Benediktas Juodka

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 °C with p‐NPB, in the range 50–60 °C with Tween 80 and in the range 50–65 °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 °C, pH 7.0–8.2, but was rapidly inactivated at 70–80 °C and at pH 7.0. The lipase was shown to be more thermolabile at 60 °C with respect to other two substrates. Using the emulsified substrate, no activity was obtained after preincubating the enzyme for 30 min at 70 °C. The enzyme was found to be pH‐tolerant when stored at 20 °C, pH 6.3–10.3 (100 mM Briton–Robson buffer) as the half‐life (t1/2) 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.

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.

Post-exposure processes in Temoporfin-photosensitized cells in vitro: reliance on energy metabolism

The progressive responses to photodynamic treatment (lambda > 590 nm) mediated by Temoporfin have been investigated in vitro on two rodent cell lines: BHK and murine hepatoma MH22 cells. Comparisons are made of two light exposure/post-exposure incubation media: Dulbeccos minimal essential medium (DMEM) and phosphate-buffered saline (DPBS) depleted of energy sources. Enhancement of lipid peroxidation is an early response to Temoporfin photosensitization in either experimental set. It is restored to the initial level by subsequent incubation in DMEM, but not in DPBS. The decrease in MTT specific activity and especially lactate dehydrogenase leakage from the cells are faster in DPBS and continue to proceed during the post-exposure incubation in the both media. The intracellular ATP pool is completely depleted within 3 h of post-exposure incubation in DPBS, but not in DMEM where, in contrast, an initial increase in ATP is observed. Based on these preliminary observations, it is presumed that ATP synthesized by injured mitochondria and activated glycolysis is being used to restore the deteriorated cell functions and/or to allow reactions involved in apoptosis to proceed.

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.

The protective effects of dihydrolipoamide and glutathione against photodynamic damage by Al-phtalocyanine tetrasulfonate.

In spite of well‐known ability of lipoamide/dihydrolipoamide (LipS2NH2/Lip(SH)2 NH2) and oxidized/reduced glutathione (GSSg/GSH) couples to scavenge singlet oxygen (1O2), the possible protective effects of these compounds against photodynamical damage by Al‐phtalocyanine tetrasulfonate (Al‐PcS4) were examined. Using erythrocyte glutathione reductase, pig heart lipoamide dehydrogenase and hamster kidney fibroblast culture as model systems, we have found that protective effects of Lip(SH)2NH2 and LipS2NH2 were close to that of azide, far exceeding the effects of GSH and GSSG, and paralleling the rates of Al‐PcS4‐sensitized photooxidation of these compounds. We have failed to observe a previously described (Devasagayam, T.P.A., et al. (1991) Biochim. Biophys. Acta 1088, 409‐412) enhancement of damaging action of 1O2 by GSH. These findings point out to the possibility of LipS2NH2/Lip(SH)2NH2 to neutralize the side‐effects of photodynamic therapy, and to a minor but definitely protective role of GSH.

Production of environmentally friendly biodiesel by enzymatic oil transesterification

Abstract In this study, the ability of commercial lipolytic enzyme Lipoprime 50T to catalyze the biotechnologically and environmentally important processes of rapeseed oil hydrolysis and transesterification were investigated and the optimal process conditions were determined using simple and accurate thin layer chromatographic, titrimetric and computer analysis (Micro image 4.0) methods. Lipoprime 50T lipase-catalyzed transesterification of rapeseed oil with several alcohols for fatty acid alkyl esters (biodiesel) production in n-hexane – a common solvent for lipolytic reactions – and in t-butanol – a novel promising solvent for biodiesel production – was investigated. The effects of molar ratio of substrates, reaction temperature and time on the constitution of the methanolysis reaction mixture were analyzed. Lipoprime 50T lipase-catalyzed rapeseed oil methanolysis in n-hexane was determined to be undesirably slow and inefficient process, whereas the lipolytic potential of the studied enzyme was determin...

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

BackgroundThe 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 α-amylase A and endosperm specific β-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.ResultsIn 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.ConclusionPlant development is followed by redistribution of TBP along individual genes and chromosomes.

Fluence-rate-dependent photosensitized oxidation of NADH

The photosensitizing activity of dimethoxyhaematoporphyrin, excited by a laser pulse at 532 nm (YAG-Nd3+), was investigated using reduced nicotinamide adenine dinucleotide (NADH) as a substrate. The photo-oxidative modification of NADH was monitored by measuring the absorbance at 340 nm. The use of nanosecond pulses (15 and 0.5 ns) resulted in photosensitized NADH oxidation which depended on the fluence but not on the fluence rate up to a peak fluence rate of 10(7) W cm-2. At higher fluence rates a decrease in NADH photo-oxidation was observed, as well as on irradiation with picosecond pulses (35 ps). Stern-Volmer assay of the quenching by sodium azide revealed a decrease in quenching efficiency with increasing peak fluence rate. Oxidation of NADH was not suppressed by the addition of 20 mM sodium azide at peak fluence rates above 6 x 10(9) W cm-2. This observation, as well as the significant bleaching of dye absorption, indicates excitation of the photosensitizer into higher lying excited singlet states and the involvement of processes other than photodynamic action.

Stable DNA—protein complexes in yeast Saccharomyces cerevisiae. Protein composition analysis@@@ Saccharomyces cerevisiae mielių stabilūs DNR baltymų kompleksai. Baltymų sudėties analizė

* Corresponding author. E-mail: lida.bagdoniene@gf.vu.lt Proteins tightly bound to DNA (TBP) are a protein group that remains attached to DNA by covalent or non-covalent bonds after its deproteinisation [1, 2]. These proteins are released only after DNA digestion with DNase I or benzonase. TBP have been found in DNA of numerous evolutionary distant species [3, 4]. Only a few of these TBP have been characterized. In the present work, we have identified some proteins that are present in Saccharomyces cerevisiae TBP–DNA protein complexes by two-dimensional electrophoresis and MALDI-TOF MS analysis. Most of these proteins (chromatin assembly factor 1, NNF1 protein, DNA damage tolerance protein RHC31, DNA repair protein RAD 7, SOH1 protein, etc.) have been found to participate in chromatin rearengament and regulation processes.

Porphyrins as radiosensitizers in 60Co-irradiated E. ascites tumor cells: possibility to combine with PDT

Efficiency of Ehrlich ascites cells radiosensitization by porphyrins is a function of irradiation dose. The maximal radiosensitization was reached at the dose--2Gy. Moreover, efficiency of cell radiosensitization depends on extracellular porphyrins concentration Hematoporphyrin (HP) and hemaporphyrin dimethylether (HPde) have different radiosensitizing effect on cells. One of the reasons of such phenomenon may be the different degree of hydrofobicity of those porphyrins. The studies of intracellular porphyrins concentration show that they differ in the case of HP and HPde in the same order as cell radiosensitization efficiencies.