Biomedical subjects

M Leitner

Publications and source records attributed to M Leitner.

At least 19 recordsLinked to original sources

PubMed2005

Drift compression of an intense neutralized ion beam.

Longitudinal compression of a velocity-tailored, intense neutralized beam at 300 keV, 25 mA has been demonstrated. The compression takes place in a 1-2 m drift section filled with plasma to provide space-charge neutralization. An induction cell produces a head-to-tail velocity ramp that longitudinally compresses the neutralized beam, enhancing the beam peak current by a factor of 50 and producing a pulse duration of about 3 ns. This measurement has been confirmed independently with two different diagnostic systems.

Journal Article↗

PubMed2005

Sertindole for schizophrenia.

BACKGROUND: Sertindole is an atypical antipsychotic, which is thought to give a lower incidence of extrapyramidal side effects at clinically effective doses than typical antipsychotic drugs. In December 1998, Lundbeck Ltd., the manufacturers of sertindole, voluntarily suspended the availability of the drug due to concerns about cardiac arrhythmia and sudden cardiac death associated with its use. However, based on the advice of an appointed expert group, the Committee for Proprietary Medicinal Products (CPMP) lifted the suspension of sertindole in October 2001, a decision that was ratified by the European Commission on the 26th of June 2002. Lundbeck have committed to the CPMP to carry out two post-marketing surveillance (PMS) studies (which were initiated in July 2002) to provide additional epidemiological data under conditions of normal drug usage. Initial marketing of the product will be restricted and Lundbeck is currently in discussions with the US health authorities (FDA) to investigate whether, and if so when, it would be possible to launch Serdolect in the US market. OBJECTIVES: To determine the effects of sertindole compared with placebo, typical and other atypical antipsychotic drugs for schizophrenia and related psychoses. SEARCH STRATEGY: Our Initial searches included electronic searches of Biological Abstracts (1980-1999), The Cochrane Library (Issue 1, 1999), The Cochrane Schizophrenia Group's Register (August 2000), EMBASE (1980-1999), LILACS (1982-1996), MEDLINE (1966-1999), PSYNDEX (1977-1995) and PsycLIT (1974-1999). In addition, we searched pharmaceutical databases on the Dialog Corporation Datastar and Dialog services. We searched references of all identified studies for further trials. We contacted the manufacturer of sertindole and authors of trials. We updated the literature search by searching the Cochrane Schizophrenia Group's Trials Register in April 2003. SELECTION CRITERIA: All randomised controlled trials that compared sertindole to placebo or other antipsychotic (atypical or typical) drug treatments for patients with schizophrenia or related psychosis . DATA COLLECTION AND ANALYSIS: We independently inspected citations and, where possible abstracts; ordered papers for re-inspection and quality assessment and independently extracted data. For homogeneous dichotomous data, we calculated the risk ratio (RR), 95% confidence interval (CI) and, where appropriate, the number needed to treat (NNT) or number needed to harm (NNH) on an intention-to-treat basis. For continuous data, we calculated weighted mean differences (WMD). We inspected all data for heterogeneity. MAIN RESULTS: Currently the review includes three studies with a total of 1,104 participants. One was a medium term (eight weeks) placebo controlled study that examined three different doses of sertindole (8, 12 and 20mg/day). The remaining two studies compared the use of sertindole with haloperidol (10mg/day). One was a short term study (six weeks) that looked at four different doses of sertindole (8, 16, 20, 24mg/day) and the other was a long term study (one year) that evaluated the use of sertindole 24mg/day in participants attending outpatients. We excluded two large important studies because they did not report any usable data. (Both had greater than 50% loss to follow-up and data on 'leaving the study early' was inadequately reported). SERTINDOLE VERSUS PLACEBO: Sertindole at 20mg/day was found to be more effective than placebo in terms of BPRS total scores (1 study, n=78, MD 6.2, CI -11.8 to -0.6) and CGI total end point scores (1 study, n=78, MD -0.9, CI -1.6 to -0.2). A marginally statistically significantly greater number of participants that were treated with 20 mg of sertindole were reported to have been 'very much improved' as compared to those taking placebo (1 study, n=102, RR 7.6, CI 1.0 to 57.9, NNT 7.9, CI 4.3 to 41.1). There was no statistically significant difference between sertindole at 8 or 12 mg/day and placebo for these three outcome measures. There were no statistically significant differences between sertindole (8, 12 or 20 mg) and placebo for the incidence of extrapyramidal symptoms, extrapyramidal related events or use of medication to avoid extrapyramidal symptoms. There were no statistically significant differences found between sertindole and placebo for the movement disorders akathisia, cogwheel rigidity, hypertonia and tremor or somnolence. At eight weeks a statistically significant difference between placebo and all