Di-mannosylation enhances the adjuvant properties of adamantane-containing desmuramyl peptides in vivo.

Muramyl dipeptide (MDP) is the smallest essential peptidoglycan substructure capable of promoting both innate and adaptive immune responses. Herein, we report on the design, synthesis, and in vivo study of the adjuvant properties of two novel MDP analogs containing an achiral adamantyl moiety attached to the desmuramyl dipeptide (DMP) pharmacophore and additionally modified by one mannosyl subunit (derivative 7) or two mannosyl subunits (derivative 11). Mannose substructures were introduced in order to assess how the degree of mannosylation affects the immune response and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) binding affinity, compared to the reference compound ManAdDMP. Both mannosylated MDP analogs showed improved immunomodulating properties, while the di-mannosylated derivative 11 displayed the highest, statistically significant increase in anti-OVA IgG production. In this study, for the first time, the di-mannosylated DMP derivative was synthesized and immunologically evaluated. Derivative 11 stimulates a Th-2-polarized type of immune reaction, similar to the reference compound ManAdDMP and MDP. Molecular dynamics (MD) simulations demonstrate that 11 has a higher NOD2 binding affinity than 7, indicating that introducing the second mannose significantly contributes to the binding affinity. Mannose interacts with key amino acid residues from the LRR hydrophobic pocket of the NOD2 receptor and loop 2.

Synthesis and Immunological Evaluation of Mannosylated Desmuramyl Dipeptides Modified by Lipophilic Triazole Substituents.

Muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP) is the smallest peptidoglycan fragment able to trigger an immune response by activating the NOD2 receptor. Structural modification of MDP can lead to analogues with improved immunostimulating properties. The aim of this work was to prepare mannosylated desmuramyl peptides (ManDMP) containing lipophilic triazole substituents to study their immunomodulating activities in vivo. The adjuvant activity of the prepared compounds was evaluated in the mouse model using ovalbumin as an antigen and compared to the MDP and referent adjuvant ManDMPTAd. The obtained results confirm that the α-position of D-isoGln is the best position for the attachment of lipophilic substituents, especially adamantylethyl triazole. Compound 6c exhibited the strongest adjuvant activity, comparable to the MDP and better than referent ManDMPTAd.

Regulation of ssb Gene Expression in Escherichia coli

Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential DNA processes such as replication, transcription, and repair. The prototypic Escherichia coli SSB protein is encoded by an ssb gene. Although the ssb gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether ssb gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the ssb gene expression kinetics in UV- and γ-irradiated E. coli and revealed that ssb gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the sulA gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the ssb gene was overexpressed in the absence of DNA damage. Furthermore, we measured ssb gene expression by droplet digital PCR during unaffected bacterial growth and revealed that ssb gene expression was equal in wild-type and SOS- bacteria, whereas sulA expression was higher in the former. This study thus reveals a complex pattern of ssb gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The E. coli ssb gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.

The role of regulatory T lymphocytes in immune control of MC-2 fibrosarcoma.

The role of T regulatory lymphocytes (Treg) particularly in cancer is well known. The goal of the present study was to determine the contribution of these lymphocytes in the regulation of anti-tumor immunity of CBA/HZgr mice against MC-2 fibrosarcoma (4th generation of methylcholanthrene induced tumor). The levels of T lymphocytes (CD4+, CD8+ and CD4+CD25+) were determined 8 and 20 days after tumor transplantation. Further, the role of CD4+CD25+ (Tregs) in tumor-host interaction was evaluated in vitro and in vivo by using specific monoclonal antibodies. We found that splenocytes of both control and Treg depleted tumor bearing mice strongly but differently inhibited growth of tumor cells in vitro. While splenocytes of untreated mice exhibited significant decrease of this activity (from 74.4% to 62.6% and 32.95%), the splenocytes of Treg depleted mice showed increase of this activity (from 79.5% to 84.3% and 86.2%) from day 6 to day 13 and day 21 after tumor grafting, respectively. Further, upon i.v. injecting specific monoclonal anti-Treg antibody tumor immediately prior to tumor cell intracutaneous transplantation, the tumor was rejected after initial growth. In treated mice, the incidence of Treg cells was very low initially, reaching normal values two weeks later. These animals were shown to be resistant to tumor transplantation four months later.

