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Biomedical subjects

P Kovác

Publications and source records attributed to P Kovác.

At least 19 recordsLinked to original sources
PubMed2006

[Expert witness and expert testimony in New Slovak Criminal Procedure Code].

As of 1st January 2006, new criminal law consisting of the Act No. 300/2004 Coll. (Penal Code) and the Act No. 301/2005 Coll. (Criminal Procedure Code) became effective in the Slovak Republic. These two acts replaced Criminal Law originating from 1961. The author analyses impact and changes introduced with new Criminal Procedure Code in the regulation of expert witness in general and expert witness testimony.

Criminal Law↗
PubMed2005

[New legislation on expert witnesses in the Slovak Republic].

Legal framework for expert witnesses in the continental legal system differs from that of common law in many aspects. The position of an expert witness in continental legal system is more formalized and the processual rights and duties of an expert witness differs from rights and duties of witness in general. As of 1st September 2004 the Act No. 417/2002 Coll. on Expert Witnesses, Translators and Interpreters became effective in the Slovak Republic. The Act repealed expert witnesses legislation that originated from 1967 which is still effective in the Czech Republic. The authors present a short overview of the Act.

Expert Testimony↗
PubMed2003

[Legal regulations of forensic DNA analysis in the Slovak Republic].

As of 1st January 2003 the Law No. 417/2002 Coll. on the Usage of Deoxyribonucleotic Acid Analysis for the Identification of Persons became effective in the Slovak Republic. The Law sets the conditions to take samples due to carry out the forensic DNA analysis, laid down regulations for laboratories approved to perform DNA analysis and at the same time establishes national database of DNA profiles. The author presents short overview of the Law.

DNA Fingerprinting↗
PubMed2001

Synthetic explorations towards 3-deoxy-3-fluoro derivatives of D-perosamine.

Based on a literature precedent, preparation of methyl 4-azido-3,4,6-trideoxy-3-fluoro-alpha-D-mannopyranoside (18) was attempted via fluorination of methyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-altropyranoside with diethylaminosulfur trifluoride (DAST). Contrary to expectations, the reaction took place with retention of configuration at the site of the fluorination yielding methyl 4-azido-2-O-benzyl-3,4,6-trideoxy-3-fluoro-alpha-D-altropyranoside. Treatment with DAST of methyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-allopyranoside (8), or its 2-(p-methoxybenzyl) analog 9 resulted in fluorination with inversion of configuration at position 3, to give the corresponding 3-deoxy-3-fluoro glucopyranosides 10 and 11, respectively. Accordingly, compound 18 was prepared from 11, by de-p-methoxybenzylation at O-2, followed by inversion of configuration at C-2 in the resulting methyl 4-azido-3,4,6-trideoxy-3-fluoro-alpha-D-glucopyranoside. The 2-O-methyl analog of 18 (19) was prepared by methylation of 18. Compounds 18 and 19 were converted, conventionally, into the 3-fluoro analogs of the terminal determinants of the O-PS of Vibrio cholerae O:1, serotype Inaba and Ogawa, respectively.

Diethylamines↗
PubMed2001

Conjugating oligosaccharides to proteins by squaric acid diester chemistry: rapid monitoring of the progress of conjugation, and recovery of the unused ligand.

Samples that are periodically withdrawn from the mixture of a conjugation reaction can be analyzed on a picomolar scale without any work-up or pre-purification using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in combination with the ProteinChip System. The technique provides rapid information about the increasing molecular mass of the glycoconjugate formed, thereby allowing termination of the process when the desired incorporation of the ligand onto the carrier protein is achieved. The excess oligosaccharide used at the onset of conjugation can be recovered and used in preparation of a similar neoglycoconjugate. The overall economy of conjugations, which often involve labor-intensive linker-equipped oligosaccharides, can be markedly increased in this way.

Animals↗
PubMed2001

Syntheses of the L-manno and some other analogs of the terminal determinants of the O-PS of Vibrio cholerae O:1.

Analogs of the methyl alpha-glycosides of the terminal residues of the O-specific polysaccharides (O-PS) of Vibrio cholerae O:1, serotype Inaba and Ogawa, have been prepared as probes to study their interaction with anti V. cholerae O:1 antibodies. They differ from the termini of the respective O-PSs in anomeric or absolute configuration of perosamine, position of the O-methyl group in D-perosamine, and nature of the N-acyl side chain.

Antigens, Bacterial↗
PubMed2000

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: molecular basis for serotype specificity.

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab-Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Animals↗
PubMed1999

Studies on vaccines against cholera. Synthesis of neoglycoconjugates from the hexasaccharide determinant of Vibrio cholerae O:1, serotype Ogawa, by single-point attachment or by attachment of the hapten in the form of clusters.

