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Biomedical subjects

E H Lee

Publications and source records attributed to E H Lee.

At least 19 recordsLinked to original sources
PubMed1992

Features of replication fork blockage by the Escherichia coli terminus-binding protein.

Blockage of the progress of a DNA replication fork in Escherichia coli can be ascribed to an inhibition of helicase action at the orientation-specific binding of a termination sequence (ter) by the ter-binding protein (Lee, E.H., Kornberg, A., Hidaka, M., Kobayashi, T., and Horiuchi, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9104-9108). These observations have been extended to include the PriA helicase, thus confirming that blockage is general for helicases. The site of arrest of synthesis by a replication fork is at the very first nucleotide of the 22-base pair E. coli-terB sequence. Strand displacement by DNA polymerases is also inhibited, but is less profound and is orientation-specific. The ter sequences of plasmids R1-terR and -terL and of plasmids R6K and R100 have been compared with those of E. coli-terA and -terB.

Adenosine Triphosphatases↗
PubMed1992

Role of asparagine-111 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as explored by site-directed mutagenesis.

Crystallographic studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum suggest that active-site Asn111 interacts with Mg2+ and/or substrate (Lundqvist, T., and Schneider, G. (1991) J. Biol. Chem. 266, 12604-12611). To examine possible catalytic roles of Asn111, we have used site-directed mutagenesis to replace it with a glutaminyl, aspartyl, seryl, or lysyl residue. Although the mutant proteins are devoid of detectable carboxylase activity, their ability to form a quaternary complex comprised of CO2, Mg2+, and a reaction-intermediate analogue is indicative of competence in activation chemistry and substrate binding. The mutant proteins retain enolization activity, as measured by exchange of the C3 proton of ribulose bisphosphate with solvent, thereby demonstrating a preferential role of Asn111 in some later step of overall catalysis. The active sites of this homodimeric enzyme are formed by interactive domains from adjacent subunits (Larimer, F. W., Lee, E. H., Mural, R. J., Soper, T. S., and Hartman, F. C. (1987) J. Biol. Chem. 262, 15327-15329). Crystallography assigns Asn111 to the amino-terminal domain of the active site (Knight, S., Anderson, I., and Brändén, C.-I. (1990) J. Mol. Biol. 215, 113-160). The observed formation of enzymatically active heterodimers by the in vivo hybridization of an inactive position-111 mutant with inactive carboxyl-terminal domain mutants is consistent with this assignment.

Amino Acid Sequence↗
PubMed1992

Cholinergic and GABAergic mediations of the effects of apomorphine on serotonin neurons.

Apomorphine (APO) has been shown to elevate tryptophan, serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the dorsal raphe (DR) and its corresponding projection site, the striatum, but not in the median raphe (MR) and its terminal area, the hippocampus. We have previously demonstrated that these effects are indirectly mediated through dopamine (DA) autoreceptors in the substantia nigra and possibly gamma-aminobutyric acid (GABA) neurons in or near the DR. In the present study, we have further found that the effects of APO on 5-HT neurons are also mediated through both nicotinic and M1 muscarinic cholinergic receptors as well as GABAA receptors in the DR. This suggestion is based on the findings that both atropine and mecamylamine antagonized the effects of APO, while carbachol at a high dose exerted an effect opposite to that of APO. Besides, pirenzepine and bicuculline at low doses also antagonized, whereas saclofen did not alter the influence of APO on 5-HT in the striatum. Bicuculline at a higher dose enhanced tryptophan and 5-HT measures by itself. None of the drugs studied had a significant effect on tryptophan, 5-HT, or 5-HIAA in the hippocampus. These results together suggest that DA, ACh, and GABA neurons are all involved in the actions of APO on 5-HT, while the direct synaptic relationships among these neurotransmitters and the precise anatomical locus for these interactions to occur are still unknown. It is possible that APO, by inhibiting DA neuron firing in the substantia nigra and through the GABA disinhibition mechanism, therefore indirectly activates 5-HT neurons in the DR and the striatum. While the above neuronal firing model well explains the elevation of 5-HIAA, the simultaneous increases of tryptophan and 5-HT, especially tryptophan, may be more readily explained by a mechanism of tryptophan uptake upon APO administration. Further anatomical, biochemical, and electrophysiological studies are ongoing to test this hypothesis and to clarify the circuit and the anatomical locus (loci) for these interactions to occur.

Acetylcholine↗
PubMed1992

Fluoxetine and 8-OH-DPAT in the lateral septum enhances and impairs retention of an inhibitory avoidance response in rats.

