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Biomedical subjects

M Leitner

Publications and source records attributed to M Leitner.

At least 37 records · Page 2Linked to original sources
PubMed1992

Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.

Acetylcholinesterase↗
PubMed1992

Case report: fatal seronegative rickettsial infection diagnosed by the polymerase chain reaction.

A previously healthy man presented with a five day history of high fever and headache, later followed by rash and the appearance of jaundice. On the second hospital day, he suddenly developed seizures, lapsed into a coma, and died. Polymerase chain reaction (PCR) amplification revealed a 434 base pairs DNA fragment common to the genome of typhus and spotted fever group rickettsiae in the patient's blood (estimated at about 1 x 10(2) organisms/ml), and to a lesser degree in the cerebrospinal fluid. However, serological tests for rickettsiae remained negative. PCR techniques may confirm the diagnosis at an early stage, even though the rickettsemia may be minimal and the patient seronegative.

DNA, Bacterial↗
PubMed1992

Divergent envelope E2 alphavirus sequences spanning amino acids 297 to 352 induce in mice virus-specific protective immunity and antibodies with complement-mediated cytolytic activity.

We have proposed a general algorithm for identification of potential immunoprotective domains (cassettes) on the envelope E2 polypeptide of alphaviruses (H. Grosfeld, B. Velan, M. Leitner, S. Cohen, S. Lustig, B.E. Lachmi, and A. Shafferman, J. Virol. 63:3416-3422, 1989). To assess the generality of our approach, we compared analogous E2 cassettes from Sindbis virus (SIN) and Semliki Forest virus (SFV), two alphaviruses which are philogenetically very remote. The antigenically distinct SFV E2 and SIN E2 cassettes exhibit comparable immunological characteristics. Most significantly, the SIN E2 LMN cassette cluster (E2 amino acids 297 to 352 fused to beta-galactosidase), like the analogous SFV E2 LMN cassettes, elicited high titers of antivirus antibodies in mice and proved to be highly effective in protection against lethal challenge. Mice immunized with SIN E2 LMN were completely protected against intracerebral challenge of 10 to 100 50% lethal doses of different neurovirulent SIN strains. Anti-SIN LMN antibodies, like anti-SFV LMN antibodies, lacked in vitro neutralizing activity, yet both exerted protection against homologous challenge upon transfer to mice. The two antibody preparations exhibited virus-specific complement-mediated cytolysis of cells infected with the homologous but not heterologous virus. These results suggest a possible mechanism for virus-specific E2 LMN-induced protection and demonstrate the generality of our methodology for deciphering immunogenic and protective domains in alphavirus systems. Results suggest also that the E2 LMN sequence of any given alphavirus should be considered as a component of a synthetic vaccine against that specific virus.

Algorithms↗
PubMed1992

Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture.

The pharmacological properties of glutamate agonists were compared in astrocyte-rich and astrocyte-poor cultures derived from embryonic rat cerebral cortex. The object of this investigation was to determine the extent to which glutamate uptake might influence the receptor-mediated neurotoxic actions of these compounds. In astrocyte-rich cultures, using 30 min exposures, we observed that the potencies of the poorly transported agonists NMDA (35 microM) and D-glutamate (89 microM) were higher than that of L-glutamate (205 microM). In astrocyte-poor cultures, L-glutamate was much more potent, with an EC50 of 5 +/- 4 microM (3-12 microM), for a 30 min exposure, whereas the potencies of NMDA and D-glutamate were essentially unchanged. L- and D-aspartate were also more effective in astrocyte-poor cultures, again with EC50 values of approximately 6-10 microM, as compared with 130 and 108 microM, respectively, in astrocyte-rich cultures. In other experiments, blocking sodium-dependent glutamate uptake in astrocyte-rich cultures, by using a sodium-free medium, made glutamate as potent an agonist as in astrocyte-poor cultures. Finally, we directly assessed the glutamate uptake system in astrocyte-rich and astrocyte-poor cultures and found that uptake was reduced approximately 25-fold in the astrocyte-poor cultures. These results show that in the presence of abundant astrocytes the neurotoxic potencies of L-glutamate, L-aspartate, and D-aspartate are substantially under-estimated.

Animals↗
PubMed1991

The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant.

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.

Acetylcholinesterase↗
PubMed1991

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme.

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

Acetylcholinesterase↗
PubMed1991

Delineation of protective epitopes on the E2-envelope glycoprotein of Semliki Forest virus.

