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Biomedical subjects

M Koziołkiewicz

Publications and source records attributed to M Koziołkiewicz.

16 recordsLinked to original sources
PubMed2001

Stereochemistry of cleavage of internucleotide bonds by Serratia marcescens endonuclease.

Endonuclease from Serratia marcescens hydrolyzes internucleotide phosphorothioate linkages of R(P) configuration with inversion of configuration at P-atom. This observation supports a reported architecture of the active site, with 3'-bridging and pro-S(P) non-bridging oxygen atoms of the scissile phosphate group involved in direct contact with hydrated magnesium cation, while His-89 activates a water molecule which attacks the phosphorus atom according to a one-step in-line mechanism. The presence of a phosphorothioate bond of S(P) configuration downstream to that one being cleaved reduces the rate of hydrolysis. This suggests participation of the pro-S(P) oxygen atom of that phosphate bond in the mechanism of action of the enzyme, which was not detected in published crystallographic analyses.

Catalysis↗
PubMed1999

Enzymatic assignment of diastereomeric purity of stereodefined phosphorothioate oligonucleotides.

Enzymatic hydrolysis of stereoregular oligodeoxyribonucleoside phosphorothioates (PS-oligos) synthesized via the oxathiaphospholane method has been used for assignment of their diastereomeric purity. For this purpose, two well-known enzymes of established diastereoselectivity, nuclease P1 and snake venom phosphodiesterase (svPDE) have been used. However, because of some disadvantageous properties of svPDE, a search for other [Rp]-specific endonucleases was undertaken. Extracellular bacterial endonuclease isolated from Serratia marcescens accepts PS-oligos as substrates and hydrolyzes phosphorothioate bonds of the [Rp] configuration, whereas internucleotide [Sp]-phosphorothioates are resistant to its action. Cleavage experiments carried out with the use of unmodified and phosphorothioate oligonucleotides of different sequences demonstrate that the Serratia nuclease is more selective in recognition and hydrolysis of oligodeoxyribonucleotides than previously reported. The substrate specificity exhibited by the enzyme is influenced not only by the nucleotide sequence at the cleavage site but also by the length and base sequence of flanking sequences. The Serratia nuclease can be useful for analysis of diastereomeric purity of stereodefined phosphorothioate oligonucleotides, but because of its sequence preferences, the use of this enzyme in conjunction with svPDE is more reliable.

Endodeoxyribonucleases↗
PubMed1999

Inhibition of plasminogen activator inhibitor release in endothelial cell cultures by antisense oligodeoxyribonucleotides with a 5'-end lipophilic modification.

A series of conjugates containing residues of lipophilic alcohols covalently bound to 5' end of oligodeoxyribonucleotides targeted against human plasminogen activator inhibitor (PAI-1) mRNA was synthesized via the oxathiaphospholane approach. The highest anti-PAI-1 activity in EA.hy 926 endothelial cell cultures was found for conjugates containing menthyl or heptadecanyl groups linked with an oligonucleotide complementary to a segment of human PAI-1 mRNA. The phosphodiester antisense oligonucleotides, which otherwise exhibit only limited anti-PAI-1 activity, were found to be more active than phosphorothioate oligonucleotides when conjugated to lipophilic alcohol residues. For menthyl conjugates an evidence of antisense mechanism of inhibition was found.

Base Sequence↗
PubMed1998

Effect of P-chirality of oligo(deoxyribonucleoside phosphorothioate)s on the activity of terminal deoxyribonucleotidyl transferase.

Phosphorothioate analogues of oligonucleotides (PS-oligos) of predetermined chirality at the phosphorus atom at each internucleotide linkage have been used as primers for terminal deoxyribonucleotidyl transferase (TdT, EC 2.7.7.31). The enzyme catalyzes efficient elongation of PS primers in which all phosphorothioate internucleotide linkages are uniformly of the [R(P)] configuration, while the presence of the linkage(s) of the [S(P)] configuration significantly decreases or completely inhibits the primer extension. Our results indicate that for the elongation of phosphorothioate oligomers the most important is the internucleotide bond located between the second and the third nucleoside from the 3'-end. The presence of [S(P)] linkage at this position strongly reduces the enzyme activity while the [R(P)] bond allows for effective elongation of the primer. The activity of the enzyme is also influenced by base composition and sequence of phosphorothioate primer as well as the dNTP used for elongation process.

DNA Nucleotidylexotransferase↗
PubMed1998

Synthesis, biochemical and biological studies on oligonucleotides bearing a lipophilic dimethoxytrityl group.

Dimethoxytritylphosphono-oligonucleotide conjugates have been prepared. They are totally resistant to nucleases present in human serum and do not affect cleavage of a complementary oligoribonucleotide by RNase H. Conjugates possessing a phosphate backbone gave better antisense inhibition of expression of plasminogen activator inhibitor type-1 within endothelial cells as compared with unconjugated oligonucleotides.

Cells, Cultured↗
PubMed1997

Stability of stereoregular oligo(nucleoside phosphorothioate)s in human plasma: diastereoselectivity of plasma 3'-exonuclease.