sertindole groups (8, 12 and 20 mg) for mean change from baseline in the QT and QTc intervals were observed (p values and SD were not reported). There was a statistically significant greater mean weight gain among participants taking sertindole (20 mg, mean weight gain of 3.3 kg) as compared to placebo (mean weight gain of 0.8 kg; p<0.05). SERTINDOLE VERSUS HALOPERIDOL: At one year, a greater number of participants who were treated with haloperidol as compared to sertindole (24mg/day) were leaving the study early due to any reason (1 study, n=282, RR 0.6, CI 0.4 to 1.0, NNH 8.8, CI 4.7 to 74.0) or non-compliance (1 study, n=282, RR 0.2, CI 0.0 to 0.7, NNH 12.8, CI 7.7 to 37.8). However, at six weeks, there was no statistically significant difference between sertindole (at 8, 16, 20, or 24mg) and haloperidol for this latter outcome. The incidence of EPS was higher among those treated with haloperidol than sertindole at 8, 16, 20 or 24mg/day (8mg: 1 study, n=245, RR 0.1, CI 0.0 to 0.7, NNH 11.4, CI 7.1 to 29.8; 16mg: 1 study, n=252, RR 0.3, CI 0.1 to 1.0, NNH 15.5, CI 8.0 to 217.9; and 20mg: 1 study, n=253, RR 0.2, CI 0.1 to 0.8, NNH 13.7, CI 7.7 to 68.3; 24mg: 2 studies, n=524, RR 0.6, CI 0.4 to 0.8, NNH 8.7, CI 5.4 to 23.0). More participants treated with haloperidol experienced akathisia, tremor and hypertonia than those treated with sertindole (Akathisia - 8mg: 1 study, n=245, RR 0.2, CI 0.1 to 0.5, NNH 6.0, CI 4.1 to 11.2; 16mg: 1 study, n=252, RR 0.1, CI 0.0 to 0.3, NNH 5.4, CI 3.9 9.0; 20mg: 1 study, n=253, RR 0.3, CI 0.2 to 0.7, NNH 7.3, CI 4.6 to 17.9; 24mg: 2 studies, n=524, RR 0.5, CI 0.3 to 0.7, NNH 8.6, CI 5.6 to 18.3. Tremor - 8mg: 1 study, n=245, RR 0.3, CI 0.1 to 0.7, NNH 8.5, CI 5.2 to 24.0; 16mg: 1 study, n=252, RR 0.2, CI 0.1 to 0.5, NNH 7.3, 4.8 to 15.6; 20mg: 1 study, n=253, RR 0.2, CI 0.1 to 0.6, NNH 7.8, CI 4.9 to 18.1; 24mg: 2 studies, n=524, RR 0.4, CI 0.2 to 0.6, NNH 8.2, CI 5.6 to 15.3. For Hypertonic - 24mg: 2 studies, n=524, RR 0.5, CI 0.3 to 0.8, NNH 12.4, CI 7.5 to 35.0; for sertindole 8, 16 and 20mg there was no statistically significant differences between the treatment groups). One study reported that at six weeks, there was a statistically significant greater increase from baseline to final value in mean QTc interval in the sertindole 16, 20 and 24mg groups (20, 26, and 24msec, respectively) than in the haloperidol group (0msec; p value was not reported), but no SD or any other measure of variance for the effect sizes were reported. For one long term study only one participant from the sertindole group (24mg) had a QT interval that exceeded 500msec (1 study, n=282, RR 3.0 CI 0.1 to 73.0), but 11participants treated with Sertindole had QTc intervals of at least 500msec, compared to none in the haloperidol treated group (1 study, n=282, RR 23.0, CI 1.4 to 386.6, NNH 12.8, CI 8.2 to 29.6). At six weeks, fewer participants treated with sertindole at 8mg or 24mg were affected by somnolence than those treated with haloperidol (sertindole 8mg: 1 study, n=245, RR 0.1, CI 0.0 to 0.7, NNH 11.4, CI 7.1 to 29.8; 24mg: 2 studies, n=524, RR 0.6, CI 0.4 to 1.0, NNH 14.8, CI 7.7 to 205.2). The incidence of rhinitis was found to be statistically significantly higher among those taking sertindole at 16 or 24mg as compared to haloperidol (16mg: 1 study, n=252, RR 10.8, CI 1.4 to 82.6, NNH 12.7, CI 7.7 to 36.7; 24mg: 2 studies, n= 524, RR 2.1, CI 1.4 to 3.1, NNH 8.7, CI 5.6 to 18.6). At one year, 33 participants treated with sertindole (24mg) had experienced the sexual adverse event of decreased ejaculatory volume, compared with six participants treated with haloperidol. However the number of included male participants was not reported and therefore the RR could not be calculated. At one year, more participants taking sertindole (24mg/day) had put on weight compared to those taking haloperidol (1 study, n=282, RR 6.3, CI 1.9 to 20.9, NNH 8.8, CI 5.7 to 19.1). At six weeks, all of the sertindole groups showed an increase in body weight from baseline to final evaluation ranging from 1.3kg to 1.9kg, all of which represented a statistically significantly different weight change than that recorded for the haloperidol treatment group (-0.1Kg). However, the actual weight gain for each sertindole dosage group was not reported and no SD or any other measure of variance was given. AUTHORS' CONCLUSIONS: Sertindole at a dose of 20mg/day was found to be more antipsychotic than placebo. When used at 8, 12 or 20mg/day it appears to be as acceptable as placebo (in terms of various adverse events including movement disorders and somnolence), but seems to be associated with more cardiac problems (8, 12 or 20mg/day) and an increase in weight gain (20mg/day) than placebo. Sertindole at a dose of 24mg/day was better tolerated than haloperidol (in terms of participants leaving the study early). It was also found to be was associated with fewer movement disorders (at 8, 16, 20 or 24mg/day) and sedation (8 or 24mg/day) than haloperidol. However, it was shown to cause more cardiac anomalies (16, 20 or 24mg/day), weight gain (all doses combined), rhinitis (16 or 24mg/day), and problems with sexual functioning (24mg/day) than haloperidol. One short term study reported that sertindole 16mg/day was the most optimal dose.

