Purification of protein AP-toxin from Arthrinium phaeospermum causing blight in Bambusa pervariabilis × Dendrocalamopisis grandis and its metabolic effects on four bamboo varieties.

Bambusa pervariabilis × Dendrocalamopisis grandis blight is caused by a toxin produced by the fungus Arthrinium phaeospermum. In this study, a toxin fraction (P1-2-2) with an estimated molecular mass of 31 kDa was purified from a culture filtrate of this fungus by ammonium sulfate precipitation, Sephadex G-50 gel chromatography, Q Sepharose Fast Flow anion exchange resin, and Sephadex G-75 chromatography. The N-terminal amino acid sequence (i.e., H(2)N-Gln-Val-Arg-Asp-Arg-Leu-Glu-Ser-Thr) determined by Edman degradation showed homology to known serine alkaline proteases. The purified protein was named AP-toxin. Effects of the purified protein toxin on total phenol, flavonoid, total nucleic acid, DNA, RNA, soluble protein, and soluble sugar content, as well as DNase and RNase activities and disease index, were analyzed in different bamboo varieties by the impregnation method. The toxin had a significant effect on each parameter tested. In addition, a significant correlation was observed among the metabolic index, treatment time, bamboo resistance, and disease index. These data suggest that AP-toxin plays an important role in mediating the phytotoxic activities of A. phaeospermum. This study also indicates that metabolic indices could reflect the resistance indices of hybrid bamboo to blight.

Cu2+-controlled hybridization of peptide nucleic acids.

We have prepared cyclic peptide nucleic acids (PNAs). These compounds do not bind complementary nucleic acids. One carboxylic ester group was introduced in the backbone of the cyclic PNAs. This group is cleaved in the presence of Cu(2+) or coordinatively unsaturated Cu(2+) complexes. The cleavage products are linear PNAs. In contrast to the cyclic PNAs, they are efficient nucleic acid binders. The rate of formation of the linear PNAs is proportional to the concentration of the cleaving agents. Therefore, one may apply highly sensitive methods of detection of linear PNAs for determination of Cu(2+) concentration. In particular, we have demonstrated that both fluorescent spectroscopy in combination with molecular beacons and MALDI-TOF mass spectrometry are suitable for the detection of Cu(2+). A range of related divalent metal ions and Eu(3+), Ln(3+), Pr(3+), Ce(3+), and Zr(4+) do not interfere with Cu(2+) detection.

Curcumin induces cell death without oligonucleosomal DNA fragmentation in quiescent and proliferating human CD8+ cells.

UNLABELLED: Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemotherapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective chemopreventive agent. METHODS: We treated human quiescent or proliferating CD8+ cells with 50 microM curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell viability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observation, Western blotting and flow cytometry. RESULTS: Curcumin decreased cell viability, activated caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease. However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes characteristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be particularly undesirable.

The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis.

The HNH motif was originally identified in the subfamily of HNH homing endonucleases, which initiate the process of the insertion of mobile genetic elements into specific sites. Several bacteria toxins, including colicin E7 (ColE7), also contain the 30 amino acid HNH motif in their nuclease domains. In this work, we found that the nuclease domain of ColE7 (nuclease-ColE7) purified from Escherichia coli contains a one-to-one stoichiometry of zinc ion and that this zinc-containing enzyme hydrolyzes DNA without externally added divalent metal ions. The apo-enzyme, in which the indigenous zinc ion was removed from nuclease-ColE7, had no DNase activity. Several divalent metal ions, including Ni2+, Mg2+, Co2+, Mn2+, Ca2+, Sr2+, Cu2+ and Zn2+, re-activated the DNase activity of the apo-enzyme to various degrees, however higher concentrations of zinc ion inhibited this DNase activity. Two charged residues located at positions close to the zinc-binding site were mutated to alanine. The single-site mutants, R538A and E542A, showed reduced DNase activity, whereas the double-point mutant, R538A + E542A, had no observable DNase activity. A gel retardation assay further demonstrated that the nuclease-ColE7 hydrolyzed DNA in the presence of zinc ions, but only bound to DNA in the absence of zinc ions. These results demonstrate that the zinc ion in the HNH motif of nuclease-ColE7 is not required for DNA binding, but is essential for DNA hydrolysis, suggesting that the zinc ion not only stabilizes the folding of the enzyme, but is also likely to be involved in DNA hydrolysis.

