mTHPC-mediated photodynamic treatment up-regulates the cytokines VEGF and IL-1alpha.

Photodynamic therapy (PDT) of cancer induces oxidative stress, which intervenes in the expression of cytokines by tumor cells. The cytokines might have either a positive or a negative impact on tumor eradication. Here, we studied the expression of cytokines vascular endothelial growth factor (VEGF) and interleukin-1alpha (IL-1alpha) in the human epidermoid carcinoma A-431 cells following m-tetra(3-hydroxyphenyl)-chlorin (mTHPC)-mediated PDT in vitro and assessed the IL-1alpha effect on VEGF expression. Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay revealed the enhanced production of VEGF and IL-1alpha both on mRNA and protein levels by mTHPC-loaded cells after light exposure. The silencing of IL1A by small interfering RNA resulted in decreased production of IL-1alpha and a reduced amount of VEGF. Furthermore, exogenous recombinant IL-1alpha stimulated the VEGF expression after PDT. Thus, in addition to the cytotoxic action on the A-431 cells, mTHPC-mediated PDT stimulated the production of VEGF and IL-1alpha, and IL-1alpha contributed to the VEGF overexpression. These data establish IL-1alpha as a possible target of combined cancer treatment.

Synthesis of trimethylolpropane esters of oleic acid by Lipoprime 50T.

The ability of the commercial lipolytic enzyme Lipoprime 50T to catalyze the biotechnologically important synthesis of the biodegradable and environmentally acceptable trimethylolpropane (2-ethyl-2-(hydroxymethyl)-1,3-propanediol) ester of oleic acid was investigated. Simple and accurate thin-layer chromatography and computer analysis methods were used that enable one to follow changes of all reaction mixture components simultaneously. The processes of transesterification and esterification were compared. The effects of the molar ratio of the substrates, reaction temperature, time, and medium on the composition of the reaction mixture were analyzed. Esterification was determined to be more preferable than transesterification in both studied solvents. Under the optimal conditions identified (15% w/w water, temperature 60°C, trimethylolpropane to oleic acid molar ratio 1:3.5, and reaction time 72 h), the highest trimethylolpropane trioleate yield of around 62% and trimethylolpropane mono-, di-, and trioleate overall yield of about 83% were obtained. Although the yields are not high enough for industrial application, the process shows the potential to be optimized for higher yields in the near future as the conversions were obtained at ambient pressure, whereas many other processes described in the literature are conducted under vacuum at a specific pressure.

Rat serum carboxylesterase partly hydrolyses gamma-butyrobetaine esters.

Although described some time ago, gamma-butyrobetaine esters and related compounds have not gained much attention from researchers, and their physiological function remains obscure. Formerly we detected GBB-esterase enzymatic activity in rat blood serum using phenylated gamma-butyrobetaine as an artificial substrate of the enzyme and HPLC. The aim of the present work was to develop an assay that would enable spectrophotometric or colorimetric determination of the reaction products of GBB-esterase activity and to reveal individual proteins performing GBB-esterase activity in rat blood serum. For this purpose gamma-butyrobetaine 1-naphthyl ester was synthesised. Hydrolysis of this ester releases 1-naphthol, which increases the optical absorbance at 322 nm. We have shown that the enzymatic hydrolysis of GBB 1-naphthyl ester to 1-naphthol in rat blood serum is due to GBB-esterase activity. An attempt was done to purify the enzyme from rat blood serum. By combining DEAE Sepharose at pH 4.2 and affinity chromatography with procainamide we achieved a 68-fold enrichment of GBB-esterase activity in our preparations. Separation of fraction proteins in 2D protein electrophoresis with following mass-spectrometry indicated that GBB esterase activity in rat blood serum is performed in part by carboxylesterase.

Development-dependent changes in the tight DNA-protein complexes of barley on chromosome and gene level.