Antipsychotic Agents↗

PubMed2005

Psychosocial assessment of patients who attend an accident and emergency department with self-harm.

It has been recommended that all patients attending hospital accident and emergency departments in the United Kingdom (UK) with self-harm should be offered adequate psychosocial assessment to identify those at risk of completed suicide or repeated self-harm. This paper focuses on the extent to which this recommendation has been implemented in a large district general hospital in Wales, and compares the characteristics and hospital management of patients who received no specialist assessment with those who were assessed by a psychiatric liaison nurse or a psychiatrist. Data were routinely collected as part of the hospital annual audit procedure and confirm previous findings that guidelines for the general hospital management of self-harm patients have not been well implemented. Whilst we found some evidence that certain high risk patient groups were more likely to receive specialist assessment, this was far from universal. We conclude that if there is to be a reduction in the rate of suicide in Wales and the UK as a whole, it is crucial that services for people who self-harm are more appropriately targeted on patients who are at greatest risk.

Adolescent↗

PubMed2004

Short shrift for the sane? The hospital management of self-harm patients with and without mental illness.

In recent years the British government has sought to target service provision both on people with severe enduring mental illness and on those considered to be at risk of suicide. The study reported here suggests these policies may have had unforeseen repercussions on services for people who self-harm but have no identifiable mental illness. This paper compares the hospital management of self-harm patients with and without mental illness. Over the years 1996-2000, a total of 4,329 self-harm patient attendances were recorded at the accident and emergency department of a British hospital. Whilst patients in 63.7% of attendances could reasonably have been considered to have some form of mental illness, we found that in the remaining 36.3% of attendances (where patients had no indicator of mental illness) a particularly poor standard of service was given. We suggest that if government targets for a reduction in the suicide rate are to be met, it is crucial that there should be marked improvements in services for all self-harm patients presenting at accident and emergency departments. However, in improving services we must be vigilant to the danger that targeting those with a mental illness may mean we fail to provide even basic levels of care for others.

Community Mental Health Services↗

PubMed2003

Morphological changes of the endometrial epithelium in the bitch during metoestrus and anoestrus.

