Biomedical subjects

T A Patterson

Publications and source records attributed to T A Patterson.

At least 37 records · Page 2Linked to original sources

PubMed1995

Recombinant human dihydroorotate dehydrogenase: expression, purification, and characterization of a catalytically functional truncated enzyme.

An N-terminally truncated cDNA for human dihydroorotate dehydrogenase (DHODase) was placed under the control of the inducible T7 lac promoter in a pyrimidine auxotrophic strain of Escherichia coli lacking the endogenous enzyme. Induction of gene expression rescued growth in media lacking exogenous pyrimidines. The recombinant enzyme was purified to homogeneity from detergent extracts of bacterial membranes by two chromatographic steps. The purity of the resulting enzyme was judged to be > 95% based on SDS-PAGE with Coomassie staining. The enzyme displays an apparent molecular weight of ca. 40 kDa on SDS-PAGE and ca. 120 kDa on native size-exclusion chromatography, suggesting that the native enzyme is multimeric. Recombinant DHODase displayed a specific activity and Km for dihydroorotate that were similar to those for the enzymes from bovine and human liver tissue. The pH dependence of the activity of the recombinant enzyme was likewise similar to that of the enzyme from human liver and may indicate the involvement of a critical histidine residue in catalytic turnover; only eight histidine residues remain in the truncated version of DHODase used here. The catalytic activity of the recombinant enzyme is inhibited in a dose-dependent fashion by the histidine-selective modifying agent diethylpyrocarbonate. These results further suggest a potential role for histidine in enzyme turnover. Brequinar sodium, an experimental drug which has been shown to be a nanomolar noncompetitive inhibitor of mammalian DHODases, inhibited the activity of the purified recombinant enzyme with a Ki value similar to that for enzyme derived from human liver tissue. The recombinant DHODase thus displays enzymatic behavior similar to the 50-kDa full-length human liver enzyme, illustrating that the catalytically essential structural features of the enzyme, as well as the site of Brequinar binding, are contained within the 40-kDa truncated version of the enzyme that was expressed here.

Amino Acid Sequence↗

PubMed1995

Endogenous excitatory amino acid release from brain slices and astrocyte cultures evoked by trimethyltin and other neurotoxic agents.

Trimethyltin (TMT) is a toxic alkyltin compound that is known to produce neuronal necrosis in the CNS. The present study examined the effects of TMT on the release of excitatory amino acids (EAA) from cortical slices prepared from adult and aged (24 months old) rats. The calcium dependence of TMT-induced EAA efflux was evaluated and compared to other neurotoxic agents. The actions of TMT were also evaluated in an astrocyte culture model to assess glial contributions to TMT-induced EAA efflux. TMT (10-1000 microM) evoked a dose-related increase in GLU and ASP efflux during a 30 min incubation period and this efflux was sustained or slightly higher during a 15 min recovery period. TMT-stimulated GLU efflux was not altered in aged rats. TMT-induced GLU efflux was significantly reduced by removing extracellular calcium and including 10 microM EGTA in the incubation media. Calcium channel blockers (nifedipine, verapamil, flunarizine, amiloride, neomycin) and MK-801 did not significantly attenuate TMT-induced GLU efflux. Diltiazem (25 microM) produced modest but inconsistent reductions in TMT-induced GLU efflux from brain slices, and significantly inhibited the leakage of lactate dehydrogenase (LDH) from TMT-treated astrocyte cultures. TMT did not increase GLU efflux from glial cultures during a 30 min incubation period, but did significantly elevate GLU efflux during the 15 min recovery period. TMT evoked the release of EAA by both calcium dependent and independent mechanisms in brain slices. TMT at high concentrations also produced a delayed increase in glial GLU efflux. These studies suggest that excitotoxic mechanisms may contribute to TMT-induced neurotoxicity.

Aging↗

PubMed1995

Glutamate efflux from rat brain slices and cultures: a comparison of the depolarizing agents potassium, 4-aminopyridine, and veratrine.