The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA-metabolizing proteins with toroidal structure.

Herpesvirus DNA packaging involves binding and cleavage of DNA containing the specific DNA-packaging motifs. Here we report a first characterization of the terminase subunits pUL56 and pUL89 of human cytomegalovirus (HCMV). Both gene products were shown to have comparable nuclease activities in vitro. Under limiting protein concentrations the nuclease activity is enhanced by interaction of pUL56 and pUL89. High amounts of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole partially inhibited the pUL89-associated nuclease activity. It was demonstrated that pUL56 is able to bind to nucleocapsids in vivo. Electron microscopy (EM) and image analysis of purified pUL56 revealed that the molecules occurred as a distinct ring-shaped structure with a pronounced cleft. EM analysis of purified pUL89 demonstrated that this protein is also a toroidal DNA-metabolizing protein. Upon interaction of pUL56 with linearized DNA, the DNA remains uncut while the cutting event itself is mediated by pUL89. Using biochemical assays in conjunction with EM pUL56 was shown to (i) bind to DNA and (ii) associate with the capsid. In contrast to this, EM analysis implied that pUL89 is required to effect DNA cleavage. The data provide the first insights into the terminase-dependent viral DNA-packaging mechanism of HCMV.

Uniformly modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets.

"Uniformly" modified phosphodiester or phosphorothioate oligonucleotides incorporating 2'-deoxy-2'-fluoroadenosine, -guanosine, -uridine, and -cytidine, reported herein for the first time, when hybridized with RNA afforded consistent additive enhancement of duplex stability without compromising base-pair specificity. CD spectra of the 2'-deoxy-2'-fluoro-modified oligonucleotides hybridized with RNA indicated that the duplex adopts a fully A-form conformation. The 2'-deoxy-2'-fluoro-modified oligonucleotides in phosphodiester form were not resistant to nucleases; however, the modified phosphorothioate oligonucleotides were highly nuclease resistant and retained exceptional binding affinity to the RNA targets. The stabilizing effects of the 2'-deoxy-2'-fluoro modifications on RNA-DNA duplexes were shown to be superior to those of the 2'-O-methylribo substitutions. RNA hybrid duplexes with uniformly 2'-deoxy-2'-fluoro-modified oligonucleotides did not support HeLa RNase H activity; however, incorporation of the modifications into "chimeric" oligonucleotides has been shown to activate mammalian RNase H. "Uniformly" modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides afforded antisense molecules with (1) high binding affinity and selectivity for the RNA target and (2) stability toward nucleases.

Inhibition of O6-alkylguanine-DNA alkyltransferase and DNase I activities in vitro by some alkylating substances and antineoplastic agents.

The specificities of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase from brain and liver cells of the chick embryo and of DNase I were demonstrated in vitro by their response to substrate DNA pretreated with monofunctional alkylating agents of different O6-guanine alkylating ability and some antineoplastic agents. Treatment of DNA with ethidium bromide, Hoechst 33258, doxorubicin, Fe2+/bleomycin, and suramin resulted in a dose-dependent diminution of alkyltransferase activity (DE50 approximately 5 micrograms/ml, 15 micrograms/ml, 5 micrograms/ml, 5 micrograms/ml, 100 micrograms/ml, respectively). Apart from bleomycin, comparable results were obtained with DNase I. Thermal denaturation of the substrate DNA reduced both alkyltransferase and DNase I activity. No effect was seen with X-irradiation. Cisplatin decreased only DNase I activity. Some topoisomerase II and/or gyrase inhibitors remained without significant effects on the alkyltransferase reaction whereas DNA catabolism by DNase I was diminished in a dose-dependent manner (DE50 between 6.5 and 19 micrograms/ml).

Physico-chemical parameters influencing DNase activity of the cyanobacterium Spirulina platensis.

The effects of temperature, Mg2+, EDTA concentration and rinsing on extra- and intra-cellular DNase activity of Spirulina platensis strain SSP-14, were investigated. The results indicate that the tested strain contains very high extra- and intracellular DNase activity, which actually hinders the transfer of foreign gene(s) to S. platensis, a cyanobacterium with multiple economic potentials. The extracellular DNase activity could easily be removed by rinsing the cells with Zarrouk medium more than once. The intracellular DNase activity could also be inhibited by (1) removal of Mg2+, (2) maintaining EDTA concentration above 1 mmol l(-1), and (3) manipulating below 0-4 degrees C, during all the incubation procedures. We suggest that, by using one or more of, or combining, all those experimental conditions, the chances of foreign DNA attempted to be introduced into S. platensis without being digested would be increased.