Although cyclic changes of the endometrium in dogs involving both stromal and glandular compartments have been described, the fate of the surface epithelium after progressive growth and secretion is still unclear. In the present study, uteri of 43 healthy bitches in metoestrus and anoestrus were examined macroscopically and histologically. Tissue biopsies were taken from three different locations (cranial and middle parts of uterine horns and bifurcation). The stage of the oestrous cycle was determined by evaluation of progesterone and oestradiol levels in plasma hormone and was also confirmed clinically. Crypts formed in the luteal phase were covered with a columnar epithelium which gradually underwent fatty degeneration. In addition, the stromal part of the crypts disappeared and finally, in early anoestrus, epithelial sheaths desquamated and shed off into the uterine lumen. The surface epithelium was replaced by new cuboidal cells proliferating and migrating from the glandular openings. These findings were confirmed by oil red O staining and immunohistochemical detection of proliferation with Ki-67 marker.

Anestrus↗

PubMed2003

Lectin binding patterns in normal canine endometrium and in bitches with pyometra and cystic endometrial hyperplasia.

Cystic endometrial hyperplasia (CEH) and pyometra in the bitch are dioestral syndromes, supposed to be caused by hormonal disturbances and changes in endometrial steroid hormone receptor levels. Histologically, the endometria show cystic dilated glands and, if bacteria succeed in invading the uterus, pyometra may develop in the following metoestrus. In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of uterine glands and surface epithelium in healthy dogs and in dogs with CEH and pyometra. Lectin binding is a useful tool to identify glycoconjugates, especially of the glycocalyx, which has essential functions in the endometrium during reproduction. Uterine tissue was obtained from 18 healthy bitches in metoestrus or anoestrus and 18 bitches with a clinical diagnosis of CEH or pyometra. Normal endometria showed cycle-dependent changes in SBA, PNA, HPA and UEA binding during metoestrus and anoestrus. LCA did not show cycle-dependent changes and WGA bound to Golgi regions in the apical parts of surface epithelial cells only in metoestrous. Endometria with inflammatory alterations lost cycle-specific lectin binding patterns and, with increasing severity of pathological changes, showed a marked decrease in binding intensity to the glandular and surface epithelial glycocalyx and secretions. In dogs with CEH, unaltered glands with generally strong lectin binding to the glycocoalyx and Golgi regions were found adjacent to altered glands. The decrease of lectin binding in pyometra cases is supposed to be a result of glandular exhaustion after cystic hyperplasia. In addition, bacterial adhesion to sugar residues on the uterine surface epithelium might impede lectin binding.

Animals↗

PubMed2000

Sertindole for schizophrenia.

Antipsychotic Agents↗

PubMed2000

Loxapine for schizophrenia.

BACKGROUND: Loxapine is a drug with D2/D3 receptor antagonist activity and a higher affinity for D3 than D2. Some authors have suggested that loxapine is more effective than typical antipsychotics in reducing the negative symptoms of schizophrenia, that extrapyramidal side-effects are not usually seen at clinically effective antipsychotic doses and that it should therefore be classed as atypical. OBJECTIVES: To determine the effects of loxapine compared with placebo, typical and other atypical antipsychotic drugs for schizophrenia and related psychoses. SEARCH STRATEGY: Electronic searches of Biological Abstracts (1980-1999), The Cochrane Library (Issue 1, 1999), The Cochrane Schizophrenia Group's Register (January 1999), EMBASE (1980-1999), MEDLINE (1966-1999), LILACS (1982-1996), PSYNDEX (1977-1995) and PsycLIT (1974-1999) were undertaken. In addition, pharmaceutical databases on the Dialog Corporation Datastar and Dialog services were searched. References of all identified studies were searched for further trials. Pharmaceutical companies and authors of trials were contacted. SELECTION CRITERIA: All randomised controlled clinical trials relevant to the care of schizophrenia that compared loxapine to other treatments. DATA COLLECTION AND ANALYSIS: Citations and, where possible, abstracts were independently inspected by reviewers, papers ordered, re-inspected and quality assessed. Data were independently extracted but excluded if loss to follow up was greater than 50%. For homogeneous dichotomous data the risk ratio (RR), 95% confidence interval (CI) and, where appropriate, the number needed to treat (NNT) were calculated on an intention-to-treat basis. For continuous data, weighted mean differences were calculated (WMD). All data were inspected for heterogeneity. MAIN RESULTS: Compared to placebo, loxapine is antipsychotic (Global effect - not improved at 6 weeks, n=66, RR 0.6 CI 0.4-0.9 NNT 4 CI 2-62) with similar adverse effect profile to typical drugs. Is as effective as typical drugs in the short term (4-12 weeks) (Global effect - not improved, n=411, RR 0.89 CI 0.7-1.2). Very limited heterogeneous data suggest that, given intramuscularly (IM), loxapine may be at least as sedating as IM haloperidol and thiothixene. REVIEWER'S CONCLUSIONS: Loxapine is antipsychotic but its effects are under researched. It is not clearly different from typical drugs in either its positive or adverse effects.