The major excitatory amino acid neurotransmitter in the mammalian brain is glutamate (GLU). GLU release from nerve terminals is both calcium-dependent and -independent, yet these mechanisms of release are not fully understood. Potassium, 4-aminopyridine (4-AP) and veratrine are commonly used depolarizing agents that were studied for their ability to stimulate GLU efflux from brain slices. These agents produced significant regional variations in GLU efflux from rat brain slices. Potassium was the most potent of the three secretogogues tested. 4-AP produced a significant GLU efflux only in the cerebellum. Veratrine produced consistent stimulation of GLU efflux from all brain regions tested. Potassium was the only depolarizing agent tested that stimulated GLU release from primary astroglial cultures of rat cerebral cortex. All three agents also demonstrated an ability to inhibit GLU reuptake in brain slice preparations. This data suggest that both GLU release and uptake are modulated in a regionally selective manner, and that commonly used depolarizing agents affect not only calcium-dependent neuronal release, but also uptake and glial responses.

4-Aminopyridine↗

PubMed1995

Immunocytochemical localization of delta opioid receptors in mouse brain.

An affinity-purified anti-peptide antibody generated against the carboxy-terminal region of the delta opioid receptor was used to localize delta opioid receptors in mouse brain. delta Opioid receptor immunoreactivity was found in axons and nerve terminals in regions of the olfactory bulb, hippocampal formation, cerebral and cerebellar cortex, midbrain and hindbrain. The immunocytochemical distribution correlated well, though not completely with autoradiographic distribution of delta opioid receptors in mouse brain using either [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin (DPDPE) or [3H]naltrindole. Confocal microscopy of double-labeled tissue provided direct evidence that delta opioid receptors are principally expressed on GABAergic terminals in the hippocampus. These anatomical findings complement extensive physiological studies to provide a more detailed description of endogenous opioid circuitry.

Animals↗

PubMed1995

Structural and functional analyses of the transcription-translation proteins NusB and NusE.

The NusB and NusE (ribosomal protein S10) proteins function in transcription and translation. The two proteins form a complex that binds to the boxA sequence found in the leader RNA of rrn operons; boxA is required for transcription antitermination in rrn operons. Although binding of these two proteins to the boxA RNA of the bacteriophage lambda nut site has not been observed, both NusB and NusE as well as the RNA boxA sequence are required for lambda N-mediated antitermination. Studies identifying the amino acid changes caused by mutations in nusB and nusE and relating these changes to altered function are reported. It is concluded that boxA is essential for an effective NusB contribution to N-mediated antitermination and that by mutation NusB may be changed to allow more-effective binding to boxA variants.

Alleles↗

PubMed1995

Colocalization of mu opioid receptors with GIRK1 potassium channels in the rat brain: an immunocytochemical study.

Affinity-purified anti-peptide antibodies generated against the carboxy-terminal region of the mu opioid receptor and the GIRK1 (Kir 3.1) ion channel were used to localize these two proteins in the rat brain. Mu opioid receptor immunoreactivity was detected in brain regions that were previously found to contain mu opioid binding sites using autoradiography. The distribution of GIRK1 immunoreactivity in the brain correlated well with a previous in situ hybridization study. Confocal microscopy of rat brain sections double-labelled with anti-mu opioid receptor and anti-GIRK1 antibodies revealed colocalization of GIRK1 and mu opioid receptor immunoreactivities in somata of subpopulations of neurons in the cerebral cortex, anterior olfactory nucleus, nucleus accumbens, globus pallidus, substantia nigra, peripeduncular nucleus, hippocampal formation, diagonal band, thalamus, locus coeruleus, dorsal raphe, red nucleus, nucleus of the trapezoid body, reticular nucleus, vestibular nucleus, inferior colliculus and the mesencephalic trigeminal nucleus. These anatomical findings suggest that the mu opioid receptor may couple to GIRK1 in some but not all regions of the rat brain.

Amino Acid Sequence↗

PubMed1994

Oocytes from Xenopus laevis contain an intrinsic sigma 2-like binding site.