[The nuclease activity of the cell nuclei in 2 lines of murine myeloma sp2/0 differing in their resistance to cytostatics in adriamycin-induced apoptosis]. / Nukleaznaia aktivnost' iader kletok dvukh linii myshinoi mielomy sp2/0, razlichaiushchikhsia po ustoichivosti k tsitostatikam, pri indutsirovannom adriamitsinom apoptoze.

The endonuclease activity of two drug-sensitive and drug-resistant mouse myeloma cell lines during cytotoxic drug-induced apoptosis was studied. It was shown that internucleosomal fragmentation of DNA in drug-sensitive line sp2/0, undergoing apoptosis in the presence of adriamycin and colchicine, was not dependent on intracellular calcium content and was associated with activation of both Ca(2+)-Mg(2+)-dependent and acidic cation-independent endonucleases. In contrast, in multidrug resistant spEBR-5 cells, treated with the same drugs, only Ca(2+)-Mg(2+)-dependent endonuclease activity was detected. These data suggest that the differences in the pattern of endonuclease activity revealed in these cells are linked to drug-resistant phenotype and do not depend on the apoptosis-inducing agent used.

[The chronobiological aspects of lysosomal enzyme activity in the synovial fluid and of remission in rheumatoid inflammation]. / Khronobiologicheskie aspekty aktivnosti lizosomnykh fermentov sinovial'noi zhidkosti i remissii revmatoidnogo vospaleniia.

A study of activity of 4 lysosomal enzymes (LE) (deoxyribonuclease, acid phosphatase, acid cathepcines and beta-galactosidase) in synovial fluid (SF) depending on month of obtaining of the sample was conducted in 112 patients with rheumatoid arthritis (RA). A statistically significant chronobiological difference in this parameter was shown for all investigated enzymes with its increasing in spring and autumn and decreasing in summer and winter. The mean duration of the disease was 9.05 +/- 0.51 years. The dependence of duration of the effect after single intraarticular injection of glucocorticosteroids (methylprednisolone acetate, triamcinolone acetonide, the composition of the latest with cyclophosphamide and dymethylsulfoxide) upon the month of the puncture was investigated in 194 patients with RA. A statistically significant fluctuation (r < 0.05) of remission duration after single injection was shown only for triamcinolone acetonide +/- cyclophosphamide, but the analysis of graphic distribution gives an opportunity to imagine such a chronobiological law for other lysosomal enzymes. The identical distribution of extremes of LE activity and duration of effect after single intraarticular injection of glucocorticosteroids suggests the chronobiological link of these two parameters.

[The effect of the prolonged action of cytostatics and hormones on the properties of Escherichia coli]. / Vplyv tryvaloï diï tsytostatykiv i hormoniv na vlastyvosti Escherichia coli.

Cytostatics and hormones were studied for their effect on biological peculiarities of Escherichia coli--the main agent of inflammatory processes of kidneys and urinary tracts in children and adults. It was established that low doses of glucocorticoids, cytostatics and their combinations lead to changes in morphological properties in the strain of E. coli: M- and R-forms appeared in some variants. Besides, changes in a number of the biochemical peculiarities of M- and R-forms were found. These peculiarities are taken into account during identification of these microorganisms. The decrease of adhesion activity in almost all variants of E. coli 2417 was registered under long-term cultivation with cytostatics and prednisolone; hemolytic activity and ability to hydrolyze DNA were also established in certain variants.

Apoptotic DNA fragmentation is not related to the phosphorylation state of histone H1.

Changes in chromatin structure, histone phosphorylation and cleavage of DNA into nucleosome-size fragments are characteristic features of apoptosis. Since H1 histones bind to the site of DNA cleavage between nucleosomal cores, the question arises as to whether the state of H1 phosphorylation influences the rate of internucleosomal cleavage. Here, we tested the relation between DNA fragmentation and H1 phosphorylation both in cultured cells and in vitro. In Jurkat cells, hyperosmotic mannitol concentration resulted in apoptosis, including nucleosomal fragmentation, whereas apoptosis induction by increased NaCl concentration was not accompanied by DNA fragmentation. However, both treatments induced dephosphorylation of H1 histones. In contrast, treatment of Raji cells with alkylphosphocholine led to induction of apoptosis with internucleosomal fragmentation, albeit without notable histone H1 dephosphorylation. These results demonstrate that dephosphorylation of H1 histones is neither a prerequisite for nor a consequence of internucleosomal cleavage. Moreover, we observed with an in vitro assay that the known enhancing effect of H1 histones on the activity of the apoptosis-induced endonuclease DFF40 is independent of the subtype or the phosphorylation state of the linker histone.

