Phosphatase activity in barley proteins tightly bound to DNA and its development-dependent changes.

The tightly bound proteins (TBPs), a protein group that remains attached to DNA either covalently or noncovalently after deproteinization, have been found in numerous eukaryotic species. Some TBPs isolated from mammalian and yeast cells possess phosphatase or kinase activity. The aim of this study was to characterize further TBPs in barley (Hordeum vulgare) cells. The spectra of TBPs varied in different organs of barley shoots (first leaves, coleoptile, and roots) and at different developmental stages of the plant. Some barley TBPs manifested phosphatase, probably Ser/Thr or dual Ser/Thr/Tyr activity. MALDI-TOF mass spectrometry of barley TBPs identified several proteins involved in chromatin rearrangement and regulation processes, including transcription factors, serpins, protein phosphatases and protein kinases, RNA helicases, and DNA topoisomerase II.

Schedule-dependent interaction between Doxorubicin and mTHPC-mediated photodynamic therapy in murine hepatoma in vitro and in vivo.

PURPOSE: To evaluate cytotoxic and antitumor effects of a conventional anticancer drug Doxorubicin (Dox) and photodynamic therapy (PDT) mediated by a promising photosensitizer of second generation meta-tetra (3-hydroxyphenyl)-chlorin (mTHPC) in combination. METHODS: Murine hepatoma MH-22A was used for investigation in vitro and in vivo. In vitro, the cells were incubated with 0.15 microg/ml mTHPC for 18 h and exposed to light from LED array (lambda = 660+/-20 nm) at 0.6-2.4 kJ/m2. 0.05-0.2 microg/ml Dox was administered either 24 h prior to or immediately after light exposure (Dox-->PDT or PDT + Dox, respectively). The cytotoxicity was tested by staining with crystal violet. The character of the combined effect was assessed by multiple regression analysis. In vivo, the antitumor activity was estimated by monitoring the tumor volume over time, in mice transplanted subcutaneously with MH-22A and treated with Dox and/or PDT. For PDT, mice were exposed to light from diode laser (lambda = 650+/-2 nm) at 12 kJ/m2 following 24 h after administration of 0.15 mg/kg mTHPC. A 3 mg/kg Dox was administered either within 15 min prior to mTHPC or within 15 min after light exposure (Dox-->PDT or PDT + Dox, respectively). RESULTS: Both in vitro and in vivo, the combination of mTHPC-mediated PDT and Dox was evaluated to be more effective than each treatment alone. In vitro, the difference between cell viability curves after photodynamic treatment as a single modality and after combination of photodynamic treatment with Dox was statistically significant under most of the applied conditions (P < or = 0.02). In the case of PDT + Dox, the combination had an additive character, and the sequence Dox-->PDT caused a sub-additive interaction. In vivo, both regimens of combination were more effective in inhibiting tumor growth than any single treatment (P < 0.09). The antitumor activity of PDT + Dox regimen was more prominent than that of Dox-->PDT; however, significance of the difference was not high (P = 0.08). CONCLUSIONS: These results indicate that Dox potentiates therapeutic efficacy of mTHPC-mediated PDT and vice versa, and the degree of potentiation is influenced by the combination schedule: administration of Dox immediately after light exposure is preferable to administration of Dox at 24 h prior to light exposure.

Intracellular location and nuclear targeting of the Spi-1, Spi-2 and Spi-3 gene-derived serine protease inhibitors in non-secretory cells.

Proteases and their inhibitors are indispensable for the regulated activation and/or degradation of structural and functional proteins involved in basic cellular processes, e.g. in cell cycle control, cell growth, differentiation and apoptosis. In this context the serine protease inhibitors derived from the murine Spi-1, Spi-2 and Spi-3 genes, and their human homologs, deserve reconsideration. Microsequencing data indicate that a fraction of the three serpins has the capability to constitute a well characterized proteinase K, high salt and SDS-stable complex which coisolates with DNA under salting out conditions from various cell and tissue types. This tight association with DNA isolated under conditions designed to deproteinize DNA efficiently points to an in situ preformed chromatin complex. Accordingly, in addition to their well known functions as 'serum protease inhibitors' the Spi-1 and Spi-2 gene-derived proteins appear to have intracellular functions as well. The involvement of the three serpins in chromatin complexes requires their nuclear translocation. Application of (enhanced) green fluorescent protein technology and optical section microscopy reveals that truncation of the N-terminal signal sequences of the Spi-1 and Spi-2 gene-encoded proteins is a prerequisite for their nuclear translocation while non-truncated fusion proteins are enriched at the nuclear indentation which is the site of the Golgi apparatus and the centrosome. The identification of new species of intracellular serpins is of potential interest with respect to accumulating evidence for serine protease inhibitor-dependent inhibition or prevention of apoptosis.

Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D.

Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12-24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.

Partial characterization of the ribonuclease P from Tetrahymena pyriformis.

Ribonuclease P activity from infusoria Tetrahymena pyriformis has been isolated and purified more than 1000-fold over cytosol crude extract. Purified tRNA 5' endonuclease processes in vitro heterologous substrates, precursors of the human tRNA(Tyr) and Drosophila melanogaster tRNA(Leu), exactly at the 5' end of the mature molecules. The activity was abolished by micrococcal nuclease and protease treatment indicating that both RNA and protein components are essential for its activity. The most abundant polypeptides in the purified enzyme fractions have molecular masses of about 100, 44 and 35 kDa. The enzyme requires divalent cations for its activity and shows optimal activity in the presence of the low concentrations of the monovalent salts. Substrate structural requirements for the purified enzyme were analyzed with different tRNA precursor models. The analysis of the derivatives of tRNA(Leu) precursors with altered aminoacyl stem structures reveals that end of the stem is important for substrate 5' end processing with purified enzyme.

