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Advantage of a baculovirus expression system for protein-protein interaction studies. Involvement of posttranslational phosphorylation in the interaction between wt p53 protein and poly(ADP-ribose) polymerase-1

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Schmid G., Wojciechowski J., Węsierska-Gądek J.
Acta Biochimica Polonica
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2005
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vol. 52
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issue 3
713-719
EN
We recently observed an interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 protein. However, more extensive studies on both proteins, especially those on characterization of their domains involved in the interaction were difficult due to very low expression levels of p53 in mammalian cells. Therefore, we generated recombinant proteins for such studies. To clarify which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction, we generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 each were sufficient to confer binding to PARP-1, whereas the amino-terminal part harbouring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were both necessary for complex formation with p53 protein. Since the most important features of p53 protein are regulated by phosphorylation, we addressed the question whether its phosphorylation is essential for the binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomers were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1, indicating that complex formation between the two proteins was regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export.
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Clinical Evaluation of Near-Infrared Cerebral Oximetry in the Awake-Patient Carotid Endarterectomy

64%
Wojciechowski J., Sidorowicz M., Szyndler K., Znaniecki Ł., Trenkner M., Halena G., Brzeziński M., Rogowski J.
Polish Journal of Surgery
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2007
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vol. 79
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issue 8
519-525
EN
The aim of the study was to evaluate the usefulness of continuous monitoring of regional cerebral oxygen saturation (rSO2) for detection of brain ischemia during carotid endarterectomy.Material and methods. We performed 44 carotid endarterectomies using regional anesthesia, with simultaneous regional cerebral oxygen saturation monitoring in both hemispheres of the brain.Results. Oxygen saturation in the hemisphere ipsilateral to the operated carotid artery dropped from 65.1±8.1 to 58.2±10.7 after carotid artery cross-clamping. The difference was statistically significant (p<0.005). Oxygen saturation in the hemisphere contralateral to the operated artery did not demonstrate a difference between that before or after carotid artery closure (65.7±9.2 and 66.1±10.2, respectively, p=0.1). In five patients (11.4%) carotid artery clamping was associated with the appearance of neurological deficits. Shunt usage was necessary in four cases; the rSO2 decreased by 19.2±14% in this group. In the group without neurological deficit during carotid clamping, the rSO2 decreased by 9.7±10.3% (the difference between groups with and without neurological deficit was not statistically significant, p=0.5). In patients with a rSO2 drop above 20%, the sensitivity of the cerebral oximetry was 20% and specificity 97.5%, while the negative predictive value was 90.7%.Conclusions. Continuous cerebral oximetry is a simple and non-invasive method of patient monitoring during carotid endarterectomy. The rSO2 decreases significantly after the ICA clamping. The sensitivity of cerebral oximetry in prediction of neurological deficit during the procedure is low. Defining the threshold value of rSO2 decrease after ICA clamping as an indication for shunt was not possible with the results of this study.
3
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Development of a new, simple and cost-effective diagnostic tool for genetic screening of hereditary colorectal cancer - the DNA microarray assay

52%
Stojcev Z., Banasiewicz T., Kaszuba M., Sikorski A., Szczepkowski M., Bobkiewicz A., Paszkowski J., Krokowicz Ł., Biczysko M., Szmeja J., Jurkowska M., Majewski P., Mackiewicz A., Lamperska K., Drews M., Wojciechowski J.
Acta Biochimica Polonica
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2013
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vol. 60
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issue 2
195-198
EN
Detection of mutations in families with a hereditary predisposition to colon cancer gives an opportunity to precisely define the high-risk group. 36 patients operated on for colon cancer, with familiar prevalence of this malignancy, were investigated using the DNA microarrays method with the potential detection of 170 mutations in MLH1, MSH2, MSH6, CHEK2, and NOD2 genes. In microarrays analysis of DNA in 9 patients (25% of the investigated group), 6 different mutations were found. The effectiveness of genetic screening using the microarray method is comparable to the effectiveness of other, much more expensive and time-consuming methods.
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