In preparation for expression studies for rat brain sigma-binding sites, Xenopus oocytes were tested for the presence of [3H]di-o-tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [3H]DTG-binding sites, a high-affinity site (Kd = 32 +/- 6 nM, Bmax of 45.7 +/- 19 pmol/mg protein) and a low-affinity binding site (Kd = 1.3 +/- 0.7 microM, Bmax of 3.2 +/- 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar Kd to that of the mammalian sigma 2-binding site (32 vs. 38 nM). Comparison of the IC50 values for inhibition of [3H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (-)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP), (+)-pentazocine and Zn2+, showed similarity in rank (r2 = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [3H]DTG-binding site in oocytes represents a sigma 2-like binding site.

Animals↗

PubMed1994

Increases in neuronal bursting recorded from the chick lobus parolfactorius after training are both time-dependent and memory-specific.

Day-old-chicks can be trained in one trial to avoid a methylanthranilate-coated bead (methyl-chicks). The lobus parolfactorius of the chick forebrain is an important structure for memory of this avoidance response. To examine training-induced electrophysiological changes in this structure, spontaneous neuronal bursting activity was measured from the lobus parolfactorius of anaesthetized, day-old methyl- and water-chicks (the latter chicks trained to peck at a water-coated bead) over the period 1-10 h post-test. Bursting was significantly higher in methyl-chicks over this period. This post-test increase was time-dependent: bursting in methyl-chicks was significantly higher only during the period 4-7 h post-test. In a second experiment, methyl-chicks were subjected to brief, subconvulsive electroshock 5 min post-training. When tested 1 h later about half of these chicks showed recall (avoided the bead) and half were amnesic (pecked the bead). These chicks were anaesthetized and bursting was recorded from the lobus parolfactorius. Chicks that showed recall exhibited a significantly higher level of bursting over the period 1-10 h post-test when compared to chicks that were amnesic. The time course of bursting was similar to that seen in non-electroshocked methyl-chicks. These results suggest that passive avoidance training induces a memory-specific, time-dependent increase in neuronal activity within the lobus parolfactorius of day-old chicks. This increase may be directly associated with long-term consolidation of memory for the task.

Animals↗

PubMed1994

Bacteriophage lambda N-dependent transcription antitermination. Competition for an RNA site may regulate antitermination.

Bacteriophage lambda controls the expression of its early genes in a temporal manner by a series of transcription termination and antitermination events. This antitermination requires the lambda N protein as well as host proteins called Nus, and cis-acting sites called nut. Following transcription of the nut site, N and Nus proteins bind to the nut RNA and modify the transcription complex to a termination-resistant form. The nut site is a composite of at least two components; one is the boxB hairpin structure which interacts with N. The other is boxA, a nine-nucleotide sequence upstream of boxB. To understand more about the formation of the antitermination complex, we have characterized the effect of point mutations in and deletions of boxA on antitermination. Point mutations in boxA were found to either enhance or reduce N-mediated antitermination. Several boxA deletions, on the other hand, had little effect on antitermination other than to eliminate the requirement for the NusB host protein. To explain these observations, we propose that at least two factors compete to interact with boxA, NusB and an inhibitor of the antitermination reaction. In addition, we propose that NusB is required to prevent the inhibitor from binding at boxA. The results with various nusB and boxA mutations can be explained by this model of competition between NusB and an inhibitor for boxA RNA.

Bacterial Proteins↗

PubMed1994

Modification of the duration of action and pharmacodynamics of arecoline by tetraisopropylpyrophosphoramide.

Tetraisopropylpyrophosphoramide (ISO-OMPA) pretreatment in the mouse was examined for its ability to prolong the time course of arecoline (ARE) concentration using brain ARE concentration as an index. Brain ARE levels were also correlated with acetylcholine (ACh) and choline concentrations. Pretreatment with 40 mg kg-1 ISO-OMPA increased ARE brain concentrations from 3 nmoles g-1 to 76 nmoles g-1 15 min after i.p. administration of 25 mg kg-1 ARE. ARE (25 mg kg-1) alone significantly increased brain ACh and choline levels. In time course studies, administration of ARE (15 mg kg-1) produced a peak brain level of 7.9 nmoles g-1 at 3 min. ISO-OMPA pretreatment increased the peak level of ARE to 46.5 nmoles g-1 and the peak time to 7-15 min. The time of maximum brain elevation of ACh and choline produced by ARE alone lagged slightly behind the peak level time observed for ARE. ARE alone produced a maximal increase in ACh and choline levels to 34.1 and 57.1 nmoles g-1, respectively. In the presence of ISO-OMPA, ARE further increased ACh and choline levels to 48.2 and 103 nmoles g-1, respectively. After i.p. administration the highest concentration of ARE was found in the cortex, followed by the subcortex, and cerebellum. A significant elevation of ACh was observed in the cortex.