The stability of stereoregular oligo(nucleoside phosphorothioate)s (PS-oligos) in human plasma has been studied. 3'-Exonuclease present in human plasma appeared to be RP specific, that is, it cleaves internucleotide phosphorothioate linkages of [RP]-configuration and not those of [SP]-configuration. Therefore, PS-oligos containing all phosphorothioate internucleotide linkages of [RP]-configuration [RP-PS-oligos]) are more effectively degraded by the enzyme than PS-oligos prepared via nonstereo-controlled methods (so-called random mixture of diastereomers [Mix-PS-oligos]), whereas oligo(nucleoside phosphorothioate)s of [S(P)]-configuration remain intact. The enzyme activity depends on the sequence of nucleobases. The presence of deoxycytidine units (three or more residues) at the 3'-end of PS-oligo substrate significantly inhibits the enzyme activity.

Drug Stability↗
PubMed1997

Stereodependent inhibition of plasminogen activator inhibitor type 1 by phosphorothioate oligonucleotides: proof of sequence specificity in cell culture and in vivo rat experiments.

Hexadecadeoxyribonucleotides complementary to a fragment of human PAI-1 mRNA located upstream of the start codon and their phosphorothioate analogs were studied in cultured HUVECs as sequence-dependent inhibitors of PAI-1 expression. The activity of the random mixture of diastereomers of phosphorothioate hexadecanucleotide PS-16H has been compared with that of isosequential, stereoregular [All-Sp] and [All-Rp] isomers. The highest inhibitory effect on PAI-1 synthesis was observed with the [All-Sp] diastereomer. Stereorandom phosphorothioate oligonucleotide PS-16R complementary to the same region of rat PAI-1 mRNA, when injected into tail vein of rats, substantially decreased the level of PAI-1 in blood plasma.

Animals↗
PubMed1994

The epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets.

To examine whether the epidermal growth factor (EGF)-like domain Pro47-Asp87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E. coli. The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met1-Val444 at high yield in eight clones of E. coli. The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro47-Asp87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC. After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro47-Asp87 peptide fragment reduced fluidity of the membrane lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements. Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.

Base Sequence↗
PubMed1992

Synthesis of a gene encoding bovine substance P precursors and its expression in Escherichia coli.

Using an efficient Escherichia coli expression system, we have been able to obtain the precursor of substance P, alpha-preprotachykinin (alpha PPT). The alpha PPT protein is produced in E. coli as a fusion to beta-galactosidase, and accumulates in the cytoplasm as insoluble inclusion bodies. We also produced protachykinin (alpha PT), i.e., alpha PPT without a signal peptide. Further purification and characterization of the alpha PPT and alpha PT polypeptides strongly suggest that fully purified products can be obtained using our procedures.

Amino Acid Sequence↗
PubMed1991

Synthesis and expression in E. coli of the gene for human tumor necrosis factor (h alpha TNF).

A gene coding for human tumor necrosis factor (h alpha TNF) has been assembled by ligating short oligodeoxyribonucleotides and cloning into plasmid vectors. These oligonucleotides were prepared by the modified phosphoramidite methodology using isopropoxyacetyl (IPA) as a protecting group for exoamino- functions of nucleosides. Gene was expressed in E. coli and the protein product was purified to homogeneity by ion-exchange chromatography.

Amino Acid Sequence↗
PubMedDate not supplied

Modulation of plasminogen activator inhibitor type-1 biosynthesis in vitro and in vivo with oligo(nucleoside phosphorothioate)s and related constructs.

Oligonucleotides with a nucleotide sequence complementary to various regions of human plasminogen activator inhibitor type-1 (PAI-1) mRNA have been studied as antisense inhibitors of expression of PAI-1 protein in cultured cells [human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells, human hybrid endothelial cells]. Hexadeca(deoxyribonucleoside phosphorothioate) 13 complementary to a fragment of a signal peptide PAI-1 mRNA was found to be most active, giving ca. 70% inhibition of PAI-1 release in a time- and dose-dependent way. The stereo-regular All-S(P) and All-R(P) diastereomers of 13 were studied and found to inhibit PAI-1 synthesis in HUVEC in a stereo-dependent manner, with the All-S(P) diastereomer considerably more active than the stereo-random construct and All-R(P) isomer. The observed stereo-dependent activity of oligonucleotide phosphorothioate constructs is presumably governed by their resistance to nucleases. The corresponding phosphodiester analogue of 13 was not active unless covalently bound at its 5'-end to a lipophilic alcohol residue (menthol, heptadecanol). The observed antisense activity of phosphodiester oligonucleotide bioconjugates in cultured human hybrid endothelial cells was paralleled by their increased stability in human plasma with respect to unconjugated oligonucleotide. The oligo(deoxyribonucleoside phosphorothioate) complementary to the same signal peptide region of rat PAI-1 mRNA was found to reduce the PAI-1 level in blood plasma of rats after intravenous administration into the tail vein. The effect was both time- and dose-dependent. The same oligonucleotide was found to protect against arterial thrombus formation in the rat (lower incidence of venous thrombosis, lower thrombus weight, and increased occlusion time in experimentally induced thrombosis). An anti-PAI-1 inhibitory activity has been independently reported for a 20-mer oligo(2'-O-methyl-ribonucleoside phosphorothioate) complementary to a 3'-untranslated region of human PAI-1 mRNA in cultured HUVEC and human aortic smooth muscle cells.

Animals↗