The present study investigated the role of lateral septal serotonin (5HT) in memory consolidation and the subtype of 5HT receptors involved in this process. Rats with cannulae implanted bilaterally into the lateral septum were trained in an inhibitory avoidance task. Immediately after training, the septal serotonergic function was manipulated by pharmacological agents selectively blocking 5HT reuptake (fluoxetine and zimelidine), antagonizing 5HT2 receptors (ketanserin and ritanserin), or activating 5HT1A receptors, respectively. Results indicated that direct fluoxetine infusions into the lateral septum at a dose of 6 micrograms/0.5 microliter and zimelidine at a dose of 5 micrograms/0.5 microliter both markedly enhanced memory. Intralateral septal injections of ketanserin (0.3 microgram/0.5 microliter and 0.5 microgram/0.5 microliter) and ritanserin (0.3 microgram/0.5 microliter and 0.6 microgram/0.5 microliter) did not have a significant effect by themselves on memory, and neither did they attenuate the memory-facilitating effect of fluoxetine in the same area. Intralateral septal infusions of 8-hydroxy-2-(di-n-propylamino)tetralin at 5 micrograms/0.5 microliter significantly impaired memory retention. These findings altogether support the notion that the lateral septal nuclei of rats are involved in the memory processes of inhibitory avoidance learning. Furthermore, postsynaptic 5HT receptor activation (not the 5HT2 receptor subtype) probably exerts a facilitatory effect while presynaptic 5HT1A receptor activation exerts an impairing effect on the memory consolidation process, probably due to autoreceptor inhibition of 5HT release.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

CRF increases protein phosphorylation and enhances retention performance in rats.

This study examines the roles of PKC and protein phosphorylation in the retention performance of a passive avoidance learning (PAL) task in rats. Results revealed that H7 injected into the dentate gyrus (DG) of the hippocampus impaired retention in a dose-dependent manner. Corticotropin-releasing factor (CRF) injected into the DG improved retention and this facilitation was antagonized by H7 pretreatment. CRF increased phosphorylation of five proteins, whereas H7 decreased phosphorylation in three of these proteins in both the cytosol and the membrane fractions of hippocampus. These effects were shown not to be associated with stress. These results demonstrate that CRF increased protein phosphorylation associated with enhanced retention of PAL task in rats.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Differential effects of deprenyl and MPTP on catecholamines and activity in BALB/c mice.

The present study examined the effects of deprenyl (1.5 mg kg-1) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg kg-1) on dopamine (DA), norepinephrine (NE) and motor activity in BALB/c mice. Results indicate that pre-treatment with deprenyl injections protected against MPTP's toxicity on DA, NE and motor function. However, if deprenyl was given 3 or 7 days after the MPTP injection (for 3 days in succession), it could not reverse MPTP's toxicity. However, if deprenyl was given for an extended period of 7 days, it reversed MPTP's toxicity on NE, but not on DA and behaviour. If deprenyl was given soon after MPTP each day for 10 days, it protected against MPTP's toxicity on DA and NE, but not on locomotor activity. These results suggest that only prior and repeated deprenyl injection has a satisfactory protective effect against MPTP's neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

Diffusion of beta-lactam antibiotics into proteoliposomes reconstituted with outer membranes of isogenic imipenem-susceptible and -resistant strains of Enterobacter cloacae.

The influence of outer membrane (OM) permeability on carbapenem susceptibility was studied in strains of Enterobacter cloacae, a species in which carbapenem resistance depends upon the conjunction of overproduction of the chromosomal cephalosporinase and reduction of OM permeability. Relative trans-OM diffusion rates were measured using the liposome swelling assay. Proteoliposomes were reconstituted with OM from the members of an isogenic set of E. cloacae strains, selected in vivo or in vitro, which produced either porins F and D (wild-type), or F or D only, or neither. For all but one mutant, and compared with the wild-type strain, the respective increases in MICs and decreases in trans-OM diffusion of carbapenems were: nil and 13 to 18%; 4- to 32-fold and 33 to 50%; > or = 64-fold and > or = 90%. Our results suggest (i) that carbapenems (and other beta-lactam antibiotics) diffuse through porins F and D, but more rapidly through porin F, and (ii) that OM permeability is the critical factor in determining the level of MICs of carbapenems for cephalosporinase-overproducing strains of E. cloacae. The OM of one particular low-level carbapenem-resistant and porin F- and D-deficient mutant was at least five times more permeable to carbapenems than the similarly porin-deficient high-level resistant mutants. We infer from this observation the possible existence of an alternative carbapenem penetration pathway which could be associated with two as yet uncharacterized overproduced OM proteins of about 22 and 47 kDa.

