Interleukin-2 and Cancer - Physisological and Pharmacological Uses
Full text. 111-112
Necrobiotic Process Causing Burn Wound Conversion May Be Prevented
by Allogeneic Keratinocytes Delivered by the Recombined Human/Pig Skin
(necrobiosis / cultured keratinocytes / delivery system for keratinocytes / recombined human/pig skin /
grafting upside-down / woound healing)
E.MATOUŠKOVÁ1, L.BROŽ2, E.POKORNÁ1, R.KÖNIGOVÁ2.................................................135
1Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
2Prague Burn Center, 3rdMedical Faculty, Charles University Hospital, Prague, Czech Republic
Full text. 135-136 137 138 139-140 141-142
New Monoclonal Antibodies Recognizing p53 Protein
Phosphorylated by Casein Kinase II at Serine 392
Š. POSPÍŠILOVÁ1, K. KAŇKOVÁ2, M. SVITÁKOVÁ1, R.NENUTIL2, B. VOJTĚŠEK1
1Department of Experimental Oncology, Masaryk Memorial Cancer Institute, Brno, czech Republic
2Department of Pathology, Faculty Woman,s Hospital Brno, Brno, Czech Republic
Abstract. The heparin-binding activity of bull seminal plasma proteins was inhibited by D-fructose, D-glucose, inulin and glycogen: D-galactose, dextran and mannan had no effect. While the ejaculated spermheparin interaction was not influenced by the presence of saccharides, the heparin-binding activity of epididymal sperm was inhibited by D-fructose. The results of the binding studies were confirmed by affinity chromatography on immobilized heparin followwed by elution with monosaccharides. Proteins adsorbed to a heparin-polyacrylamide column and eluted with D-fructose were analyzed by RP HPLC, SDS electrophoresis and by determination of the N-terminal amino-acid sequence. RNAase dimer, PDC-109 and metalloproteinase inhibitor (TIMP -2) were identified.
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Abstract. BRCA1 is a tumour suppressor gene with a caretaker function in the DNA-damage repair and the maintenance of genome integrity. The human BRCA1 and NBR2 genes and homologous Brca1 and Nbr1 mouse genes are situated head-to-head on human chromosome 17q21 and on mouse chromosome 11, respectively. Their transcription start sites, located on opposite DNA strands, are separated by 218 bp in humans, and by 289 bp in mice. Because of this intimate contact and because of our previous observation of a quasireciprocal expression pattern of BRCA1, NBR1 (next-to-BRCA1) and NBR2 genes in a panel of permanent cell lines and primary cell cultures derived from human breast cancer or normal mammary tissue.The analysis revealed highly significant downregulation of BRCA1 in 11 out of 12 examined tumour cell lines and primary cell cultures as compared to non-malignant mammary cells. Two isoforms of NBR1 (1A) and the classical NBR1(1B) transcripts were found in cells from malignant mammary tissues, all of them downregulated in respect to normal cells. The expression of NBR2 differed, being incrased in three permanent tumour cell lines and slighty decreased in all primary breast cancer cell cultures. The in silico analysis revelaed two new putative domains of the predicted NBR1 protein, duggesting its role in the ubiquitin pathway. The recent identification of the ubiquitin protein ligase activity of BRCA1 implies a possible functional connection between both genes.
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RHPS, composed of conflluent allogeneic kerationocytes cultured on cell-free pig dermis, stimulates wound healing when applied with thekeratinocyte layer facing the wound. So far it has not been clarified whether the confluent keratinocytes implanted "upside-down" can "take" or only stimulate healing by producing growht factors. Confluent male keratinocytes were grafted onto donor sites of three female patiens. Biopsies were taken on days 4,6 and 9 afteer grafting. The fate of donor cells was followed in paraffin sections by FISH for the Y chromosome and by persisting expression of vimentin taken as a marker of cultured keratinocytes. Histological evaluation was complemented by detection of keratin 10 and involucrin. All three donor sites healed within one week. On day 4 the early neoepidermis was multilayered but ddisorder after transplantation. A large proportion of cells were apparently of donor origin as indicated by the presence og Y chromosomes, irregular morphology, expression of vimentin in the bottom and upper layers of the neoepidermis, and by irregular expression of involucrin and keratin 10 only in the central layer of the neoepidermis. From day 6 owards, the new epidermis acquired an ordered stratification. Involucrin and keratin 10 renewed normal distribution in suprabasal layers. Concomitantly, vimentin expression was decreasing. The Y chromosome was still found on day 6 but not on day 9. We concluded that confluent allogeneic keratinocytes temporarily "take" to the wound and contribute to rapid wound closure, being replaced by the patient's epidermal cells after about one week.