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)2NH2) and oxidized/reduced glutathione (GSSG/GSH) couples to scavenge singlet oxygen (1O2), the possible protective effects of these compounds against photodynamical damage by Alphtalocyanine 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.

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: Dulbecco's 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.

Activation and enzyme characteristics of a DNA-restrained phosphatase in chromatin-associated complexes.

DNA-bound polypeptide complexes composed of several non-histone polypeptides that resisted harsh DNA deproteinization procedures were characterized. The three major polypeptides of these complexes have molecular masses of 62, 52, and 40 kDa. They constitute supramolecular structures that reside on isolated DNA in dense clusters. The supramolecular complexes were released from DNA as globular 12.8 +/- 0.8-nm particles; these particles were gradually disassembled to form smaller supramolecular structures. The DNA-bound complexes comprise of an encrypted adenosinetriphosphatase/phosphatase activity, which is a minor but intrinsic component of the complexes. The enzyme remained inactive as long as the complexes were bound to DNA. However, the enzyme was activated concomitantly with the progression of DNA digestion, which indicated that DNA was involved in the downregulation of the enzyme. The inactive DNA-restrained complex could not be restored in vitro, which indicated its non-trivial nature. Once released from DNA, the enzyme was inactivated over a period of several hours. However, in the DNA-associated complexes its potential to become activated during DNA digestion was conserved for several months. In the activated state, the enzyme showed an optimum activity at pH 9.5, was stimulated by Mg2+, inhibited by vanadate and EDTA, but was not significantly inhibited by okadaic acid. The active enzyme, which consists of two subunits of 56 kDa and 59 kDa, can be released from the supramolecular structures by agarose gel electrophoresis. A regulatory mechanism therefore exists for the downregulation of this phosphatase by DNA.

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei.

Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei.

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.

Chemical and enzymatic analysis of covalent bonds between peptides and chromosomal DNA.

DNA from Ehrlich ascites tumor (EAT) cells and from human placenta was examined for covalent bonds between hydroxy amino acid residues in peptides and nucleotide phosphate groups. The residual proteinaceous material in highly purified DNA was radiolabelled with 125Iodine and the linking-groups between peptides and nucleotides released by combined protease and nuclease treatment were investigated with respect to their chemical and enzymatic stabilities. The residual nucleotide(s)-peptide(s) fraction from DNA isolated after prolonged alkaline cell lysis and phenol extraction contains mainly alkali and acid-stable but phosphodiesterase-sensitive peptide-nucleotide complexes which indicates phosphodiesters between tyrosyl residues in peptides and nucleotide phosphates. In contrast, the linking-group fraction from DNA isolated under native conditions contains additional peptide components. (a) Phospho-peptides that co-purify with DNA but that are not covalently bound to nucleotides. (b) A fraction of peptides that is released from nucleotides by alkali in a time and concentration-dependent reaction. Evidence is presented indicating that the latter fraction involves phospho-triesters between hydroxy amino acid residues in peptides and internucleotide phosphates. The phosphodiesters between hydroxy amino acids and nucleotide phosphates representing the predominant class of peptide-nucleotide complexes in alkali-denatured DNA are most likely side products of peptide-nucleotide phospho-triester hydrolysis.

Synthesis and selected properties of oligonucleotidyl-(Pm----O)-amino acids.

N-Cbz-Ser(OMe)-(Pm----O)-d(TpT) was synthesized as a diastereomeric mixture of approx. (1:1) by reacting d(TpT) with 2,4,6-triisopropylbenzenesulfonyl chloride and N-Cbz-Ser-OMe. The phosphoesteric bond of N-Cbz-Ser (OMe)-(Pm----O)-d(TpT) was found to be stable in acid (1 N HCl, 1 h, 37 degrees C), but labile in alkaline solution (0.1-1 N NaOH, 1 h, 37 degrees C). The products of alkaline hydrolysis were determined to be d(TpT) and the amino acid derivatives. Furthermore, the phosphotriester N-Cbz-Ser(OMe)-(Pm----O)-d(TpT) was more labile than the diesteric analogue N-Cbz-Ser (OMe)-pdT. The internucleotide phosphotriester linkage of N-Cbz-Ser (OMe)-(Pm----O)-d(TpT) was also found to be resistant to enzymatic digestion with spleen and snake venom phosphodiesterases.

The synthesis and characteristics of adenylyl-(5'----N epsilon)-lysyl peptides.

The dipeptides Boc-Thr-Lys(Cbz)-OMe and Boc-Lys(Cbz)-Glu(OEt)-OEt were prepared by the classical method of peptide synthesis. The Cbz protecting group was subsequently removed via hydrogenolysis. AMP was then covalently joined to the free lysine epsilon-amine by the carbodiimide (DCC) method. The resulting RNA-ligase active center model compounds, the adenylyl-(5----N epsilon)-lysylpeptides were then used to study the participation of the amino acid residue functional groups in the hydrolysis of the phosphoamide center.