Two short linear peptides, 17 and 14 amino acids long, on the Semliki Forest virus (SFV) E2-envelope polypeptide are shown to be involved in the protection of mice against lethal challenge with SFV. Peptides corresponding to these two regions, designated H and L, were selected for study on the basis of our model for prediction of protective epitopes on E2 polypeptide of alphaviruses. These peptides were produced in Escherichia coli as recombinant proteins fused to the amino terminus of beta-galactosidase. Both the H epitope (amino acid positions 227-243 on E2) and L epitope (amino acid positions 297-310) are recognized by antibodies raised against SFV, and both trigger antibodies that interact with native SFV-E2. Vaccination of mice with the H-beta-galactosidase polypeptide confers 64-87% protection against a lethal viral challenge (250 LD50), and immunization with L-beta-galactosidase leads to 23-66% protection of challenged mice. The efficacy of the L-based synthetic vaccine could be improved further (up to 100% protection) by presentation of this epitope as a dimer fused to beta-galactosidase. These results provide evidence that the algorithm and the methodology proposed by us previously are effective tools for identification of linear protective epitopes on E2-envelope of SFV.

Amino Acid Sequence↗
PubMed1989

Expression of Shiga toxin epitopes in E. coli immunological characterization.

Synthetic oligodeoxynucleotides encoding for two peptides corresponding to residues 9-21 and 19-31 in the amino acid sequence of the B subunit of Shiga toxin were prepared and inserted into pTOZ plasmid, in phase with the lacZ gene. The resultant vectors were used for transfection of E. coli. The bacteria containing the recombinant DNA expressed the respective peptides in the form of fusion proteins with beta-galactosidase. The N-terminal region of the Shiga toxin B subunit, containing the two peptides, was previously identified as a relevant epitope of the toxin, leading to the induction of neutralizing antibodies. The present study demonstrates that the bacterial extracts containing the fusion proteins as the products of the recombinant vectors, when used for immunization of rabbits, elicited a humoral immune response which was specific toward the respective peptides. Furthermore, the antibodies cross-reacted with the intact Shiga toxin.

Amino Acid Sequence↗
PubMed1989

Production of monoclonal antibodies toward bovine interferons-alpha suitable for immunopurification.

Five murine hybridoma clones, producing monoclonal antibodies (MAbs) to bovine interferon-alpha (BoIFN-alpha) were established. One of these, F12, secreted MAbs giving high titers, in ELISA tests, neutralizing both BoIFN-alpha, -alpha C, and BoIFN-alpha D activities, belonging to the mouse IgG1 class, and having a binding affinity constant of 10(8) M-1. F12 MAbs were used for immunoaffinity purification of BoIFN-alpha, and recombinant BoIFN-alpha C from Escherichia coli extracts was purified to homogeneity in SDS-PAGE analysis and to a specific activity of 2 X 10(8) U/mg with 90% recovery of activity.

Animals↗
PubMed1989

Semliki Forest virus E2 envelope epitopes induce a nonneutralizing humoral response which protects mice against lethal challenge.

Along the 422 amino acids of the Semliki Forest virus (SFV) E2 envelope glycoprotein, we identified 13 peptide cassettes (ranging in size from 15 to 25 amino acids and designated A through N) that contain hydrophilic sequences flanked by amino acid sequences conserved in the E2 envelopes of the alphavirus family. Six peptide blocks containing either a single cassette or two to three contiguous cassettes (A, BC, DE, FG, HIK, and LMN) were produced in Escherichia coli as recombinant proteins fused to the N terminus of beta-galactosidase. All of the SFV E2 recombinant polypeptides except A-beta-galactosidase were recognized on Western blots (immunoblots) by anti-SFV polyclonal antisera. In addition, these five recombinant proteins induced in mice antibodies that interacted specifically with SFV E2 protein on Western blots as well as with the intact virions in an enzyme-linked immunosorbent assay. The six hybrid proteins were used to vaccinate mice and were tested for the ability to confer resistance against lethal doses of SFV. Peptides BC and HIK, located at amino acid positions 114 to 149 and 216 to 288, respectively, of E2, protected partially (40 to 60%) against SFV challenge. A third peptide, LMN, located between amino acid positions 289 and 352, rendered mice totally resistant to an SFV challenge of 250 50% lethal doses. The partially protective effects of the BC and HIK cassettes and the high efficacy of the LMN cassette were consistently demonstrated, independent of the adjuvant (complete Freund or alum), immunization protocol, and strain of mice used. None of the antisera raised against any given cassette could neutralize the virus in an in vitro tissue culture assay or in a plaque reduction neutralization test. Nevertheless, passive transfer experiments demonstrated that in the case of LMN, the protective effect was mainly of a humoral nature.

Amino Acid Sequence↗
PubMed1988

Typology of mood assessment curves by means of cluster analysis of computerized mathematical descriptions of time-series mood data collected from depressive inpatients treated with antidepressants.