Anti-Bacterial Agents↗
PubMed1992

Comparative studies of the neurotoxicity of MPTP in rats of different ages.

The present study investigated the neurotoxic effects of repeated MPTP injections on monoamine neurotransmitters and locomotor activity in rats of different ages. We also examined the mortality of MPTP-treated rats at different ages. Male Sprague-Dawley rats were used in all experiments. In the first experiment, we examined the mortality of rats (11-12 month old) subject to different doses of MPTP. In the second experiment, rats of 2-3 months old were randomly divided into five groups. Group 1 served as the control; Groups 2,3,4 and 5 received daily MPTP injections (30 mg/kg, ip) for a continuation of 7 days. Biochemical and behavioral assays were conducted at 1,7,14 and 28 days after withdrawal of MPTP, respectively. In the third and fourth experiments, the same experimental design was adopted except that rats of 5-6 months old and rats of 11-12 months old were used, respectively. Besides, the doses of MPTP used were 22.5 mg/kg and 12.5 mg/kg, respectively. Immunohistochemical experiments were always conducted 7 days after withdrawal of MPTP. Results indicated that, in young rats, repeated MPTP injections did not significantly decrease DA, and 5HT levels as well as TH and DBH immunoreactivities although it impaired locomotor activity. The same treatment significantly depleted DA, NE and 5HT levels in the middle-aged rats. It also decreased the density of TH and DBH immunoreactivities and altered the morphology of DA and NE neurons. Meanwhile, it impaired locomotor activity. In old rats, MPTP injections produced effects similar to those observed in the middle-age rats except that the hippocampal serotonergic system was also affected. However, all these effects recovered 28 days after withdrawal of MPTP injection. Finally, the dose of MPTP required to exert similar extent of neurotoxicity decreased as the age of rats increased, and the dose required to result in mortality markedly decreased in old rats. These results together suggest that MPTP does exert a toxicity on DA, NE and 5HT neurons and impair motor activity in rats. These effects are age-dependent while the irreversibility of MPTP's toxicity in rats requires further investigation.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

Biomechanical study on the load-bearing characteristics of the fibula and the effects of fibular resection.

The objective of this research was to investigate the load-bearing function of the fibula in relation to donor leg morbidity in patients who have had fibular resections. Biomechanical loading experiments were performed on ten anatomic specimens. Force transducers were mounted in place of resected tibial and fibular segments to allow load transmission to be measured. Load transmission through the fibula varied with ankle position. With the ankle at neutral position, the load distribution to the fibula averaged 7.12% of the total force transmitted through the tibia and fibula. Maximum loads occurred at full dorsiflexion and eversion. Resection of the proximal fibula results in a significant reduction of load through the distal fibular remnant. The values varied between 0.62% and 0.81% of the total force transmitted. When a cortex screw was introduced to anchor the distal fibula remnant to the tibia, the load distribution to the distal fibula remnant was partially restored with values ranging from 1.71% to 5.14% of the total force transmitted depending on the different ankle positions. These observations suggest that more consideration of the loading characteristics of the fibula should be taken into account in planning resection operations.

Biomechanical Phenomena↗
PubMed1992

Value of gait analysis in the assessment of surgery in cerebral palsy.

Twenty-three ambulatory children with cerebral palsy were assessed preoperatively by a detailed clinical examination and by gait analysis using a video-based gait analysis system (VICON). Surgery was then performed based on either the clinical assessment alone or a combination of clinical evaluation and gait analysis. About one year after surgery, a postoperative clinical and gait analysis assessment was performed. Sixteen children had improved and seven children had not improved after surgery. Most of the children who had not improved were found to have had operations that differed from those recommended by gait analysis. Dynamic EMG studies were found to be useful in preoperative planning but did not show any consistent improvement even in the children with good results. The combination of a careful clinical assessment and gait analysis can produce better results in surgery for children with cerebral palsy.

Adolescent↗
PubMed1991

Replication deficiencies in priA mutants of Escherichia coli lacking the primosomal replication n' protein.

