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1

Capillary electrophoretic analysis of the stability of RNA-peptide complex in human blood plasma

Mucha P., Szyk A., Rekowski P., Barciszewski J.

Chemia Analityczna

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2004

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Vol. 49, No. 6

845-853

EN

Capillary electrophoresis mobility shift assay (CEMSA) was employed to qualitatively study the stability of RNA and RNA-peptide complex in human blood plasma. RNA-protein interactions play an important role in the replication cycle of the human immunodeficiency virus type 1 (HIV-1). Thus, the CEMSA method was adapted to study the interactions between viral trans-activation response element (TAR RNA) and the arginine-rich fragment (49-57) of viral trans-activation protein Tat HIVn1. The stability of the free and peptide- complexed TAR in human blood plasma was investigated using a polyacrylamide (LPA)-coated capillary and a sieving matrix with the separation buffer. Under the applied conditions the studies on micromolar concentrations of TAR could be performed without labelling in less than 30 min. In the presence of Tat peptide, a significant increase of the migration time of TAR from 18.66 min to 20.12 min was observed. The differences between the behaviour of the free and complexed TAR in human blood plasma were apparent during CEMSA analysis. Both species degraded progressively in time. The first products of degradation were detected immediately after spiking the plasma sample with the free TAR. Migration times of the degradation products were longer than for the free TAR. Free TAR was degraded completely after 60 min. Since TAR was unlabelled, the products of its degradation could not be identified. TAR complexed with the Tat-peptide was much more stable in plasma compared to the free TAR. Even after 180 min of incubation a large amount of the complexed TAR still could be detected. This work is the first to present the application of CE to the stability studies of the free and peptide-complexed RNA in human blood plasma.

PL

Przedstawiono metodę elektroforezy kapilarnej do jakościowej oceny stabilności RNA i kompleksu RNA-peptyd w plazmie ludzkiej krwi. Z powodu dużego znaczenia oddziaływań RNA-białko w cyklu replikacyjnym wirusa HIV-1, metodę zaadaptowano do badań oddziaływań slruktury TAR RNA z bogatym w argininę fragmentem (49-57) wirusowego biafka transaktywowanego Tat HIV-1. Oddziaływanie TAR-Tat jest odpowiedzialne za efektywną clongację wirusowego mRNA. Używając pokrytej poliakryloamidem (LPA) kapiiary i buforu zawierającego czynnik przesiewający badano stabilność wolnego i skomplcksowanego z peptydem TAR. Zastosowane warunki pozwoliły na badanie stabilności kompleksu TAR-Tat bez znakowania RNA, w stężeniu mikromolowym w czasie krótszym niż 30 min. W obecności Tat pcptydu zaobserwowano znaczące przesunięcie czasu migracji TAR od 18.66 do 20.12 min. Zachowanie wolnego i skompleksowancgo TAR było łatwo obser-wowalne przy zastosowaniu CE. Pierwsze produkty degradacji zaobserwowano natychmiast po zmieszaniu wolnego TAR z próbką plazmy. Czasy migracji tych produktów były dłuższe niż wolnego TAR. Całkowita degradacja TAR nastąpiła po l godz. TAR skompleksowany z Tat pcptydem był znacznie stabilniejszy w plazmie w porównaniu do wolnego TAR, Czasy migracji produktów degradacji przypominały te obserwowane w wypadku wolnego TAR. Prezentowana procedura opisuje po raz pierwszy zastosowanie CE do badania zachowania wolnego i skompleksowancgo zpeptydem RNA w ludzkiej plazmie krwi.

2

New Galanin Analogues with Contractile Activities on Rat Gastric Smooth MusclesR

Ruczyński J., Sawuła A., Konstański Z., Korolkiewicz R., Rekowski P.

Polish Journal of Chemistry

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2002

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Vol. 76 / nr 1

57-66

EN

We synthesized new 15-amino-acid-residue analogues of porcine galaninmodified in positions 2, 6, 8 or 14 and studied their activity on isolated rat gastric smooth muscles. Thus, we intended to characterize the molecular domains of GAL responsible for binding to its receptors and biological activity in the gastric fundus. All peptides were synthesized by the solid phase peptide synthesis with the use of Fmoc strategy. All galanin analogues contracted rat gastric fundus strips in a concentration-dependent manner with significantly increased or decreased activities as compared to GAL(1-15)NH2. As expected, the modifications introduced into the amino acid sequence of galanin caused changes in the interaction of GAL(1-15)NH2 with its receptors. Thus, residues: Trp2, Ser6, Gly8 and His14 in the amino acid sequence of GAL(1-15)NH2 play an important roles in the high-affinity binding of GAL to its receptors and biological activity in rat gastric smooth muscle cells.

3

Interaction of a Transcription Factor IIIA Peptide Containing Two Zinc Fingers with 5S rRNA

Giel-Pietraszuk M., Mucha P., Rekowski P., Kupryszewski G., Barciszewska M. Z.

Polish Journal of Chemistry

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2002

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Vol. 76 / nr 6

815-822

EN

The interaction of lupin ribosomal 5S RNA with a chemically synthesized peptide containing 60 amino acid, derived from Xenopus laevis transcription factor IIIA, is analyzed. The results show that such short fragment retains the ability of binding to 5S rRNA molecule, as shown by electrophoretic gel shift and RNase footprint assay. The peptide protects from hydrolysis with specific nucleases helix II and V of 5S rRNA.

4

Galanin and Its Analogues Modified in Position 14: Chemical Synthesis and Biological Activity

Ruczyński J., Szyk A., Konstański Z., Korolkiewicz R., Rekowski P.

Polish Journal of Chemistry

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2000

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Vol. 74, nr 10

1375-1384

EN

In search of a specific galanin receptor antagonist, 15-amino-acid-residue analogues of porcine galanin modified in position 14 were synthesized and their activity was studied on isolated rat smooth muscles. It was shown that all galanin analogues contracted rat gastric fundus strips in a concentration-dependent manner with significantly increased activities as compared to GAL(1-15)NH2. The results suggest that position 14 in the amino acid sequence of GAL(1-15)NH2 is important for the biological activity.

5

Identification of selected siderophores in the Baltic Sea environment by the use of capillary electrophoresis

Kosakowska A., Kupryszewski G., Mucha P., Rekowski P., Lewandowska J., Pazdro K.

Oceanologia

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1999

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No. 41 (4)

573-587

EN

Extracts from seawater and sediment pore water samples were characterised by capillary electrophoresis (CE). Siderophores of the ferrioxamine family were identified. Ferrioxamine E is the dominant siderophore in both seawater and sediment pore water samples from different regions of the Baltic Sea. Ferrioxamine G was identified in subsurface seawater samples from the Gdansk Deep. Rhodotorulic acid was also identified in seawater samples from the euphotic zone (0-30 m) of Puck Bay and in sediment pore water from Puck Bay and the Bornholm Deep. Ferrioxamine B was not found. The presence of catechol siderophores was not investigated.