Highly potent photoinactivation of bacteria using a water soluble, cell permeable, DNA-binding photosensitizer

Antimicrobial photodynamic therapy (APDT) employs a photosensitizer, light, and molecular oxygen to treat infectious diseases via oxidative damage, with a low likelihood for the development of resistance. For optimal APDT efficacy, photosensitizers with cationic charges that can permeate bacteria cells and bind intracellular targets are desired to not limit oxidative damage to the outer bacterial structure. Here we report the application of brominated DAPI (BrDAPI), a water-soluble, DNA-binding photosensitizer for eradication of both gram negative and gram positive bacteria (as demonstrated on N99 E. coli and B. subtilis, respectively). We observe intracellular uptake of BrDAPI, ROS mediated bacterial cell death via 1 and 2 photon excitation, and selective photocytotoxicity of bacteria over mammalian cells. Photocytotoxicity of both N99 E. coli and B. subtilis occurred at sub-micromolar concentrations (IC50 = 0.2 to 0.4 micromolar) and low light doses (5 minute irradiation times, 4.5 J cm-2 dose) making it superior to commonly employed APDT phenothiazinium photosensitizers such as methylene blue. Given its high potency and 2 photon excitability, BrDAPI is a promising novel photosensitizer for in vivo APDT applications.

Download Full-text

Activity of Indenoisoquinolines against African Trypanosomes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00650-07 ◽

2008 ◽

Vol 53 (1) ◽

pp. 123-128 ◽

Cited By ~ 11

Author(s):

Rahul P. Bakshi ◽

Dongpei Sang ◽

Andrew Morrell ◽

Mark Cushman ◽

Theresa A. Shapiro

Keyword(s):

Anticancer Agents ◽

Mammalian Cells ◽

Sleeping Sickness ◽

Water Soluble ◽

African Trypanosomes ◽

In Vivo Experiments ◽

Topoisomerase Ib ◽

Topoisomerase Poisons

ABSTRACT African trypanosomiasis (sleeping sickness), caused by protozoan Trypanosoma brucei species, is a debilitating disease that is lethal if untreated. Available drugs are antiquated, toxic, and compromised by emerging resistance. The indenoisoquinolines are a class of noncamptothecin topoisomerase IB poisons that are under development as anticancer agents. We tested a variety of indenoisoquinolines for their ability to kill T. brucei. Indenoisoquinolines proved trypanocidal at submicromolar concentrations in vitro. Structure-activity analysis yielded motifs that enhanced potency, including alkylamino substitutions on N-6, methoxy groups on C-2 and C-3, and a methylenedioxy bridge between C-8 and C-9. Detailed analysis of eight water-soluble indenoisoquinolines demonstrated that in trypanosomes the compounds inhibited DNA synthesis and acted as topoisomerase poisons. Testing these compounds on L1210 mouse leukemia cells revealed that all eight were more effective against trypanosomes than against mammalian cells. In preliminary in vivo experiments one compound delayed parasitemia and extended survival in mice subjected to a lethal trypanosome challenge. The indenoisoquinolines provide a promising lead for the development of drugs against sleeping sickness.

Download Full-text

The activity of superoxide-dismutase in animal cell culture CHO-K1 after treatment with fullerenol and mytomicine C

Hemijska industrija ◽

10.2298/hemind0903143b ◽

2009 ◽

Vol 63 (3) ◽

pp. 143-149 ◽

Cited By ~ 1

Author(s):

Visnja Bogdanovic ◽

Marija Slavic ◽

Jasminka Mrdjanovic ◽

Slavica Solajic ◽

Aleksandar Djordjevic

Keyword(s):