A recently developed method for the computerized description of mood curves was applied to self-evaluation mood data of a large sample of 136 depressive inpatients. The values of the mathematical parameters from this description were analysed by cluster analysis to determine different types of mood courses of patients undergoing therapy with antidepressants. In general, the most unfavorable type of mood course was overrepresented in the group of neurotic depressive patients, the most favorable one in the group of endogenous depressive patients. But in this latter diagnostic group, a remarkable proportion of patients with the unfavorable mood course was observed, indicating the well-known phenomenon of non-response to antidepressants. The results can be interpreted as a validation of the new method of computerized description of mood curves. This method might lead to interesting possibilities in drug treatment evaluation.

Antidepressive Agents↗
PubMed1987

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects.

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

A linear mathematical model for computerized analyses of mood curves. An empirical investigation on mood courses in depressive and schizophrenic inpatients.

The repetitious measurement of actual mood alterations becomes more and more common in research oriented psychiatric departments. In this article a new method for the mathematical description and statistical evaluation of time series data is presented. This computerized approach is less restrictive than other procedures and seems to be very appropriate to the complex structure of time series. First results on mood courses of depressive and schizophrenic patients are presented to demonstrate the usefulness and validity of this new method.

Computer Simulation↗
PubMed1987

Optimizing a nonlinear mathematical approach for the computerized analysis of mood curves.

A nonlinear mathematical model for computerized description of mood curves is presented. This model reaches a high goodness of fit to the real data. It seems superior to two other models recently proposed. Using this model in a computer program for describing the mood data of a large sample of inpatients, significant and clinically meaningful group differences between the mood curves of schizophrenic, endogenous-depressive, and neurotic-depressive inpatients could be demonstrated. The application of the methodology might be helpful, e.g. in the field of evaluative research.

Affect↗
PubMed1985

Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.

A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of CTP3 and beta-galactosidase, followed by a booster injection of a sub-immunizing amount (1 microgram) of cholera toxin, led to a substantial level of neutralizing antibodies against both cholera toxin and the heat-labile toxin of Escherichia coli.

Animals↗
PubMed1985

Bovine interferon alpha genes. Structure and expression.

The bovine genome contains a gene family of interferon-alpha s (bIFN-alpha) that consists of at least five distinct members. Four of the bIFN-alpha genes isolated show a high degree of homology (97% in the nucleotide sequence and 93% in amino acid sequence). The overall homology in amino acid sequence of bIFN-alpha to human, murine, and rat IFNs-alpha is approximately 60%. Yet there are amino acid clusters (positions 28-41 and 118-146) which are highly conserved throughout the mammalian evolution and in which the overall homology can be as high as 86%. Within the C terminus conserved cluster there is a sequence containing 9 amino acids completely conserved in 16 mammalian IFNs-alpha and of these, 7 are also shared with a similar domain in some bacterial toxins, implying a common functional role for these domains. One of the genes, IFN-alpha C, was expressed in Escherichia coli. The purified bacterial IFN (specific activity, 2 X 10(8) units/mg) exhibited antiviral activity on bovine cells but no detectable activity was demonstrated on human and simian cells.

Amino Acid Sequence↗
PubMed1985

Cloning, expression and biological activity of a new variant of human interferon alpha identified in virus induced lymphoblastoid cells.

A synthetic oligonucleotide complementary to a highly conserved sequence in the IFN-alpha gene family, was used to screen a Namalva cDNA library. Among the cDNA clones having typical IFN-alpha traits, one was distinct from previously characterized IFN-alpha cDNAs. E. coli cells carrying this recombinant cDNA plasmid express an alpha-interferon activity. The sequence of this IFN-cDNA is extremely homologous (99.5%) to that of the IFN-alpha J gene and is designated IFN-alpha J1. Several E. coli trp expression plasmids were constructed for efficient transcription and translation of the mature IFN-alpha J1. The maximal level of expression (5 X 10(3) molecules/cells) was obtained from plasmid pJ1-4. A synthetic consensus translation initiation sequence coupled to the trp p/o region (in pJ1-5) proved to be 10 times less effective in promoting metIFN production in bacteria, than the in-vitro mutated trpL initiation sequence carried on pJ1-4. The bacterial IFN-alpha J1 was purified (to over 90% purity) to a specific activity of 1.3 X 10(8) units/mg. The antiviral activity of the purified IFN-alpha J1 was compared with other highly purified IFN-alpha species (bacterial IFN alpha A and alpha C, leukocyte IFN-alpha 1, leukocyte IFN mixture and Namalva IFN preparation) on a large panel of mammalian cell cultures. IFN-alpha J1 exhibits a distinct antiviral activity.

Amino Acid Sequence↗