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Biomedical subjects

T B Johnson

Publications and source records attributed to T B Johnson.

At least 19 recordsLinked to original sources
PubMed2001

GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry.

GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABA(A) receptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an alpha5 subunit-selective ( approximately 75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5-20 microgram) and unilateral (0.01-40 microgram) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 microgram). The competitive BDZ antagonist ZK 93426 (ZK) (7 microgram) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABA(A) receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of alpha1 and alpha2 subunits, respectively, we determined whether RY exhibited a "negative" or "neutral" pharmacological profile at recombinant alpha1beta3gamma2, alpha2beta3gamma2, and alpha5beta3gamma2 receptors expressed in Xenopus oocytes. RY produced "classic" inverse agonism at all alpha receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the alpha5-containing GABA(A) receptors in the hippocampus play an important role in regulating EtOH-seeking behaviors.

Animals↗
PubMed1999

Serotonergic neuronal activity in the dorsal raphe nucleus of selectively bred alcohol-preferring and alcohol-nonpreferring rats and unselected wistar rats.

BACKGROUND: Selectively-bred alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) than do alcohol-nonpreferring (NP) rats. The present study was designed to test the hypothesis that the remaining 5-HT neurons in P rats compensated for their reduced number by increasing neuronal activity. METHODS: Spontaneous activity was recorded from single-spiking and bursting 5-HT neurons in the DRN of unanesthetized paralyzed, alcohol-naive P, NP, and Wistar rats. Firing frequencies, the percentages of action potentials in bursts, and the percentages of bursting neurons were evaluated. RESULTS: There were no significant differences among the three groups of rats in any of the parameters measured. Power analyses were performed on preliminary data to determine the sample sizes necessary for detection of significant differences. The mean firing frequencies of single-spiking 5-HT neurons averaged 1.8 (37 neurons), 1.7 (17 neurons), and 1.8 (41 neurons) spikes per second in P, NP, and Wistar rats, respectively. For bursting 5-HT neurons, the percentages of action potentials in bursts for P, NP, and Wistar rats were 55.0% (24 neurons), 49.7% (18 neurons), and 55.1% (21 neurons). The mean percentages of bursting 5-HT neurons encountered per electrode penetration were 44% for P rats (n = 28), 44% for NP rats (n = 14), and 34% for Wistar rats (n = 26). CONCLUSIONS: The results indicate that the sample of 5-HT neurons recorded in the DRN of P rats had not compensated for a reduced number by altering neuronal activity.

Action Potentials↗
PubMed1997

More real-time visual and haptic interaction with anatomical data.

The University of Colorado's Center for Human Simulation has developed fundamental algorithms for real-time visual and haptic interaction with polygonal and voxel base data sets, including those derived from the Visible Human Dataset. These algorithms are currently being used to create prototype simulators for surgery, needle insertion (anesthesiology, radiology, and rheumatology), dentistry, and ophthalmology. This paper briefly discusses our segmentation and classification effort as well as our ability to create texture-mapped polygonal models directly from the data: both are fundamental to the creation of anatomically based simulators.

Algorithms↗
PubMed1996

Lack of proarrhythmia as assessed by Holter monitor after atrial radiofrequency ablation of supraventricular tachycardia in children.

The purpose of this study was to assess the short-term arrhythmogenicity of atrial radiofrequency (RF) ablation lesions in children. Patients with the greatest exposure to RF energy comprised the study group. Holter data on 35 RF ablation procedures in 31 patients with a median age of 13.2 years (range 3 months to 20 years) was retrospectively analyzed. Patients received an average of 19.9 (SD = 13.6) RF lesions, all delivered by an atrial approach. Supraventricular ectopy and ventricular ectopy were compared immediately before and after and 4 to 9 weeks after RF ablation by serial Holter monitoring. Factors thought to possibly predispose patients to a proarrhythmic effect were used to define subgroups for separate analysis. No increase in ambient supraventricular ectopy or ventricular ectopy was observed either immediately after or 4 to 9 weeks after RF ablation compared with the baseline Holter recordings. Children exposed to relatively large doses of RF energy may demonstrate transient and asymptomatic nonsustained tachycardias in the short term. However, no new sustained tachycardias and no increase in supraventricular or ventricular ambient ectopy are detected by short-term Holter monitoring.