Superoxide Dismutase ◽

Mammalian Cells ◽

Animal Cell ◽

Water Soluble ◽

Cytotoxic Agents ◽

Antioxidant Defenses ◽

Free Radical Scavengers ◽

Sod Activity

Eukaryotic cell survives in predominantly reduced conditions. Homeostasis of cellular redox system is an imperative of cell surviving and its normal metabolism. ROS are well recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. These species are mutagenic compounds known to lead to DNA damage, favor cell transformation, and contribute to the development of a variety of malignant diseases. All the effects of oxidants are influenced by the cellular antioxidant defenses. This multilayer system consists of low molecular weight components and several antioxidant enzymes. Superoxide dismutases (SODs) are the only enzymes dismuting superoxide radicals. Mitomycin C, a cross-linking agent, demonstrated genotoxicity in all in vitro and in vivo test systems in mammalian cells and animals. Water-soluble fullerenes are well known as cytotoxic agents for many cell lines in vitro. At the other side, fullerenols are good free radical scavengers and antioxidants both in vitro and in vivo. This paper investigates the effects of fullerenol on survival and fullerenol/ /mytomicine (MMC) treatment on superoxide-dismutase (SOD) activity in CHO-K1 cells. Samples were treated 3 and 24 h with fullerenol (C60(OH)24) at concentration range 0.01-0.5 mg/mL and survival was monitored with dye exclusion test (DET). The activity of total SOD was estimated in samples treated with chosen concentrations of fullerenol and MMC (0.5 and 0.1 mg/mL) after 3 and 24 h of cell incubation. Increasing of C60(OH)24 concentration leads to decreasing of percent of surviving cells 3 and 24 h after incubation. The activity of total SOD enhanced with higher concentration of fullerenol, while decreased in the highest concentration at both experimental points. In samples treated with MMC, as well as in samples treated with fullerenol (0.0625 mg/mL) + MMC was noticed boost in total SOD activity in comparison with controls. Treatment with fullerenol decreased SOD activity in rest of samples treated with MMC. Decreased activity of superoxide-dismutase in almost all samples treated with fullerenol and MMC might be contributed to antioxidative properties of fullerenol. Increased enzyme level at concentration of 0.0625 mg/mL may be due to its prooxidative activity.

Download Full-text

Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5309-5317.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5309-5317

Author(s):

S P Murphy ◽

J J Gorzowski ◽

K D Sarge ◽

B Phillips

Keyword(s):

Transcription Factors ◽

Heat Shock ◽

Dna Binding ◽

Mammalian Cells ◽

Embryonal Carcinoma ◽

Embryonic Stem ◽

Binding Activity ◽

Hsp70 Promoter ◽

Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

Download Full-text

CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

Journal of Biological Chemistry ◽

10.1074/jbc.m401854200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45887-45896 ◽

Cited By ~ 45

Author(s):

Mark J. Demma ◽

Serena Wong ◽

Eugene Maxwell ◽

Bimalendu Dasmahapatra

Keyword(s):

Dna Binding ◽

Mammalian Cells ◽

P53 Protein ◽

Binding Activity ◽

Mutant P53 ◽

Dependent Manner ◽

Wild Type ◽

Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

Download Full-text

Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.4.c1172 ◽

1996 ◽

Vol 271 (4) ◽

pp. C1172-C1180 ◽

Cited By ~ 786

Author(s):

B. H. Jiang ◽

G. L. Semenza ◽

C. Bauer ◽

H. H. Marti

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Functional Characterization ◽

Hypoxia Inducible Factor ◽

Binding Activity ◽

Maximal Response ◽

Hypoxia Inducible Factor 1 ◽

Dna Binding Activity

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein implicated in the transcriptional activation of genes encoding erythropoietin, glycolytic enzymes, and vascular endothelial growth factor in hypoxic mammalian cells. In this study, we have quantitated HIF-1 DNA-binding activity and protein levels of the HIF-1 alpha and HIF-1 beta subunits in human HeLa cells exposed to O2 concentrations ranging from 0 to 20% in the absence or presence of 1 mM KCN to inhibit oxidative phosphorylation and cellular O2 consumption. HIF-1 DNA-binding activity, HIF-1 alpha protein and HIF-1 beta protein each increased exponentially as cells were subjected to decreasing O2 concentrations, with a half maximal response between 1.5 and 2% O2 and a maximal response at 0.5% O2, both in the presence and absence of KCN. The HIF-1 response was greatest over O2 concentrations associated with ischemic/hypoxic events in vivo. These results provide evidence for the involvement of HIF-1 in O2 homeostasis and represent a functional characterization of the putative O2 sensor that initiates hypoxia signal transduction leading to HIF-1 expression.