The terminal hexasaccharide of the O-antigen of Vibrio cholerae O:1, serotype Ogawa, has been synthesized in the form of a glycoside whose aglycon (linker) allows conjugation to carrier proteins by reductive amination. The conjugate obtained from direct, single-point attachment of the linker-equipped hapten to chicken serum albumin (CSA) contained seven hapten residues/CSA. A neoglycoconjugate containing the carbohydrate antigen in the form of clusters was obtained using, as a hapten subcarrier, an oligopeptide containing 16 amino groups. It was treated with a limited amount of hapten, to give a hapten-carrying subcarrier (HCS). Subsequent conjugation of HCS to CSA, using squaric acid diethyl ester as a conjugation reagent, gave a cross-linked, glycocluster conjugate containing 51% (w/w) of the carbohydrate.

Carbohydrate Sequence↗
PubMed1999

UDP-6-deoxy-6-fluoro-alpha-D-galactose binds to two different galactosyltransferases, but neither can effectively catalyze transfer of the modified galactose to the appropriate acceptor.

The effect of substitution of the HO-6 of D-galactose with fluorine on the ability of alpha-(1-->3)-galactosyltransferase (EC 2.4.1.151) and beta-(1-->4)-galactosyltransferase (EC 2.4.1.22) to catalyze its transfer from UDP to an appropriate acceptor was determined. HPLC analyses indicated that each transferase properly catalyzed formation of the expected product [beta-D-Gal-(1-->4)-D-GlcNAc] for the beta-(1-->4)-galactosyltransferase and alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-GlcNAc for the alpha-(1-->3)-D-galactosyltransferase] when UDP-alpha-D-Gal was the substrate. When UDP-6-deoxy-6-fluoro-alpha-D-galactose (6) was used in conjunction with each transferase, no product indicative of transfer of 6-deoxy-6-fluoro-D-galactose to its respective acceptor sugar was identified. 6-Deoxy-6-fluoro-D-galactose (3) was obtained by hydrolysis of methyl 6-deoxy-6-fluoro-alpha-D-galactopyranoside, synthesized by the selective fluorination of methyl alpha-D-galactopyranoside with diethylaminosulfur trifluoride (DAST), with aqueous trifluoroacetic acid. Acetylation of 3 gave crystalline 1,2,3,4-tetra-O-acetyl-6-deoxy-6-fluoro-beta-D-galactopyranose, which was converted to the corresponding 1-alpha-phosphate and used for the synthesis of 6.

Carbohydrate Sequence↗
PubMed1999

A highly efficient preparation of neoglycoconjugate vaccines using subcarriers that bear clustered carbohydrate antigens.

A limited amount of spacer-equipped carbohydrate haptens was linked by reductive amination to a subcarrier, an oligopeptide containing 16 amino groups, to give a hapten-carrying subcarrier (HCS). It was then linked, via the remaining free amino groups, to chicken serum albumin (CSA) to give a cross-linked neoglycoconjugate bearing the haptens in the form of clusters. Alternatively, the same type of a glycoconjugate, but with higher conjugation efficiency, was obtained when HCS was treated successively with squaric acid diethyl ester and CSA.

Antigens↗
PubMed1999

A simple, nonenzymatic method for desialylating polysialylated ganglio-N-tetraose series gangliosides to produce GM1.

Dowex-50W-H+ was used to catalyze the highly selective desialylation of polysialylated ganglio-N-tetraose series gangliosides to yield primarily GM1. High performance thin-layer chromatographic analysis of recovered lipid indicated that 60-70% of the recovered ganglioside was GM1. Identification of the major product as GM1 was confirmed by proton NMR spectra and lack of sialic acid release by Vibrio cholerae sialidase.

Animals↗
PubMed1998

Conformational differences among mono- and oligosaccharide fragments of O-specific polysaccharides of Vibrio cholerae O1 revealed by circular dichroism.

The circular dichroism (CD) of synthetic mono- and oligosaccharides that represent the terminal, non-reducing group of O-antigens of Vibrio cholerae O1 from the subtypes Ogawa and Inaba was measured in various solvents. We found differences in the CD of the monosaccharides of these subtypes that decrease with increasing chain lengths of the oligosaccharides. The differences can be explained by different orientations of the N-acyl side chain of the terminal monosaccharides. The linear relationship of ellipticity versus the number of residues in an oligosaccharide chain follows the principle of optical superposition. This, together with a similar contribution by internal units to the overall ellipticity, suggests an identical, regular conformation of oligosaccharide fragments of both Ogawa and Inaba series.

Carbohydrate Conformation↗
PubMed1998

On the antigenic determinants of the lipopolysaccharides of Vibrio cholerae O:1, serotypes Ogawa and Inaba.