In vitro and in vivo antitumour effects of phenylboronic acid against mouse mammary adenocarcinoma 4T1 and squamous carcinoma SCCVII cells.

The cytotoxic activity of phenylboroxine acid was evaluated in vitro on mouse mammary adenocarcinoma 4T1, mouse squamous cell carcinoma SCCVII, hamster lung fibroblast V79 and mouse dermal fibroblasts L929 cell lines. The cytotoxic effects were dose dependent for all tested tumour and non-tumour cell lines. Under in vivo conditions, three application routes of phenylboronic acid were studied: intra-peritoneal (i.p.), intra-tumour (i.t.) and per-oral. After tumour transplantation in syngeneic mice, phenylboronic acid was shown to slow the growth of both tumour cell lines (4T1 and SCCVII) compared with the control. The inhibitory effects were pronounced during the application of phenylboronic acid. For both tested tumour cell lines, the most prominent antitumour effect was obtained by intraperitoneal administration, followed significantly by oral administration.

Phytochemical and Bioactive Potential of in vivo and in vitro Grown Plants of Centaurea ragusina L. - Detection of DNA/RNA Active Compounds in Plant Extracts via Thermal Denaturation and Circular Dichroism.

INTRODUCTION: The phytochemical composition and biological activity of non-volatile components of Centaurea ragusina L. has not been studied previously. OBJECTIVES: Our aim was to evaluate the phytochemical and bioactive potential (including interactions with polynucleotides) of C. ragusina L. depending on the origin of plant material (in vivo - leaves from natural habitats, ex vitro - leaves from plants acclimated from culture media, in vitro - leaves and calli from plants grown in culture media) and polarity of solvents used in extract preparation (80 and 96% ethanol and water combinations or single solvents). METHODOLOGY: The polyphenol composition was determined by spectrophotometric and HPLC analysis. Biological activity of extracts was evaluated by following methods: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods for antioxidative activity, 2,3,5-triphenyl tetrazolium chloride (TTC) microdilution method for antibacterial activity, crystal-violet test for cytotoxic activity and thermal denaturation (TD) and circular dichroism (CD) for DNA/RNA interactions. RESULTS: Conditions for the most efficient polyphenol extraction were determined: the 80% ethanol/water solvent system was the most suitable for callus and leaf ex vitro samples and 80 or 96% ethanol for leaf in vivo samples. Significantly higher levels of chlorogenic acid and naringenin were detected in callus tissue than in vivo plant. Ethanolic extracts exhibited the significant antibacterial activity against Staphylococcus aureus ATCC 25923. DNA/RNA active compounds in plant extracts were detected by TD and CD methods. CONCLUSIONS: Callus tissue and ex vitro leaves represent a valuable source of polyphenols as in vivo leaves. TD and CD can be applied for detection of DNA/RNA active compounds in extracts from natural resources. Copyright © 2017 John Wiley & Sons, Ltd.

Impact of calcium ion on cytotoxic effect of the boroxine derivative, K2[B3O3F4OH].

The effect of Ca2+ ions on the cytotoxic ability of boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]), on in vitro tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) was examined. At small concentrations of Ca2+ ions (0.42 mM), K2[B3O3F4OH] (3.85 mM) has a very strong cytotoxic effect on all cancer cells tested (89.1, 85.6 and 84.6%) and significantly less effect on normal cells (19.5 and 24.2%), respectively. Applying larger concentrations of Ca2+ ions (9.42-72.42 mM), at the same concentration of K2[B3O3F4OH], no significant cytotoxic effect was detected on cancer cells and normal cells investigated. The selective ability of K2[B3O3F4OH], in the medium with a low concentration of Ca2+ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

In vitro and in vivo antitumor activity of the halogenated boroxine dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]).

Dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was listed as a promising new therapeutic for cancer diseases. For in vitro and in vivo investigation of its antitumor effects 4T1 mammary adenocarcinoma, B16F10 melanoma and squamous cell carcinoma SCCVII were used. The detailed in vitro investigation undoubtedly showed that K2[B3O3F4OH] affects the growth of cancer cells. The proliferation of cells depends on the concentration so that aqueous solution of K2[B3O3F4OH], the concentrations of 10(-4) M and less, does not affect cell growth, but the concentrations of 10(-3) M or more, significantly slows cells growth. B16F10 and SCCVII cells show higher sensitivity to the cytotoxic effects of K2[B3O3F4OH] compared to 4T1 cells. Under in vivo conditions, K2[B3O3F4OH] slows the growth of all three tumors tested compared to the control, and the inhibitory effect was most pronounced during the application of the substance. There is almost no difference if K2[B3O3F4OH] was applied intraperitoneally, intratumor, peroral or as ointment. Addition of 5-FU did not further increase the antitumor efficacy of K2[B3O3F4OH].

Single-strand DNA-binding protein suppresses illegitimate recombination in Escherichia coli, acting in synergy with RecQ helicase.

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi- assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.

In vitro antifungal effect of phenylboronic and boric acid on Alternaria alternata.

The ascomycete fungus Alternaria alternata causes early blight, one of economically the most important tomato diseases. Due to frequent use of fungicides, A. alternata has developed resistance with negative economic and environmental consequences. Research of new ways to control fungal pathogens has turned its eye to environmentally friendly chemicals with low toxicity such as boronic acids. The aim of our study was therefore to test the antifungal effects of phenylboronic and boric acid in vitro on A. alternata. We isolated the pathogen from a symptomatic tomato plant and determined the minimum inhibitory concentration of phenylboronic and boric acid on A. alternata mycelial growth using the poisoned food technique. The antifungal effect was tested on a wide range of phenylboronic and boric acid concentrations (from 0.04 % to 0.3 %) applied separately to agar with mycelial disc of the pathogen. After five days of incubation, phenylboronic acid at low concentration (0.05 %) completely inhibited mycelial growth. Boric acid, in turn, did not significantly slow down mycelial growth but did reduce sporulation and confirmed its fungistatic effect. Our findings point to the potential use of phenylboronic acid to control phytopathogenic fungi. This is, to our knowledge, the first report on its antifungal effect on an agriculturally important pathogen in vitro. Moreover, since A. alternata is also a human pathogen, these results may have clinical ramifications.

Phenylboronic acid as a novel agent for controlling plant pathogenic bacteria.

BACKGROUND: Phenylboronic acid (PBA) is an environmentally non-toxic substance with antimicrobial activity. Due to increasing ecological limitations in phytopharmacy and considering the development of resistance of phytopathogenic bacteria to available antibacterial agents, here we explore a possible role of PBA as an antibacterial agent of choice. RESULTS: We determined a minimal inhibitory concentration (MIC) of PBA in vitro on the Pseudomonas syringae pv. tomato (Pst) (0.5 mg/mL) and Erwinia amylovora (0.8 mg/mL), two of the most damaging plant pathogenic bacteria. In comparison, boric acid MIC was 2.5-6-fold higher than that of PBA, indicating enhanced antibacterial efficacy of the latter. Moreover, we determined the effect of PBA on cell growth and viability of both bacteria and have shown that PBA has bactericidal effect in concentrations > 1.0 mg/mL, whereas in lower concentration it is bacteriostatic. In addition, we have shown that PBA impairs Pst ability to cause symptoms on tomato plants in a dose-dependent manner, whereas solely applied PBA did not affect plant morphology at bactericidal concentrations. CONCLUSION: We report, for the first time, that PBA is a suitable agent for controlling phytopathogenic bacteria. PBA has bacteriostatic activity in lower, and bactericidal activity in higher (> 1.0 mg/mL) concentrations. When applied on tomato plants, PBA managed to suppress symptoms caused by Pst, while having no adverse effect on plants at the bactericidal concentrations. As an additional benefit, PBA is environmentally friendly. © 2022 Society of Chemical Industry.

Control of Early Blight Fungus (Alternaria alternata) in Tomato by Boric and Phenylboronic Acid.