BACKGROUND: The tightly bound to DNA proteins (TBPs) is a protein group that remains attached to DNA with covalent or non-covalent bonds after its deproteinisation. The functional role of this group is as yet not completely understood. The main goal of this study was to evaluate tissue specific changes in the TBP distribution in barley genes and chromosomes in different phases of shoot and seed development. We have: 1. investigated the TBP distribution along Amy32b and Bmy1 genes encoding low pI alpha-amylase A and endosperm specific beta-amylase correspondingly using oligonucleotide DNA arrays; 2. characterized the polypeptide spectrum of TBP and proteins with affinity to TBP-associated DNA; 3. localized the distribution of DNA complexes with TBP (TBP-DNA) on barley 1H and 7H chromosomes using mapped markers; 4. compared the chromosomal distribution of TBP-DNA complexes to the distribution of the nuclear matrix attachment sites. RESULTS: In the Amy32b gene transition from watery ripe to the milky ripeness stage of seed development was followed by the decrease of TBP binding along the whole gene, especially in the promoter region and intron II. Expression of the Bmy1 gene coupled to ripening was followed by release of the exon III and intron III sequences from complexes with TBPs. Marker analysis revealed changes in the association of chromosome 1H and 7H sites with TBPs between first leaf and coleoptile and at Zadoks 07 and Zadoks 10 stages of barley shoot development. Tight DNA-protein complexes of the nuclear matrix and those detected by NPC-chromatography were revealed as also involved in tissue- and development-dependent transitions, however, in sites different from TBP-DNA interactions. The spectrum of TBPs appeared to be organ and developmental-stage specific. Development of the first leaf and root system (from Zadoks 07 to Zadoks 10 stage) was shown as followed by a drastic increase in the TBP number in contrast to coleoptile, where the TBPs spectrum became poor during senescence. It was demonstrated that a nuclear protein of low molecular weight similar to the described TBPs possessed a high affinity to the DNA involved in TBP-DNA complexes. CONCLUSION: Plant development is followed by redistribution of TBP along individual genes and chromosomes.

Transcription- and apoptosis-dependent long-range distribution of tight DNA-protein complexes in the chicken alpha-globin gene.

The proteins tightly bound to DNA (TBP) are a group of proteins that remain attached to DNA with covalent or noncovalent bonds after its deproteinization, and have been hypothesized to be involved in regulation of gene expression. To investigate this question further, oligonucleotide DNA arrays were used to determine the distribution of tightly bound proteins along a 100-kb DNA fragment surrounding the chicken alpha-globin gene domain in DNA from chicken erythrocytes, liver, and AEV-transformed HD3 (erythroblast) cells in different physiological conditions. DNA was fractionated into TBP-free (F) and TBP-enriched (R) fractions by separation on nitrocellulose, and these fractions were used as probes for hybridization with the microarray. In erythrocytes, the site 60 kb from the 5' end of the sequence and containing a LINE family CR1 repeat was TBP enriched, but in HD3 cells this sequence was devoid of TBPs. Thus cessation of transcription of the domain is followed by an F-R transition of this site. In apoptotic HD3 cells, TBPs remained attached to DNA only at a site situated 16 kb from the 5' end of the sequence. These data confirm and extend previous conclusions about the specificity of the DNA sequences that preferably form tight complexes with proteins and about the differentiation-specific distribution of the TBPs in different cell lineages. Binding of TBPs appears to be independent of primary DNA sequence.

Mechanism of radiosensitization by porphyrins.

According to our previous data, hematoporphyrin dimethyl ether (HPde) at concentrations useful for photodynamic therapy can radiosensitize aggressive Ehrlich ascite carcinoma (EAT) to 2Gy irradiation inducing total tumour growth inhibition. The aim of this study was to further investigate the possible mechanism of radiosensitization of EAT by dicarboxylic porphyrin-HPde. Our results reveal that HPde is inducing several rearrangements in the EAT cells: 1.2 x 10-6 M of the photosensitizer diminishes the number of cells in mitosis by a factor of 3, increases the number of cells in the S phase of the cell cycle, modifies the activities of antioxidant enzymes glutation S-transferase (GST) and DT-diaphorase (DTD), and eventually induces slight apoptosis. Moreover, it was shown that HPde is a ligand of peripheral benzodiazepine receptor (PBR). Named "house keeper," PBR is usually responsible for all these perturbations, which, in our case, act in concert with the following ionizing radiation, producing the interaction of two antiproliferative/destructive factors.

The cloning and characterization of Tetrahymena pyriformis translation elongation factor 1b alpha and gamma subunits.

The multi-subunit eukaryotic translation elongation factor 1 (eEF1) consists of two functionally distinct parts: G-protein eEF1A and guanine nucleotide exchange factor eEF1B. Here, we report on the cloning of cDNAs of both the alpha and gamma subunits of the eEF1B from the ciliated protozoan Tetrahymena pyriformis. The open reading frame of the eEF1Bgamma cDNA encodes a 399-amino acid long polypeptide with a calculated molecular mass of 45.2 kDa. The eEF1Balpha cDNA contains an open reading frame encoding a polypeptide of 228 amino acids. The calculated molecular mass of this protein is 25.2 kDa. The overall deduced amino acid sequences of eEF1Balpha and eEF1Bgamma show a considerable homology with the families of alpha and gamma proteins from other eukaryotic organisms. We demonstrated that eEF1Bgamma is an RNA-binding protein which is able to bind to different RNAs.

Distribution of tight DNA-protein complexes along the barley chromosome 1H, as revealed by microsatellite marker analysis.