Monoclonal, murine IgG1s S-20-4, A-20-6, and IgA 2D6, directed against Vibrio cholerae O:1 Ogawa-lipopolysaccharide exhibited the same fine specificities and similar affinities for the synthetic methyl alpha-glycosides of the (oligo)saccharide fragments mimicking the Ogawa O-polysaccharide (O-PS). They did not react with the corresponding synthetic fragments of Inaba O-PS. IgG1s S-20-4 and A-20-6 have absolute affinity constants for synthetic Ogawa mono- to hexasaccharides of from approximately 10(5) to approximately 10(6) M-1. For IgG1s S-20-4, A-20-6, and IgA 2D6, the nonreducing terminal residue of Ogawa O-PS is the dominant determinant, accounting for approximately 90% of the maximal binding energy shown by these antibodies. Binding studies of derivatives of the Ogawa monosaccharide and IgGs S-20-4 and A-20-6 revealed that the C-2 O-methyl group fits into a somewhat flexible antibody cavity and that hydrogen bonds involving the oxygen and, respectively, the OH at the 2- and 3-position of the sugar moiety as well as the 2'-position in the amide side chain are required. Monoclonal IgA ZAC-3 and IgG3 I-24-2 are specific for V. cholerae O:1 serotypes Ogawa/Inaba-LPS.1 The former did not show binding with members of either series of the synthetic ligands related to the O-antigens of the Ogawa or Inaba serotypes, in agreement with its reported specificity for the lipid/core region (1). Inhibition studies revealed that the binding of purified IgG3 I-24-2 to Ogawa-LPS might be mediated by a region in the junction of the OPS to the lipid-core region of the LPS. cDNA cloning and analysis of the anti-Ogawa antibodies S-20-4, A-20-6, and 2D6 revealed a very high degree of homology among the heavy chains. Among the light chains, no such homology between S-20-4 and A-20-6 on the one hand, and 2D6 on the other hand, exists. For the anti-Inaba/Ogawa antibodies I-24-2 and ZAC-3, their heavy chains are completely different, with some homology among the light chains.

Amino Acid Sequence↗
PubMed1998

Glycosylation via locked anomeric configuration: stereospecific synthesis of oligosaccharides containing the beta-D-mannopyranosyl and beta-L-rhamnopyranosyl linkage.

cis-1,2-Stannylene acetals of D-mannose and L-rhamnose, formed preferentially from the free sugars treated with dibutyltin oxide, are capable of displacing the trifluoromethanesulfonyl (triflyl) leaving groups in carbohydrates to give, with retention of configuration at the anomeric center in the nucleophile, cis-1,2-linked oligosaccharides. In the case of secondary triflates, the new glycosidic linkage is formed with complete inversion of configuration in the electrophile. Both the reactivity of the electrophile and nucleophilicity of oxygens in the stannylene complex affect the overall outcome of the reaction. From the comparison of results of a number of glycosylations via stannylene acetals, it appears that nucleophilicity of oxygens involved in the cis-1,2-acetals decreases in the order: equatorial anomeric > equatorial non-anomeric > axial anomeric. Consequently, treatment of the stannylene acetal prepared from D-mannose (mainly the cis-1,2-stannylene compound in admixture with a small proportion of the cis-2,3-stannylene acetal) with methyl 2,3,4-tri-O-benzoyl-6-O-trifluoromethanesulfonyl-alpha-D-glucopyra noside yielded, in addition to the expected beta-D-mannopyranoside (major), a product of non-anomeric alkylation at O-3. On the other hand, glycosylation of the stannylene acetal derived from maltose with methyl 2,3,6-tri-O-benzoyl-4-trifluoromethanesulfonyl-alpha-D-galactopyra noside gave almost exclusively a non-glycosidically, (2-->4)-linked pseudo-trisaccharide. Combination of the glycosylation via locked anomeric configuration with conventional glycosylations, to yield higher oligosaccharides, is also demonstrated.

Carbohydrate Conformation↗
PubMed1998

Synthetic C-oligosaccharides mimic their natural, analogous immunodeterminants in binding to three monoclonal immunoglobulins.

The binding of three monoclonal antigalactan immunoglobulins, IgAs X24, J539 and X44 to their natural haptens methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside, and the corresponding tri- and tetrasaccharides, was compared to the binding of these immunoglobulins with the comparable C-linked oligosaccharide analogues 1-3. The near identity of affinities of the two sets of oligosaccharides indicated the absence of any hydrogen bond involvement by the intersaccharidic oxygen atoms in the natural immunodeterminants.

Antibodies, Monoclonal↗
PubMed1998

Improved synthesis of an aldobiouronic acid related to hardwood xylans, and preparation of a derivative thereof suitable for linking to proteins.

Treatment of 1,3,4-tri-O-acetyl-alpha-D-xylopyranose with methyl 2,3-di-O-benzyl-l-chloro-l-deoxy-4-O-methyl-alpha, beta-D-glucopyranuronate in the presence of silver trifluoromethanesulfonate was highly stereoselective to give the alpha-linked aldobiouronic acid derivative (4) in 86% yield, after hydrogenolysis of the crude product of the coupling and chromatography. Compound 4 was acetylated and the fully protected substance was converted to the corresponding glycosyl chloride. Reaction of the latter with p-nitrophenol under phase-transfer catalysis afforded, after deacetylation, p-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-D-glucopyranosyluronate)-beta-D-xylopyranoside.

Glycoproteins↗