The priA gene of Escherichia coli encodes the protein that initiates assembly of the promosome, the entity essential for the replication of phage phi X174 and ColE1-like plasmids in vitro. We have prepared a null priA mutant to assess its role in vivo in replication of phages, plasmids, and the host chromosome. Extracts of this mutant are inert in the initial conversion of the phi X174 viral strand to the duplex form, confirming the absence of the PriA activity. In vivo, the priA mutant fails to produce phi X174 phage and, remarkably, is unable to maintain plasmids that depend on the E. coli chromosome origin as well as those of ColE1. Deficiencies in cell growth and cell division are also manifest.

Adenosine Triphosphatases↗
PubMed1991

Nifedipine and verapamil block the memory-facilitating effect of corticotropin-releasing factor in rats.

The effects of two calcium channel blockers, nifedipine and verapamil, and the peptide corticotropin releasing factor (CRF) as well as their interactive effects on memory retention were examined in rats. Male Sprague-Dawley rats were chronically cannulated bilaterally and drugs were directly injected into the dentate gyrus of the hippocampus. Animals were trained in a one-way inhibitory avoidance task and memory was measured 24 h later. Results indicate that there was a U-shaped dose-response curve for the effects of nifedipine and verapamil with nifedipine at 8 micrograms and verapamil at 1 microgram both impaired memory formation, while CRF at 0.1 microgram enhanced this process. Nifedipine at 2 micrograms and verapamil at 0.5 microgram, which did not have significant effects on memory by themselves, antagonized the memory-enhancing effect of CRF in the hippocampus. These results suggest that under normal physiological conditions, calcium influx may play an important role in memory consolidation process in the vertebrate.

Animals↗
PubMed1991

Recombinagenic processing of UV-light photoproducts in nonreplicating phage DNA by the Escherichia coli methyl-directed mismatch repair system.

Nonreplicating lambda phage DNA in homoimmune Escherichia coli lysogens provides a useful model system for study of processes that activate DNA for homologous recombination. We measured recombination by extracting phage DNA from infected cells, using it to transfect recA recipient cells, and scoring the frequency of recombinant infective centers. With unirradiated phage, recombinant frequencies were less than 0.1%. However, recombination could be increased over 300-fold by prior UV irradiation of the phages. The dependence of recombination on UvrA function varied greatly with UV dose. With phage irradiated to 20 J/m2, recombinant frequencies in repressed infections of uvr+ bacteria were one-fifth those in uvrA infections; with phages irradiated to 100 J/m2, frequencies in uvr+ infections were thirty times higher than in uvrA infections. Most UV-stimulated recombination in uvrA infections appeared to depend on the bacterial methyl-directed mismatch-repair system: frequencies were depressed 5-20-fold in uvrA bacteria also lacking MutH, MutL or MutS functions, and recombinant frequencies decreased with increasing GATC-adenine methylation of phage stocks. The biological activity of nonreplicating UV-irradiated phage DNA declined with time after infection of uvrA cells; this decline was photoproduct-dependent, more marked for undermethylated than overmethylated phage DNA, and depended on host MutHLS functions. In uvr+ bacteria, where the UvrABC system provided an alternative, apparently less efficient, route to recombinagenic DNA, UV-stimulated recombinant frequencies were about twice as high in mutH or mutLS as in mut+ cells, in agreement with hyper-rec mut effects previously described by others.

Adenosine Triphosphatases↗
PubMed1991

Association of two resistance mechanisms in a clinical isolate of Enterobacter cloacae with high-level resistance to imipenem.

Carbapenem resistance was studied in a clinical isolate of Enterobacter cloacae, strain 201 (MIC of imipenem and meropenem, 16 micrograms/ml). This strain was analyzed comparatively with the carbapenem-susceptible parent strain 200, an equally susceptible revertant, 201-Rev, and in vitro-selected mutants with different levels of carbapenem resistance. All strains produced similarly high amounts of the same cephalosporinase (pIapp = 8.8). Strain 201 apparently lacked two major outer membrane proteins of ca. 37 and 38 kDa, while 201-Rev produced only the 37-kDa protein. The permeability coefficient, determined with cephaloridine, was reduced up to ninefold in the resistant strains which also showed a substantial reduction in the uptake of [14C]meropenem. The introduction of the plasmid-borne ampD gene (whose product decreases the expression of ampC) resulted in almost complete cessation of cephalosporinase production in all strains and a substantial decrease in the MICs of the carbapenems which remained, however, 8- to 16-fold higher than those determined for the susceptible strains containing the ampD gene. This "residual" resistance was attributed to reduced outer membrane permeability. The contribution of cephalosporinase production was verified in a reverse experiment, in which the introduction of ampC into a low-level cephalosporinase producer resulted in a fourfold increase in the carbapenem MICs. From these results, we infer that reduced outer membrane permeability and high-level cephalosporinase production can operate in conjunction in clinical isolates of E. cloacae to confer imipenem resistance.