Antipsychotic Agents↗

PubMed2000

Zotepine for schizophrenia.

BACKGROUND: Typical antipsychotic drugs are widely used as first line treatment for people with schizophrenia. The atypical class of antipsychotic drugs, however, is making important inroads into this approach and zotepine is one such compound. It is a dopamine antagonist and claimed to be to be particularly effective for negative symptoms OBJECTIVES: To determine the effects of zotepine compared with placebo, typical and other atypical antipsychotic drugs for schizophrenia and related psychoses. SEARCH STRATEGY: Electronic searches of Biological Abstracts (1980-1999), CINAHL (1982-1999), The Cochrane Library (Issue 1, 1999), The Cochrane Schizophrenia Group's Register (January 1999), EMBASE (1980-1999), Dialog Corporation Datastar service (1999), MEDLINE (1966-1999), and PsycLIT (1974-1999) were undertaken. References of all identified studies were searched for further trials. Knoll Pharmaceuticals and authors of trials were contacted. SELECTION CRITERIA: All randomised clinical trials that compared zotepine to other treatments for people with schizophrenia or other psychoses were included. DATA COLLECTION AND ANALYSIS: Citations and, where possible, abstracts were independently inspected by reviewers, papers ordered, re-inspected and quality assessed. Data were independently extracted. Data were excluded if loss to follow up was greater than 50%. For homogeneous dichotomous data the relative risk (RR), 95% confidence interval (CI) and, where appropriate, the number needed to treat (NNT) and numbers needed to harm (NNH), were calculated on an intention-to-treat basis. For continuous data, weighted mean differences were calculated (WMD). All data were inspected for heterogeneity. MAIN RESULTS: All outcomes were short term (4-12 weeks). Limited data suggest that zotepine is an antipsychotic, at least as effective as typical drugs. Mental state measures of 'no clinically important improvement' favour zotepine when compared to other active drugs (n=356, RR 0.8 CI 0.7-0.9, NNT 7 CI 4-22). About one third of people in both zotepine and control groups left the studies before trial completion. Zotepine may result in less movement disorder adverse effects than typical antipsychotic drugs. Trials have not highlighted clear differences between zotepine and other atypical drugs. REVIEWER'S CONCLUSIONS: Zotepine may be a valuable addition to the increasing ranks of atypical antipsychotic drugs. More data from already existing studies is urgently needed to increase the confidence in the findings of this review. New data from well planned, conducted and reported long term pragmatic randomised trials are necessary. Otherwise clinical use of zotepine will be based on speculation on the meaning of the findings of short explanatory trials for everyday practice.

Antipsychotic Agents↗

PubMed2000

Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax.

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.

Animals↗

PubMed1999

Cardiac rehabilitation.

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Cardiac Rehabilitation↗

PubMed1997

Too Rankian for the Freudians, or too Freudian for the Rankians: Otto Rank's contributions to psychoanalysis in the 1920s.

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Freudian Theory↗

PubMed1996

Structural modifications of the omega loop in human acetylcholinesterase.

Conformational mobility of the surface omega loop (Cys-69-Cys-96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen-bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the omega loop in the HuAChE is not involved in large lid-like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp-86 supporting the proposed coupling between the structure of the omega loop and the positioning of the Trp-86 indole moiety, in catalytic activity and in allosterism.

Acetylcholinesterase↗

PubMed1996

Alphavirus hybrid virion vaccines.

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Alphavirus↗

PubMed1993

N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis.

The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related 'peripheral-site' functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.

Acetylcholinesterase↗

PubMed1993

Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket.

Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.

Acetylcholinesterase↗

PubMed1992

Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit.

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.

Acetylcholinesterase↗

PubMed1992

Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding.

Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.

Acetylcholinesterase↗