell preparations. In this study, we investigated the effects of tyrosine phosphorylation on the ethanol sensitivity of NMDA receptor function using a recombinant cell system where levels of both NMDA subunits and protein kinases can be more carefully controlled. Human embryonic kidney (HEK 293) cells were transfected with different NMDA receptor subunits and a c-Src-green fluorescent protein (GFP) fusion protein that could be directly visualized in living cells. Agonist-stimulated calcium flux was measured in single cells using fura-2 video imaging. As expected, cells transfected with the NR1/NR2B subunits were more sensitive to inhibition by the NR2 selective antagonist ifenprodil than those transfected with NR1/ NR2A or NR1/NR2A/NR2B subunits. All receptor combinations were inhibited by ethanol (25 and 100 mM), with the NR1/NR2B combination being slightly more sensitive than NR1/NR2A or NR1/NR2A/NR2B. Control and NMDA-receptor transfected HEK 293 cells displayed a low degree of tyrosine phosphorylation as measured by immunofluorescence and Western immunoblotting using an antiphosphotyrosine antibody. Phosphorylation was markedly enhanced in cells transfected with the c-Src-GFP fusion protein. The sensitivity of NMDA receptors to either 25 or 100 mM ethanol, or 10 microM ifenprodil, was not significantly altered by co-transfection with c-Src-GFP. These results indicate that, although NMDA receptors can be a target of c-Src tyrosine kinase, tyrosine phosphorylation by this enzyme does not modulate the inhibitory effects of ethanol on NMDA-activated currents.

Animals↗
PubMed1999

Chronic ethanol upregulates NMDA and AMPA, but not kainate receptor subunit proteins in rat primary cortical cultures.

The present study examined the effects of chronic ethanol exposure on the expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA) and kainate receptor subunit proteins in rat cortical neuronal cultures grown in media containing 2 mM (high) or 0.1 mM (low) glutamine. Immunoblot analysis of NMDA (NR1, NR2A, NR2B, and NR2D), AMPA (GluR1 and GluR2/3), and kainate (GluR6/7) subunit polypeptides in 3-, 5-, 8-, 10-, and 12 day-old-cultures showed that NMDA receptor subunits NR1, NR2A, and NR2B and AMPA receptor subunits GluR2/3 progressively increased as a function of time, whereas levels of NMDA subunit NR2D were high at day 3 and progressively declined to barely detectable levels by day 12. Levels of AMPA subunit GluR1 and the kainate subunit GluR6/7 remained stable throughout the time course. Replacing the culture media with low glutamine media at culture day 5 did not alter the levels of subunit proteins measured at culture days 9 and 13. However, exposure of low glutamine cultures to 100 mM ethanol for 4 days (starting at culture day 9) significantly increased the levels of NMDA receptor subunits (NR1, NR2A, and NR2B) and AMPA receptor subunits (GluR1 and GluR2/3), but had no effect upon kainate receptor subunits (GluR6/7) or the synapse-associated proteins synapsin I and PSD-95. In contrast, chronic ethanol did not alter the levels of any of these subunit proteins in cells grown in high glutamine. These data demonstrate that under certain experimental conditions, prolonged exposure to ethanol upregulates NMDA and AMPA receptor subunit proteins, but has no effect upon kainate receptor subunit proteins. Because we have previously shown that acute ethanol can inhibit NMDA and AMPA, but not kainate, receptor function in these cultures, the increase in subunit expression likely reflects an adaptive response to the inhibitory effects of ethanol and suggests that both NMDA and AMPA receptors may play an important role in adaptation of the CNS to chronic ethanol.

Animals↗
PubMed1998

The effects of acute and repeated cocaine injections on protein kinase C activity and isoform levels in dopaminergic brain regions.

The present study was designed to assess the effects of acute and repeated cocaine exposure on protein kinase C (PKC) activity and the levels of calcium-dependent isoforms of PKC in mesocorticolimbic and nigrostriatal dopamine brain regions. Animals received repeated injections of saline or cocaine and were challenged with saline or cocaine 24 h or 7 days after the last of their daily injections. Animals were sacrificed 2, 6 or 24 h after the challenge injection and their brains were dissected and used in PKC studies. The data demonstrated that previously reported cocaine-induced increases in PKC activity in the ventral tegmental area are transient and not associated with changes in the levels of calcium-dependent isoforms of PKC. In addition, there was a decrease in membrane-associated PKC activity, with a concomitant increase in the levels of PKCbetaI in the medial prefrontal cortex 24 h after the last injection of cocaine. These data suggest that changes in PKC activity in the ventral tegmental area may be involved in the initiation of sensitization whereas changes in PKC activity in the medial prefrontal cortex may be related to the expression of the sensitized response to cocaine.

Animals↗
PubMed1998

Ethanol, stroke, brain damage, and excitotoxicity.

The N-methyl-d-aspartate (NMDA)-glutamate receptor could contribute to stroke, trauma, and alcohol-induced brain damage through activation of nitric oxide formation and excitotoxicity. In rat primary cortical cultures NMDA was more potent at activating nitric oxide formation than triggering excitotoxicity. Ethanol dose dependently inhibited both responses. In contrast, treatment of neuronal cultures with ethanol (100 mM) for 4 days significantly increased NMDA stimulated nitric oxide formation and excitotoxicity. These findings suggest that ethanol acutely inhibits but chronically causes supersensitivity to NMDA-induced excitotoxicity in neuronal cultures. To investigate ethanol's interaction with stroke induced damage models of global cerebral ischemia were studied. Transient global ischemia resulted in a loss of hippocampal CA1 pyramidal neurons over a 3- to 5-day period. Determinations of the NMDA receptor ligand binding stoichiometry or postischemic receptor binding changes did not show differences between neurons that undergo delayed neuronal death following ischemia and those that show no toxicity, for example, CA1 and dentate gyrus, respectively. Acute ethanol (3 g/kg) was found to protect against ischemia-induced CA1 hippocampal damage by lowering body temperature, but not under temperature controled conditions. These studies indicate that the factors contributing to stroke-induced brain damage are complex, although they are consistent with chronic ethanol increasing stroke-induced brain damage by increasing NMDA excitotoxicity.

Animals↗