Bacterial Outer Membrane Proteins↗
PubMed1991

Plant resistance mechanisms to air pollutants: rhythms in ascorbic acid production during growth under ozone stress.

Relationships between ozone (O3) tolerance and leaf ascorbic acid concentrations in O3-susceptible (O3-S) 'Hark' and O3-resistant (O3-R) 'Hood' soybean, Glycine max (L.) Merr., cultivars were examined with high-performance liquid chromatography (HPLC). Leaf samples were analyzed at 4 intervals during a 24 h period. Soybean cultivars grown in the greenhouse with charcoal filtered (CF) and nonfiltered (NF) air showed daily oscillations in ascorbic acid production. Highest ascorbic acid levels in leaves during light coincided with highest concentrations of photochemical oxidants in the atmosphere at 2:00 p.m. The resistant genotype produced more ascorbic acid in its trifoliate leaves than did the corresponding susceptible genotype. Under CF air (an O3-reduced environment) O3-S and O3-R cultivars showed rhythms in ascorbic acid production. In NF air (an O3 stress environment) the O3-R cultivar alone showed rhythms in ascorbic acid production. Results indicated that superior O3 tolerance in the Hood soybean cultivar (compared with Hark) was associated with a greater increase in endogenous levels of ascorbic acid. Ascorbic acid may scavenge free radicals and thereby protect cells from injury by O3 or other oxyradical products. Plants defend themselves against photochemical oxidant stress, such as O3, by several mechanisms. Experimental evidence indicates that antioxidant defense systems existing in plant tissues may function to protect cellular components from deleterious effects of photochemical oxidants through endogenous and exogenous controls.

Adaptation, Physiological↗
PubMed1991

Influence of amineptine on changes of blood pressure evoked by norepinephrine and dopamine.

The influence of amineptine, an antidepressant currently employed having mainly selective dopaminergic neurochemical activity, on the pressor responses evoked by norepinephrine (NE) and dopamine (DA) was studied in anesthetized whole rats. Amineptine at doses of 0.5, 1.5, and 5.0 mg/kg/30 min infused into the femoral vein of the rat caused a dose-related inhibition of the pressor responses of NE and DA. The hypertensive responses of NE and DA augmented by pretreatment with reserpine, a catecholamine depletor, were also clearly depressed following the infusion of amineptine with a rate of 1.5 mg/kg/30 min. Furthermore, the pressor responses of NE and DA potentiated by pretreatment with debrisoquin, a sympathetic neuron blocker, were markedly diminished after pretreatment with the infusion of amineptine at the above same rate (1.5 mg/kg/30 min). These experimental results demonstrate that amineptine causes an inhibitory effect on the pressor responses evoked by NE and DA. It is thought that the amineptine effect may be due to the blockade of the peripheral adrenergic alpha-receptors in addition to the previously described uptake inhibition of dopamine.

Animals↗
PubMed1991

Responses of neurons in paramedian reticular nucleus to chemical stimulations and alteration of blood pressure in rats.

Previous studies have shown that paramedian reticular nucleus (PRN) possessed sympathetic and parasympathetic inhibitions on autonomic nervous system. In the present study, the cardiovascular reactions of PRN by locally-applied DL-homocysteic acid (DLH), acetylcholine (ACh), monoamines and electrophysiological properties of PRN neurons responding to intravenous injection of ACh and NE were studied in adult Sprague-Dawley rats. In PRN, electrical stimulation caused hypotension and mild bradycardia while microinjection of DLH, which excites only cell body of the neurons but not passing fibers, evoked similar responses. Furthermore, direct application of ACh, norepinephrine (NE) or serotonin (5-HT) in PRN also produced hypotension, suggesting that these putative neurotransmitters may be involved in the cardiovascular responses in PRN. The electrophysiological properties of PRN neurons were studied: Neurons in PRN could be categorized into three types according to their neuronal activities in response to the changes of systemic arterial blood pressure (SAP) by ACh or NE given intravenously. Type I neurons (25/69) were activated in the same direction of SAP changes. Type II neurons (17/69) responded opposite to the direction of SAP changes. Type III neurons (27/69) responded inconsistently to the changes of SAP. All the three types of neurons were excited by locally-applied DLH and possessed a similar unfiltered action potential duration of greater than 0.5 msec.

Acetylcholine↗