Finding a suitable alternative to the small pool of existing antifungal agents is a vital task in contemporary agriculture. Therefore, intensive research has been conducted globally to uncover environmentally friendly and efficient agents that can suppress pathogens resistant to the currently used antimycotics. Here, we tested the activity of boric acid (BA) and its derivative phenylboronic acid (PBA) in controlling the early blight symptoms in tomato plants infected with pathogenic fungus Alternaria alternata. By following the appearance and intensity of the lesions on leaves of the tested plants, as well as by measuring four selected physiological factors that reflect plant health, we have shown that both BA and PBA act prophylactically on fungal infection. They did it by reducing the amount and severity of early blight symptoms, as well as by preventing deterioration of the physiological traits, occurring upon fungal inoculation. Phenylboronic acid was more efficient in suppressing the impact of A. alternata infection. Therefore, we conclude that BA, and even more so PBA, may be used as agents for controlling early blight on tomato plants, as they are both quite effective and environmentally friendly.

The characterization of blood flow changes in mouse tumor during Photofrin-based photodynamic therapy by using the color Doppler ultrasonography.

Changes in blood flow velocity through the tumors can induce damage of tumor microcirculation and thus may contribute to the final destruction of tumor masses after photodynamic therapy (PDT). The aim of this study was to evaluate the blood flow changes in a SCCVII mouse carcinoma during Photofrin-based photodynamic therapy by analyzing several quantitative spectral Doppler parameters [maximum systolic flow velocity (Vmax), end diastolic velocity (Vmin), resistance index (RI) and pulsatile index (PI)] by using the color Doppler ultrasonography. Blood flow velocities were recorded immediately prior to tumor illumination (0 h) and then 2 and 24 h after the illumination. Statistically significant increase in diastolic blood velocity (Vmin) with a corresponding decline in RI and PI was recorded in tumors of the Photofrin-injected mice prior to tumor illumination. However, 2 h after the illumination a pronounced decrease in both Vmin and Vmax was obtained. There were no changes of these parameters in controls at different times during determination. The observed changes of spectral Doppler parameters in tumors from the PDT group point to the transition of tumor blood vessels from the relaxation state recorded before tumor illumination into a state of increased contraction after the activation of Photofrin by light. Pronounced changes in tumor blood vessel tone might be an additional stress for such vessels leading to their ultimate destruction.

Biological properties of 4-methyl-2,7-diamino-5,10-diphenyl-4,9-diazapyrenium hydrogensulfate (ADAP).

OBJECTIVE: 4-Methyl-2,7-diamino-5,10-diphenyl-4,9-diazapyrenium hydrogensulfate (ADAP) is a potential antitumor compound because of its DNA and RNA intercalating ability. In this study, cellular uptake, intracellular distribution as well as mechanism of action, antitumor activity in vitro and toxicity in vivo of ADAP were investigated. METHODS: Based on the fluorescence properties of ADAP, its entry and distribution into live cells were analyzed by fluorescence microscopy. The in vitro antiproliferative activity was determined using MTT test. For screening of topoisomerase II-targeted effects of ADAP, the cell-free assay and immunoband depletion assay were used. Expression of the genes c-mos, c-N-ras, c-Ki-ras, c-H-ras, p53 and caspase 3 in Caco-2 cells treated with ADAP was examined by RT-PCR. Toxicity in vivo was determined using C3HHf/Bu Zgr/Hr mice treated by single or multiple doses of ADAP at a concentration of 25 mg/kg. RESULTS: ADAP in microM concentrations entered into MIAPaCa-2 cell's cytoplasm in 5 min and into nuclei in 60 min after administration. Intracellular distribution of ADAP depended on the period of treatment time. ADAP (0.1-100 microM) strongly inhibited the growth of both mouse (FsaR, SCCVII) and human tumor cells (HeLa, Caco-2, HT-29, MIAPaCa-2, HBL, HEp-2, SW620, MCF-7) compared to its weak cytotoxicity on controls and normal cells (WI38). Results of both topoisomerase II assays showed that ADAP is not a topoisomerase II poison. Expression of investigated genes was dependent on the incubation time, except for p53 and c-H-ras. Morphological changes in tissues and organs of mice were not observed. Results of patohistological analysis have been confirmed by hematological and clinical-chemical analysis of blood of treated and non-treated animals. CONCLUSION: ADAP is a strongly bioactive compound with antitumor potential in vitro. The antitumor potential in vivo remains to be identified.