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The spontaneous necrobiotic process frequently causes conversion of DDB (deep 2nd degree wounds) into full-thickness skin loss (3rd degree wounds). We found that this process may be positively influenced by the activity of living human allogeneic keratinocytes cultured on acellular pig dermis. This RHPS, if applied upside-down´ with the epidermal layer facing the wound, provides an opportunity for keratinocytes to influene the healing. The aim of the present study was to find conditions, in terms of timing and wound-bed preparation, for optimum healing activity of RHPS.The wound beds wwere prepared either with tangential excision, surface dermabrasion or deep dermabrasion. Out of 17 wounds grafted with RHPS after tangential excision, 15 (88%) healed in 4-10 days; early excised wounds ( up to day 5 ) healed within less than 10 days after the injury. Out of 8 wounds grafted after surface dermabrasion, only 2 (25%) healed. Out of 6 wounds grafted with RHPS after deep dermabrasion, 4 (67%) healed. The optimum healing effect of RHPS and prevention of coconversion was achieved in early tangentially excised wounds.
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Treatment of HeLa cells with low intensity ultrasound and two cytostatic drugs, cycloplatin and methotrexate, resulted in a partial disassembly of microtubules and microfilaments. This disassembly was due to depolymerization and subsequent erroneous repolymeration of essential cytoskeletal proteins, resulting in formation of unusual arrangements, mainly small, granule-like structures. The combined action of ultrasound and cytostatics had a synergistic effect dependent on both the concentration of the drug and the time of sonication. The demonstrated changes in the cytoskeleton are considered to be non-specific to ultrasound treatment, reflecting only an altered vital state of the treated cells.
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Backgroound. The p53 tumour suppressor protein is a nuclear phosprotein which plays a key role in cell-cycle regulation. The p53 protein protecs cells from undergoing tumorigenic alterations by inducing either the cell growth arrest or programmed cell death in response to a variety of cellular stress signals such as DNA damage, hypoxia, heat shock, oncogene activation or metabolic changes (Levine, 1997). One of the critical issues of p53 response to cellular stress or DNA damage is the way of its activation. It has been sugessted that posttranslational modification involving phosphorylation of the p53 protein is most likely the mechanism trought which the activity of the p53 protein may be regulated. p53 is phosphorylated on several serine residues within the N-and C-terminal regions by several cellular kinases. The N-terminal part of p53 is phosphorylated using different protein kinases including casein kinase I (CKI) (milne et al., 1992), DNA-PK (Lees-Miller et al.,1992), Chk1, Chk2, MAP kinase (Milne et al., 1994) and c-Jun kinase (Milne et al., 1995). There are also at least three phosphorylation sites on the C-terminus of human p53 at amino acids 315, 378 and 392. Serine315 of p53 is a target for cdkl and cdk2 (Price et al., 1995). Serine378 is phosphorylated by PKC (Baudier et al., 1992). Serine392 is phosphorylated by purified casein kinase II (CKII) in vitro (Blaydes and Hupp, 1998). Phosphorylation of the human p53 at Ser392 has been shown to enhance p53 sequence-specific DNA binding in vitro (Hupp and Lane, 1994), which is responsible for transcriptional activation of the p53 protein. Recentrly, it has also been shown that phosphorylation of Ser392 is important for p53-mediated trancriptional activation in vivo (Hao et al., 1996).
Phosphorylation of different proteins in cells can be studied using a range of different methods, but the primary technique for determining phosphate incorporation into the specific sites in target proteins involves labelling of cells with [32P] phosphate followed by phospho-amino-acid analysis or sequencing of the protein of interest (Van der Geer et al., 1993). The main problem associated with this technique is the incubation of cells with a radioactive precursor [32P], which itself can activate growth arrest and stress-responsive signalling pathways, obviously perturbing protein phosphorylation (Yeargin and Haas, 1995; Dover et al., 1994). Preparation of monoclonal antibodies that are specific to either phosphorylated or non-phosphorylated epitopes within the target protein provides a powerful alternative to the above techniques.
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