Acetylcholine↗

PubMed1993

Improved bacterial hosts for regulated expression of genes from lambda pL plasmid vectors.

The construction and use of a set of Escherichia coli strains with defective lambda prophages that facilitate expression of genes cloned in lambda pL-plasmid vectors is described. These bacteria allow high and regulated expression of such genes, whereas a kanamycin-resistance marker (KmR) on the prophage allows easy identification and genetic transfer from strain to strain. Optimal conditions for examining gene expression with the pL-vector systems using these strains are discussed.

Bacteriophage lambda↗

PubMed1993

Elucidation of the rapid in vivo metabolism of arecoline.

1. The metabolism of arecoline (ARE) was examined in homogenates of mouse blood, brain, kidney, and liver tissue. 2. Liver and kidney tissues exhibited the greatest rates of ARE metabolism. 3. The specific carboxylesterase inhibitor TOCP (tri-o-tolyl-phosphate) as well as ISO-OMPA (tetraisopropyl-pyrophosphoramide) completely blocked ARE metabolism in liver homogenate. 4. ISO-OMPA significantly inhibited ARE metabolism by purified porcine liver carboxylesterase. 5. The data suggest that carboxylesterase (EC 3.1.1.1) is primarily responsible for the metabolism of ARE in the mouse.

Animals↗

PubMed1993

Visual input and lateralization of brain function in learning in the chick.

Several lines of evidence (biochemical, neuroanatomical, electrophysiological, and behavioural) have indicated a critical role for the intermediate medial hyperstriatum ventrale of the chick forebrain in the acquisition of a passive avoidance response. Previous lesion studies indicated that bilateral or left, but not right, pretraining intermediate medial hyperstriatum ventrale lesions interfere with the acquisition of this task. We have further analysed this asymmetrical involvement of the intermediate medial hyperstriatum ventrale by use of a monocular learning protocol and intermediate medial hyperstriatum ventrale lesions (sham, bilateral, or unilateral). The results indicated that there is interocular transfer of information of passive avoidance learning between the two eye systems, with a tendency to be more successful from the right eye system to the left than in the opposite direction. As in binocular conditions, bilateral pretraining intermediate medial hyperstriatum ventrale lesions impair learning in monocularly trained animals. Unilateral lesions to either left or right monocularly trained experimental animals resulted in amnesia when they were made to the right intermediate medial hyperstriatum ventrale and the chicks were trained/tested with the left eye open. These results indicate that, although right intermediate medial hyperstriatum ventrale lesions do not result in amnesia in binocular animals, this region is capable of participating in memory acquisition processes. They also suggest a connection between lateralization of intermediate medial hyperstriatum ventrale function in passive avoidance learning and the behavioural and structural visual asymmetries known to occur in chicks.

Animals↗

PubMed1993

Training-induced increases in neuronal activity recorded from the forebrain of the day-old chick are time dependent.

Spontaneous neuronal bursting occurs in many areas of chick forebrain. Day-old chicks trained using a one-trial task to avoid a methylanthranilate-coated bead (methyl-chicks) show a significant increase in bursting when compared to chicks trained to peck a water-coated bead (water-chicks). This increase occurs in two forebrain areas: the intermediate medial hyperstriatum ventrale and the lobus parolfactorius. Bursting was recorded from the intermediate medial hyperstriatum ventrale of anaesthetized methyl- and water-chicks at eight time-points over the period 1-9 h post-test. Data merged over this period showed that methyl-chicks displayed an overall increase in bursting in both left and right hemispheres when compared to water-chicks. When burst activity was compared against time, bursting in methyl-chicks was significantly elevated only during the period 3-7 h post-test. Maximal bursting in methyl-chicks was seen 6-7 h post-test. These results suggest that the training-induced increase in bursting seen in the intermediate medial hyperstriatum ventrale of methyl-chicks is not a simple, generalized increase with time but rather has a significant temporal aspect. These results may have particular relevance to previously proposed models of memory formation in the chick.