Adolescent↗
PubMed1994

Management of atrial flutter after the Fontan procedure.

OBJECTIVES: The purpose of this study was to review the management of atrial flutter occurring after the Fontan procedure. BACKGROUND: Atrial flutter occurs frequently after the Fontan procedure and is often hemodynamically poorly tolerated. METHODS: The patients' charts were reviewed for relevant information. RESULTS: Between 1984 and 1992, 18 patients had atrial flutter after the Fontan procedure. The underlying heart defect was tricuspid atresia in nine, mitral atresia in six and double inlet left ventricle in three. All but three patients had undergone previous palliative surgery. The time interval from Fontan operation to atrial flutter was < 1 day to 16 years (mean 3.7 years). Seven had early atrial flutter before leaving the hospital. Electrophysiologic study in 15 showed sinus node dysfunction in 12. Atrial flutter was inducible in all patients, and 13 had > 1 flutter configuration. Digoxin and a variety of other antiarrhythmic agents (mean 2.7 drugs/patient) were tried with poor results. Only digoxin, amiodarone, flecainide and propafenone showed some benefit when used alone or in combination. Antitachycardia pacemakers were implanted in 16 patients (endocardial 14, epicardial 2) and, with drugs, were useful in 8 (50%). Because atrial flutter was resistant to treatment, right atriectomy was performed in three patients (with benefit in two, one death), successful radiofrequency catheter His bundle ablation in one patient and catheter ablation of atrial flutter in three patients (two failed, one partial success). One patient underwent heart transplantation, and two died suddenly. Another died of complications after an elective epicardial pacemaker replacement procedure. CONCLUSIONS: Atrial flutter after the Fontan procedure is difficult to control. Aggressive drug and antitachycardia pacemaker therapy help about half of the patients. When these measures fail, other options, such as atriectomy, His bundle ablation or catheter ablation of atrial flutter, need consideration. The risk of sudden death justifies the use of such aggressive treatment methods.

Adolescent↗
PubMed1994

Electrical stimulation of contractile activity accelerates growth of cultured neonatal cardiocytes.

An electrical stimulation system was designed to regulate synchronized contractile activity of neonatal rat cardiocytes and to examine the effects of mechanical contraction on cardiocyte growth. Continuous electrical stimulation at a pulse duration of 5 milliseconds and frequency of 3 Hz resulted in a time-dependent accumulation of cell protein that reached 34% above initial values, as measured by the protein-to-DNA ratio. The growth response did not occur using voltage amplitudes that were subthreshold for contraction and was independent of contraction frequencies set at > or = 0.5 Hz. The RNA-to-DNA ratio increased in parallel to cell protein, indicating that the capacity for protein synthesis was enhanced by contraction. Rates of 28S rRNA synthesis were accelerated twofold in contracting cardiocytes. By comparison, protein and RNA accumulation did not occur in electrically stimulated cardiocytes in which contraction was blocked by either 10 mumol/L verapamil or by 5 mmol/L 2,3-butanedione monoxime, an inhibitor of actomyosin crossbridge cycling. Electrical stimulation of cardiocyte contraction did not enhance alpha-cardiac actin or myosin heavy chain (alpha+beta) mRNA transcript levels relative to 28S rRNA during the period of rapid growth that occurred over the first 48 hours. It is concluded that (1) electrical stimulation of contraction accelerates cardiocyte growth and RNA accumulation, (2) mechanical contraction is involved in regulating the growth of electrically stimulated cardiocytes, and (3) the levels of alpha-actin and myosin heavy chain mRNA increase in proportion to rRNA during the growth of contracting cardiocytes.

Animals↗
PubMed1993

Echocardiographic and anatomic correlation of ventricular septal defect morphology in newborn Yucatan pigs.