The distribution of DNA complexes with proteins resistant to routine deproteinisation procedures (tightly bound proteins, TBP) was studied on the barley chromosome 1H by means of microsatellite analysis. The polypeptide spectrum of the barley shoot TBP was similar to that formerly described for other organisms. In order to reveal developmental changes in the distribution of the TBP, DNA was extracted from dry grains, coleoptiles, root tips, and young and old leaves. In the seeds, all the studied DNA sites were evenly distributed between free DNA and DNA containing the tight DNA-protein complexes. Germination made the interaction between TBP and chromosomal loci specific. In coleoptile DNA, sites containing microsatellites located in the distal part of the long arm of the chromosome were not bound to the TBP anymore, however, the centromeric markers were found exclusively in the tight DNA-protein complexes. A similar but not identical distribution of markers was observed in the root tips and young leaves. Leaf senescence was accompanied by a loss in interaction specificity between chromosomal loci and tightly bound proteins. These results are considered to reflect changes in chromatin domain interaction with the nuclear matrix during plant development.

Experimental survey of non-clonogenic viability assays for adherent cells in vitro.

Results of rapid cell viability assays were experimentally compared in order to reveal the most suitable test for in vitro investigations of the combination of photodynamic therapy (PDT) with chemotherapeutic drugs. meso-Tetra(3-hydroxyphenyl)-chlorin (m-THPC) accumulating in cell membranes and meso-tetra(4-sulfonatophenyl)-porphin (TPPS4) accumulating in lysosomes were used as photosensitisers. Doxorubicin that localises, mainly, to nucleus and vincristine that binds to microtubules were used as cytostatic drugs. Two adherent rodent cell lines, baby hamster kidney (BHK-21) and murine hepatoma (MH-22A), were used to examine the contribution of a cell. We tested cytotoxicity assays of the main groups of fast (non-clonogenic) methods of cell viability measuring. Plasma membrane integrity was estimated by trypan blue exclusion and LDH leakage, metabolic activity was tested by [3H]-thymidine incorporation and MTT assay, loss of monolayer adherence was measured by staining with crystal violet and CyQUANT. The most sensitive test in each case was the assay related to the site of the direct damage, and measurement of the loss of monolayer adherence proved to be as sensitive assay as the damage-specific one. All the assays applied, except for the LDH release, revealed a higher effect of combination of m-THPC-mediated phototreatment and doxorubicin compared to either of the single treatments.

Correlation of death modes of photosensitized cells with intracellular ATP concentration.

The impact of intensity of glycolysis and oxidative phosphorylation on death of photosensitized murine hepatoma MH22 cells in vitro has been investigated. Cells photosensitized with meso-tetra(4-sulfonatophenyl)-porphine localized to lysosomes died mostly by necrosis, and the mode of cell death did not depend on the energy metabolism. Photosensitization with 5-aminolevulinic acid-stimulated endogenous porphyrins localized mainly in mitochondria or 5,10,15,20-tetrakis(m-hydroxyphenyl)-chlorine localized to cell membranes, including mitochondria, led to cell death mostly by apoptosis. In this case, the mode of cell death depended on the medium: under conditions unfavorable to glycolysis the ratio apoptosis/necrosis decreased significantly.

Characterization and physicochemical properties of a lipase from Pseudomonas mendocina 3121-1.

The lipase from Pseudomonas mendocina 3121-1 was found to be homogeneous with a molecular mass of 30 kDa by SDS/PAGE. It is composed of two identical subunits. A molecular mass of 62 kDa was determined by gel chromatography on a Toyopearl HW-55F column. Some physicochemical properties of the lipase were investigated using p-nitrophenyl butyrate (p-NPB), Tween 80 solution and Sigma olive-oil emulsion as substrates. The optimum temperature was determined to be 52 degrees C with p-NPB, in the range 50-60 degrees C with Tween 80 and in the range 50-65 degrees C with olive-oil emulsion. The optimum pH was determined to be in the pH range 7.2-7.5, both with Tween and the emulsion, but was unusually alkaline (pH 9.5) with p-NPB. The enzyme was activated for p-NPB hydrolysis by thermal treatment up to 60 min at 60 degrees C, pH 7.0-8.2, but was rapidly inactivated at 70-80 degrees C and at pH 7.0. The lipase was shown to be more thermolabile at 60 degrees C with respect to other two substrates. Using the emulsified substrate, no activity was obtained after preincubating the enzyme for 30 min at 70 degrees C. The enzyme was found to be pH-tolerant when stored at 20 degrees C, pH 6.3-10.3 (100 mM Briton-Robson buffer) as the half-life (t(1/2)) was more than 240 h when p-NPB was used as the substrate. By contrast, the pH-stability range was more narrow (pH 8.0-10.5) with olive-oil emulsion. The effect of various metal ions and EDTA depended on the nature of the substrate.