RecF, UvrD, RecX and RecN proteins suppress DNA degradation at DNA double-strand breaks in Escherichia coli.

Double strand breaks (DSBs) in E. coli chromosome (such as those induced by gamma rays) are repaired by recombination repair, during which a certain amount of DNA gets degraded. We monitored DNA degradation in gamma-irradiated cells to assess processing of DSBs. DNA degradation in irradiated cells is regulated by RecA protein concentration and its affinity of ssDNA binding, as well as by exonucleases that trim 3'-terminated ss tails. Here we determined the effects of proteins that affect formation and stability of RecA nucleofilaments on DNA degradation and cell survival. RecF and UvrD suppressed DNA degradation through RecA protein function and SOS induction, while also improving gamma survival. RecF and UvrD function in one pathway. Acting along with RecF, RecX suppressed DNA degradation and stimulated gamma-survival, which also depends on RecA protein and SOS induction. Furthermore, we determined a role in DNA degradation of several proteins that participate in DSB repair. RecN was required for DNA repair and for degradation suppression, acting on the RecABCD pathway. Furthermore, we show that SSB protein overproduction did not affect DNA degradation. Inactivation of RecG and RuvABC, proteins that catalyze the postsynaptic phase of recombination repair of DSBs, also did not affect DNA degradation, suggesting that once formed, recombination intermediates are not subject to DNA degradation, and that the postsynaptic phase is an irreversible, single-round process, unlike the presynaptic phase, which is mostly repetitive.

Biological Activity of Flavonoids and Rare Sesquiterpene Lactones Isolated From Centaurea ragusina L.

The endemic Croatian species Centaurea ragusina L., like other species from the genus Centaurea, has been traditionally used in Croatia as an antibacterial agent and for the treatment of gastrointestinal and urogenital disorders. In several chromatographic steps, three flavonoids and three sesquiterpene lactones (STLs) were isolated and identified from the most active fractions of the ethanol extract. Two STLs, one for which we created the trivial name ragusinin, and hemistepsin A are here reported for the first time as constituents of the genus Centaurea. All six compounds were screened for their effect on several tumor and one normal cell lines. Among them, ragusinin showed the best bioactivity and high specificity to affect tumor murine SCCVII, human HeLa and Caco-2 cell lines, but not the viability of normal V79 fibroblasts. Due to these characteristics the action of ragusinin was investigated in more detail. Since DNA is the primary target for many drugs with antibacterial and anticancer activity, we studied its interaction with ragusinin. Rather moderate binding affinity to DNA excluded it as the primary target of ragusinin. Due to the possibility of STL interaction with glutathione (GSH), the ubiquitous peptide that traps reactive compounds and other xenobiotics to prevent damage to vital proteins and nucleic acids, its role in deactivation of ragusinin was evaluated. Addition of the GSH precursor N-acetyl-cysteine potentiated the viability of HeLa cells, while the addition of GSH inhibitor L-buthionine sulfoximine decreased it. Moreover, pre-treatment of HeLa cells with the inhibitor of glutathione-S-transferase decreased their viability indicating the detoxifying role of GSH in ragusinin treated cells. Cell death, derived by an accumulation of cells in a G2 phase of the cell cylce, was shown to be independent of poly (ADP-ribose) polymerase and caspase-3 cleavage pointing toward an alternative cell death pathway.

Nucleolytic degradation of 3'-ending overhangs is essential for DNA-end resection in RecA-loading deficient recB mutants of Escherichia coli.