With the use of a Yucatan micropig strain with a high incidence of ventricular septal defects (VSDs), results of two-dimensional and color-flow Doppler echocardiography of VSD morphology in newborn piglets were correlated with autopsy findings. A spectrum of perimembranous, muscular outlet, and doubly committed subarterial VSDs was found. Echocardiography was performed in 29 piglets weighing 1.2 to 4.4 (mean 2.8) kg, studied at age 4 to 18 (mean 8) days. VSD was diagnosed by means of echocardiography in 16 of 29 subjects; morphologic findings included perimembranous defects in 12, muscular outlet in two, and doubly committed subarterial defect in two. At autopsy the presence and location of defects were confirmed in all pigs. No additional defects were found. VSD diameters were 1.0 to 5.0 (mean 3.94) mm on echocardiography and 1.0 to 6.0 (mean 2.84) mm at autopsy. After aortic valve diameter was used as an internal control for tissue shrinkage during fixation, echocardiography/color Doppler imaging tended to overestimate VSD diameter by 21% (0.6 mm). In conclusion, echocardiography/Doppler imaging accurately identified the presence, morphology, and size of even the smallest VSDs in newborn Yucatan micropigs. Echocardiographic classification of VSD morphology in vivo will facilitate future research on specific types of VSDs in this animal model.

Animals↗
PubMed1992

Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species.

Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2.

Amino Acid Sequence↗
PubMed1991

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.

Animals↗
PubMed1991

Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins.

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai.

Bacillus thuringiensis subsp. aizawai EG6346, a novel grain dust isolate, was analyzed by Southern blot hybridization for its insecticidal crystal protein (ICP) gene profile. Strain EG6346 lacks previously characterized cryIA ICP genes yet does possess novel cryI-related gene sequences. A recombinant genomic plasmid library was constructed for strain EG6346 in Escherichia coli. One recombinant plasmid, pEG640, isolated from the library contained a novel ICP gene on a 5.7-kb Sau3A insert. The sequence of this gene, designated cryIF, was related to, but distinct from, the published sequences for other cryI genes. A second novel cryI-related sequence was also located on pEG640, approximately 500 bp downstream from cryIF. Introduction of cryIF into a Cry- B. thuringiensis recipient strain via electroporation enabled sufficient production of CryIF protein for quantitative bioassay analyses of insecticidal specificity. The CryIF crystal protein was selectively toxic to a subset of lepidopteran insects tested, including the larvae of Ostrinia nubilalis and Spodoptera exigua.

Amino Acid Sequence↗
PubMed1989

The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase.

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex.

Animals↗
PubMed1989

The effect of methionine on methotrexate metabolism in rat hepatocytes in monolayer culture.

The effect of methyl donors on the metabolism of methotrexate has been investigated in rat hepatocytes in monolayer culture. Pulse exposure to low concentrations of methotrexate (1 microM, 3h) in the absence of methionine results in the facile formation of the di- to pentaglutamates with the di- and triglutamate predominating. Further incubation after the removal of methotrexate (MTX) results in a shift to the tetra- and pentaglutamate at the expense of the shorter chain length derivatives. The same measurement in the presence of 1 mM methionine causes approx. an 80% inhibition in the formation of polyglutamates. This effect can be partially achieved when methionine is replaced by choline or betaine. No alteration in the formation of 7-hydroxymethotrexate could be detected by similar changes in methionine concentrations in the medium. The activity of the enzymes which synthesize and degrade methotrexate polyglutamates, folylpolyglutamate synthetase and gamma-glutamyl hydrolase, respectively, were the same in extracts of cells grown in the absence and in the presence of 1 mM methionine. Incubation of the hepatocytes with methionine causes a significant increase in 5,6,7,8-tetrahydrofolate (H4folate), 5,10-methylenehydrofolate and 10-formyltetrahydrofolate and a decrease in 5-methyltetrahydrofolate. These results suggest that the inhibition of glutamylation of methotrexate could be due in part to an elevation in reduced folates which can more effectively compete with methotrexate as a substrate for folylpolyglutamate synthetase. Inhibition in methotrexate glutamylation by methionine, betaine and choline in hepatocytes may contribute to the alleviation of hepatic toxicity by methyl donors.

Animals↗
PubMed1989

The enhancement of the activity of 10-propargyl-5,8-dideazafolate and 5,10-dideazatetrahydrofolate by inhibitors of dihydrofolate reductase.