Animals↗

PubMed1993

D2 receptors may modulate the function of the striatal transporter for dopamine: kinetic evidence from studies in vitro and in vivo.

Recently it was hypothesized by others that the D2 dopamine receptor can regulate the uptake of dopamine. However, the evidence in support of this hypothesis, although compelling, was not based on observations related to direct measures of the kinetic activity of the transporter itself. Here kinetic evidence in support of this hypothesis is shown. The apparent time-resolved initial velocity of the transport of 1.0 microM dopamine into striatal suspensions, measured using rotating disk electrode voltammetry, was found to increase in the presence of the D2 receptor agonist, quinpirole, at 100 nM. This effect was reversed by sulpiride. In separate studies it was shown that acute and chronic treatments with haloperidol at 0.5 mg/kg, i.p., reduced the reuptake transport of dopamine in vivo following intrastriatal stimulation of its release by K+. Thus, it appears that D2 receptors may influence the functioning of the striatal transporter for dopamine. These results are consistent with a model in which presynaptically released dopamine may feed back onto the function of its transporter to increase the velocity of the clearance of synaptic dopamine following an action potential, suggesting the existence of a mechanism, in addition to release and synthesis modulation, for fine-tuning dopaminergic chemical signaling.

Animals↗

PubMed1992

Construction and use of lambda PL promoter vectors for direct cloning and high level expression of PCR amplified DNA coding sequences.

A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed. The vectors contain the phage lambda PL promoter, a synthetic translation initiation region (TIR), and convenient cloning sites. The cloning sites provide all or part of an AUG translation initiation codon and facilitate the precise fusion of target DNA sequences to vector transcriptional and translational signals. The vectors were constructed with synthetic TIRs because there is evidence which suggests that the efficiency of the phage lambda cII gene TIR present in the parental vector depends strongly on information contained within the cII N-terminal coding sequence. Bovine brain 14-3-3 eta chain cDNA was PCR-amplified and used to demonstrate the expression capacity of the newly constructed vectors. A significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR. Expression levels vary from 15% to 48% of total cell protein. The effects of a reported translational enhancer from phage T7 on expression of the 14-3-3 protein are also discussed. The vectors should be generally useful for high level heterologous protein expression in Escherichia coli.

Bacteriophage lambda↗

PubMed1992

Unilateral hippocampal lesions prevent recall of a passive avoidance task in day-old chicks.

The role of the hippocampal system in learning and memory processes in the chick was investigated. A series of experiments examined the effects of lesions in the hippocampal system on the acquisition and retention of a passive avoidance task. Chicks given pretraining bilateral hippocampal lesions showed a decrease of retention of the avoidance response evaluated 3 h posttraining. When given unilaterally, left, but not right lesions, resulted in reduced avoidance. However, bilateral posttraining lesions, made 1 h after training, did not interfere with retention of the task. These results suggest an involvement of the hippocampal system in learning processes in the chick.

Animals↗

PubMed1992

Deletion of hydrophobic domains of viral glycoproteins increases the level of their production in Escherichia coli.

Overexpression of glycoprotein-encoding genes in Escherichia coli sometimes results in toxicity to the host and low protein yields. One possible explanation for this phenomenon is the presence of hydrophobic amino acid (aa) domains approx. 15-20 aa in length in the overproduced protein. As an initial test of this hypothesis, regions of hydrophobicity located within the envelope glycoproteins of HIV-1 and HTLV-1 were identified by computer analysis, and subsequently deleted by site-directed mutagenesis. The parent and modified envelope genes were expressed in bacteria using both lambda pL and T7 inducible expression systems. Removal of the hydrophobic domains reduced the apparent toxicity and significantly increased the accumulation of recombinant protein from undetectable levels to approx. 10-15% of total cellular protein.

Amino Acid Sequence↗