Photosensitizer in a molecular bowl: steric protection enhancing the photonuclease activity of copper(II) scorpionates.

Steric encumbrance caused by the tripodal ligand in the ternary tris(3-phenylpyrazolyl)borate copper(II) heterocyclic base complexes [Cu(B)(Tp(Ph))](ClO(4)) (B = dipyridoquinoxaline, dipyridophenazine) leads to efficient cleavage of supercoiled DNA to its relaxed form upon exposure to red light at 632.8 and 694 nm as a result of protection of the photosensitizer in the molecular bowl of the {Cu(Tp(Ph))} moiety, which generates singlet oxygen as the reactive species in a type-II process.

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF.

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptotic effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.

DMI-1, a new DNA methyltransferase inhibitor produced by Streptomyces sp. strain No. 560.

A new inhibitor of DNA methyltransferase named DMI-1 has been discovered in the culture filtrate of Streptomyces sp. strain No. 560. DMI-1 was purified by extraction with ethyl acetate followed by Diaion HP-20SS and silica gel column chromatography. The structure of DMI-1 was determined to be 8-methylpentadecanoic acid (C16H32O2). DMI-1 is a novel inhibitor of methyltransferase isolated from microorganisms and is structurally different from sinefungin and A9145C which are structural analogs of S-adenosylmethionine (methyl donor). DMI-1 was a strong inhibitor of N6-methyladenine-DNA methyltransferase (M. Eco RI, EC 2.1.1.72) in a noncompetitive manner and its inhibition depended on the pH and temperature in the assay media.

Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates.

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.

Time-course of programmed cell death during leaf senescence in Eucommia ulmoides.

Leaves of Eucommia ulmoidesOliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca(2+)-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves.

[Effects of pesticide fluometuron (cotoran) on template synthesis of RNA]. / Vliianie pestitsida fluometurona (kotorana) na matrichnyi sintez RNK.

The experiments on the investigation of pesticide fluometuron (cotoran) influence on nuclease sensitivity and template activity of rat liver chromatin were carried out. Cotoran was found to bind specifically with non-histone proteins of chromatin. It was shown that this pesticide considerably decreases template activity of chromatin and its sensitivity to the action of nucleases. It suggests, that certain conformation changes occur in chromatin upon the action of cotoran.

[The stability of the alkylating derivatives of oligodeoxyribonucleotides containing a cholesterol or phenazine radical added to the 3'-termination during their interaction with Acholeplasma laidlawii PG-8]. / Issledovanie stabil'nosti alkiliruiushchikh proizvodnykh oligodezoksiribonukleotidov, soderzhashchikh prisoedinennyi k 3'-kontsu ostatok kholesterina ili fenaziniia, pri vzaimodeistvii ikh s kletkami Acholeplasma laidlawii PG-8.

Stability of alkylating derivatives of decathymidylates protected on the 3'-terminal by cholesterol and phenazine residues has been studied in the process of their interaction with cells of Acholeplasma laidlawii PG-8. It is shown that the studied reagents are not split by nucleases of A. laidlawii PG-8 for the time necessary for alkylation of mycoplasma biopolymers.

Determination of some nuclear deoxyribonucleases in X-irradiated rat thymocytes.

The spectrum of nuclear nucleases in control and irradiated (4 Gy) thymocytes has been investigated. Using the method of SDS electrophoresis of nuclear proteins in 3H-DNA-polyacrylamide gels a number of polypeptides of MW. 35, 32, 17.7, 17.2 and 16.4 kDa possessing nuclease activity were found. The 35 kDa enzyme is only active in the presence of Ca2+ and Mg2+ ions. In response to cycloheximide injection (3 mg/100 g body weight) and irradiation, we did not detect the 35 kDa nuclease activity. Nucleases of 32, 17.7, 17.2 and 16.4 kDa are active in the presence of Ca2+ ions. The activities of these nucleases increases 60 min after irradiation. These nucleases were also found in the fraction of polydeoxyribonucleotide (PDN).