Degradation of a 5'-ending strand is the hallmark of the universal process of DNA double strand break (DSB) resection, which results in creation of the central recombination intermediate, a 3'-ending overhang. Here we show that in Escherichia coli recB1080/recB1067 mutants, which are devoid of RecBCD's nuclease and RecA loading activities, degradation of the unwound 3' tail is as essential as is degradation of its 5'-ending complement. Namely, a synergistic action of ExoI, ExoVII, SbcCD and ExoX single-strand specific exonucleases (ssExos) of 3'-5' polarity was essential for preserving cell viability, DNA repair and homologous recombination in the recB1080/recB1067 mutants, to the same extent as the redundant action of 5'-tail trimming ssExos RecJ and ExoVII. recB1080 derivatives lacking 3'-5' ssExos also showed a strong induction of the SOS response and greatly increased SOS-dependent mutagenesis. Furthermore, we show that ExoI and ExoVII ssExos act synergistically in suppressing illegitimate recombination in the recB1080 mutant but not in a wt strain, while working in concert with the RecQ helicase. Remarkably, 3'-5' ssExos show synergism with RecQ helicase in the recB1080 mutant in all the assays tested. The effect of inactivation of 3'-5' ssExos in the recB1080/recB1067 mutants was much stronger than in wt, recD, and recB strains. These results demonstrate that the presence of a long, reactive 3' overhang can be as toxic for a cell as its complete absence, i.e. it may prevent DSB repair. Our results indicate that coupling of helicase and RecA-loading activity during dsDNA-end resection is crucial in avoiding the deleterious effects of a long and stabile 3' tail in E. coli.

3'-Terminated Overhangs Regulate DNA Double-Strand Break Processing in Escherichia coli.

Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in Escherichia coli To study DSB processing in vivo, we induced DSBs into the E. coli chromosome by γ-irradiation and measured chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with single-stranded DNA (ssDNA). RecA decreased DNA degradation in wild-type, recB, and recD strains, indicating that it is a general phenomenon in E. coli On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos). 3'-5' ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding the 3' tail from degradation by 3'-5' ssExos. Since 3' tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3' tails emanating from DSBs, along with 3' tail's massive length, or prevention of their degradation by inactivation of 3'-5' ssExos. Thus, we conclude that 3' overhangs are crucial in controlling the extent of DSB processing in E. coli This study suggests a regulatory mechanism for DSB processing in E. coli, wherein 3' tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends.

Cytotoxicity of nanosize V(2)O(5) particles to selected fibroblast and tumor cells.

Two kinds of nanosize V(2)O(5) particles were synthesized in our own laboratory and concomitantly applied to V79 and L929 fibroblasts and SCCVII, B16F10 and FsaR tumor cells. The morphologies of the cells were monitored using an inverted inverse microscope equipped with digital camera, while quantitative determination of the cytotoxicity of nanosize V(2)O(5) particles was measured using crystal violet bioassay. Twenty four hours after the addition of nanosize V(2)O(5) particles (20muM), noticeable changes in the morphology and density of fibroblast and cancer cells were observed. Reculturing in a freshly prepared medium for the next 24h showed a high recovery effect on V79, SCCVII and B16F10 cells, while FsaR and L929 cells were seriously damaged and unable to recover. At a higher concentration of nanosize V(2)O(5) particles (100muM), the cytotoxicity of V(2)O(5) prevailed against the recovery effect in all cell types. Quantitative measurements have shown that the resistance of investigated cell cultures to the cytotoxicity of nanosize V(2)O(5) particles decreases in the order V79>SCCVII>B16F10>FsaR>L929. The high cytotoxic effect found on FsaR cells suggests that nanosize V(2)O(5) particles could be regarded as poisoning material in the treatment of FsaR fibrosarcoma cells. Possible mechanisms involved in the cytotoxicity of nanosize V(2)O(5) particles were discussed.

Combined action of virus injection and local tumor irradiation on tumor growth in mice.

The dynamics of SCCVII transplantable tumor growth in C3H/H mice was determined after local tumor irradiation and/or virus (NDV LaSota) i.p. injection. The virus applied alone significantly suppressed tumor growth, particularly until the 19th day after tumor transplantation. Local irradiation with 30 Gy resulted in tumor disappearance followed with its regrowth about 15 days later. However, if the virus was injected after the irradiation, there was no tumor growth until the end of the 31 day observation period. It should be noted that virus application prior to local irradiation did not have any additional influence on tumor growth. Thus, the pronounced efficacy of virus applied after tumor irradiation deserves attention. It is possible that the virus injected after irradiation induced a chain of cytokine production joining the action of tumor destruction induced by irradiation. This should be further studied in clarifying the approaches to combined tumor therapy with possible cell-free vaccine production.