Download Full-text

Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3576 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3576-3586 ◽

Cited By ~ 77

Author(s):

C H Yang ◽

J Tomkiel ◽

H Saitoh ◽

D H Johnson ◽

W C Earnshaw

Keyword(s):

Dna Binding ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Mammalian Cells ◽

In Vitro Assays ◽

Centromeric Heterochromatin ◽

Structural Protein ◽

Chromosomal Site

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

Download Full-text

orf4 of the Bacillus cereus sigB Gene Cluster Encodes a General Stress-Inducible Dps-Like Bacterioferritin

Journal of Bacteriology ◽

10.1128/jb.00272-09 ◽

2009 ◽

Vol 191 (14) ◽

pp. 4522-4533 ◽

Cited By ~ 8

Author(s):

Shin-Wei Wang ◽

Chien-Yen Chen ◽

Joseph T. Tseng ◽

Shih-Hsiung Liang ◽

Ssu-Ching Chen ◽

...

Keyword(s):

Dna Binding ◽

Oxidative Damage ◽

Environmental Stress ◽

Bacillus Cereus ◽

Size Exclusion ◽

Null Mutant ◽

Cell Protection ◽

Ferroxidase Activity

ABSTRACT The function of orf4 in the sigB cluster in Bacillus cereus ATCC 14579 remains to be explored. Amino-acid sequence analysis has revealed that Orf4 is homologous with bacterioferritins and Dps. In this study, we generated an orf4-null mutant and produced recombinant protein rOrf4 to establish the role of orf4. In vitro, the purified rOrf4 was found to exist in two distinct forms, a dimeric form and a polymer form, through size exclusion analysis. The latter form exhibited a unique filament structure, in contrast to the typical spherical tetracosamer structure of bacterioferritins; the former can be induced to form rOrf4 polymers immediately after the addition of FeCl2. Catalysis of the oxidation of ferrous irons by ferroxidase activity was detected with rOrf4, and the mineralized irons were subsequently sequestered only in the rOrf4 polymer. Moreover, rOrf4 exerted DNA-protective activity against oxidative damage via DNA binding in a nonspecific manner, as is seen with Dps. In vivo, deletion of orf4 had no effect on activation of the alternative sigma factor σB, and therefore, orf4 is not associated with σB regulation; however, orf4 can be significantly upregulated upon environmental stress but not H2O2 treatment. B. cereus strains with constitutive Orf4 expression exhibited a viability higher than that of the orf4-null mutant, under specific oxidative stress or heat shock. Taken together, these results suggest that Orf4 functions as a Dps-like bacterioferritin in response to environmental stress and can provide cell protection from oxidative damage through iron sequestration and DNA binding.

Download Full-text

Dual Regulation of c-Myc by p300 via Acetylation-Dependent Control of Myc Protein Turnover and Coactivation of Myc-Induced Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10220-10234.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10220-10234 ◽

Cited By ~ 113

Author(s):

Francesco Faiola ◽

Xiaohui Liu ◽

Szuying Lo ◽

Songqin Pan ◽

Kangling Zhang ◽

...

Keyword(s):

Dna Binding ◽

Cell Fate ◽

Mammalian Cells ◽

Dependent Manner ◽

Dual Roles ◽

Human Telomerase Reverse Transcriptase ◽

Lysine Residues ◽

Human Telomerase

ABSTRACT The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. Here, we report a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. We show that p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independently of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene, and p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. Our results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation.

Download Full-text

Basal phosphorylation of the PEST domain in the I(kappa)B(beta) regulates its functional interaction with the c-rel proto-oncogene product.