Treatment of H35 hepatoma cells with the lipid soluble dihydrofolate reductase inhibitors metoprine and trimetrexate cause a nearly 10-fold increase in the toxicity of the antipyrimidine folate analogue PDDF and the antipurine folate analogue DDATHF. Evaluation of these interactions by the combination index developed by Chou (17-20) yields results conforming to synergistic interactions. The capacity of PDDF to inhibit thymidylate synthase in intact cells as measured by tritium release from [5-3H]deoxyuridine was increased by approximately the same amount by preincubation with the dihydrofolate reductase inhibitors. The primary effect of the reductase inhibitors in causing greater activity may be a reduction in cellular folates which can cause 5,10-CH2H4PteGlun to decrease and cellular PDDF (polyglutamates) to increase. These conditions would favor inhibition of thymidylate synthase by PDDF by promoting formation of the stable, inhibited PDDF (polyglutamates)-thymidylate synthase-dUMP complex (12).

Animals↗
PubMed1988

Role of folylpolyglutamate synthetase in the regulation of methotrexate polyglutamate formation in H35 hepatoma cells.

The effect of culture conditions on the glutamylation of methotrexate by intact H35 hepatoma cells and folylpolyglutamate synthetase (FPGS) activity in the corresponding crude extracts has been examined. The rate of cellular glutamylation of methotrexate observed in rapidly dividing cultures was 4-fold higher than confluent cultures, and was accompanied by an increase in extract FPGS activity (2.2-fold). The depletion of cellular folates produced comparable increases in both cellular methotrexate glutamylation and extract FPGS activity (approximately 1.8-fold). Near-quantitative reductions in cellular methotrexate glutamylation were caused by media additions of reduced folates and methotrexate to confluent cultures of wild-type and folate-depleted H35 cells. However, these produced relatively modest reductions in FPGS activity in the corresponding crude extracts (approximately 50%). Methionine exclusion resulted in a greater than 50% decrease in FPGS activity in crude extracts of these cells compared to extracts of control cultures. The combination of methionine exclusion and folinic acid addition lowered the FPGS activity to less than 25% that of control. The data suggest that the changes in the glutamylation rate of methotrexate in whole cells due to culture conditions such as folate restriction, reduced folate addition, methionine exclusion, and growth state are at least in part a consequence of alterations in FPGS activity. This conclusion is consistent with the proposition that the metabolism of slow-acting substrates for FPGS (such as 4-amino antifolates and their corresponding polyglutamates) may be sensitive to changes in enzyme levels or activity (Cook et al., Biochemistry, 26: 530-539, 1987). Analysis of the products formed by FPGS from extracts using methotrexate as the substrate revealed no significant amounts of polyglutamate species higher than 4-NH2-10-CH3-PteGlu3. In contrast, when using the thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid as the starting substrate under identical assay conditions, FPGS from extracts catalyzed the formation of predominantly long chain polyglutamate derivatives (Glu4 and higher). These results reflect the relative efficacy of methotrexate and N10-propargyl-5,8-dideazafolic acid, as well as their polyglutamate derivatives, as substrates for FPGS.

Animals↗
PubMed1987

The absence of gamma-glutamyltransferase activity in transport-dependent methotrexate-resistant hepatoma cells.

A cell line derived from H35 hepatoma cells resistant to methotrexate (MTX) as a result of a defective transport system for MTX has been examined to determine how closely the variant resembles the parent cells with regard to other biochemical properties. The capacity of extracts of resistant cells to catalyze the poly-gamma-glutamylation of MTX was approximately twice as great as that of wild-type cell extracts. Evidence of similarity between wild-type and H35 R0.3 cells was derived from the equitoxic activity to both cell lines of nonclassical antifolates and other miscellaneous antineoplastics which act by a variety of mechanisms. Two phenotypic markers of hepatic cell function, alpha-aminoisobutyric acid (AIB) transport and tyrosine aminotransferase (TAT) activity inducibility, were present in both cell types, demonstrating the maintenance of these phenotypic properties in the H35 R0.3 cells. gamma-Glutamyltransferase (GGT, EC 2.3.2.2) activity differed in that it was present in wild-type cells and barely detectable in H35 R0.3 cells. The GGT activity reappeared in the H35 cells when they regained MTX sensitivity after incubation for 14-20 weeks in MTX-free media. Although defective MTX transport appeared to be correlated with the disappearance of GGT activity in an H35 variant cell line, no functional relationship between them is apparent at this time. It is possible that a lack of GGT activity may be evidence of a more differentiated phenotype in the transport-resistant cell line.

Aminoisobutyric Acids↗