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.5974 ◽

1996 ◽

Vol 16 (11) ◽

pp. 5974-5984 ◽

Cited By ~ 58

Author(s):

Z L Chu ◽

T A McKinsey ◽

L Liu ◽

X Qi ◽

D W Ballard

Keyword(s):

Dna Binding ◽

Mammalian Cells ◽

Peptide Mapping ◽

Binding Activity ◽

Phosphoryl Group ◽

I Kappa B Alpha ◽

Dna Binding Activity ◽

Antigenic Properties

The product of the c-rel proto-oncogene (c-Rel) belongs to the NF-kappaB/Rel family of polypeptides and has been implicated in the transcriptional control of cell proliferation and immune function. In human T lymphocytes, c-Rel is sequestered in the cytoplasmic compartment by constitutively phosphorylated inhibitors, including I(kappa)B(alpha) and I(kappa)B(beta). Studies with bacterially expressed forms of these inhibitory proteins revealed that unphosphorylated I(kappa)B(alpha) but not I(kappa)B(beta) assembles with c-Rel and inhibits its DNA binding activity. Furthermore, latent I(kappa)B(beta)-c-Rel complexes derived from mammalian cells were sensitive to phosphatase treatment, whereas I(kappa)B(alpha)-c-Rel complexes were resistant. We have identified a constitutive protein kinase in unstimulated T cells that associates with and phosphorylates I(kappa)B(beta) in vitro. The substrate specificity, electrophoretic mobility, and antigenic properties of this I(kappa)B(beta)-associated kinase (BAK) suggest identity with casein kinase II (CKII), an enzyme known to mediate basal phosphorylation of I(kappa)B(alpha). Phosphorylation of recombinant I(kappa)B(beta) by either BAK or CKII restored the capacity of this inhibitor to antagonize the DNA binding activity of c-Rel. Peptide mapping and mutational analyses localized the bulk of the basal phosphorylation sites in I(kappa)B(beta) to the C-terminal PEST domain, which contains two potential acceptors for CKII-mediated phosphoryl group transfer (Ser-313 and Ser-315). Point mutations introduced into the full-length inhibitor at Ser-313 and Ser-315 led to a significant reduction in the phosphorylation of I(kappa)B(beta) and severely impaired its c-Rel inhibitory function in vivo. Taken together, these findings strongly suggest that basal phosphorylation of the PEST domain of I(kappa)B(beta) at consensus CKII sites is required for the efficient formation of latent I(kappa)B(beta)-c-Rel complexes.

Download Full-text

Plasmon-Enhanced Antibacterial Activity of Chiral Gold Nanoparticles and In Vivo Therapeutic Effect

Nanomaterials ◽

10.3390/nano11061621 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1621

Author(s):

Yuelong Xu ◽

Hongxia Wang ◽

Min Zhang ◽

Jianhao Zhang ◽

Wenjing Yan

Keyword(s):

Gold Nanoparticles ◽

Antibacterial Activity ◽

Intestinal Microflora ◽

Antibacterial Properties ◽

Au Nps ◽

E Coli ◽

Bacterial Cell Death ◽

Steric Configuration

d-cysteine (d-cys) has been demonstrated to possess an extraordinary antibacterial activity because of its unique steric configuration. However, inefficient antibacterial properties seriously hinder its wide applications. Here, cysteine-functionalized gold nanoparticles (d-/l-Au NPs) were prepared by loading d-/l-cysteine on the surface of gold nanoparticles for the effective inhibition of Escherichia coli (E. coli) in vitro and in vivo, and the effects on the intestinal microflora in mice were explored during the treatment of E. coli infection in the gut. We found that the antibacterial activity of d-/l-Au NPs was more than 2–3 times higher than pure d-cysteine, l-cysteine and Au NPs. Compared with l-Au NPs, d-Au NPs showed the stronger antibacterial activity, which was related to its unique steric configuration. Chiral Au NPs showed stronger destructive effects on cell membrane compared to other groups, which further leads to the leakage of the cytoplasm and bacterial cell death. The in vivo antibacterial experiment illustrated that d-Au NPs displayed impressive antibacterial activity in the treatment of E. coli-infected mice comparable to kanamycin, whereas they could not affect the balance of intestinal microflora. This work is of great significance in the development of an effective chiral antibacterial agent.

Download Full-text