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1.
Eur Biophys J ; 52(6-7): 545-557, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37507591

ABSTRACT

8-azaguanine is a triazolopyrimidine nucleobase analog possessing potent antibacterial and antitumor activities, and it has been implicated as a lead molecule in cancer and malaria therapy. Its intrinsic fluorescence properties can be utilized for monitoring its interactions with biological polymers like proteins or nucleic acids. In order to better understand these interactions, it is important to know the tautomeric equilibrium of this compound. In this work, the tautomeric equilibrium of all natural neutral and anionic compound forms (except highly improbable imino-enol tautomers) as well as their methyl derivatives and ribosides was revealed by quantum chemistry methods. It was shown that, as expected, tautomers protonated at positions 1 and 9 dominate neutral forms both in gas phase and in aqueous solution. 8-azaguanines methylated at any position of the triazole ring are protonated at position 1. The computed vertical absorption and emission energies are in very good agreement with the experimental data. They confirm the validity of the assumption that replacing the proton with the methyl group does not significantly change the positions of absorption and fluorescence peaks.


Subject(s)
Azaguanine , Protons , Spectrum Analysis , Proteins/chemistry , Quantum Theory
2.
Life Sci ; 288: 120182, 2022 Jan 01.
Article in English | MEDLINE | ID: mdl-34843735

ABSTRACT

BACKGROUND: Sjögren's syndrome (SjS) is an autoimmune disease with a strong genetic association. To date, no vaccine or therapeutic agent exists to cure SjS, and patients must rely on lifelong therapies to treat symptoms. Human leukocyte antigens (HLA) are primary susceptibility loci that form the genetic basis for many autoimmune diseases, including SjS. In this study, we sought to determine whether blocking MHC class II IAg7 antigen presentation in the NOD mouse would alleviate SjS by preventing the recognition of autoantigens by pathogenic T cells. METHODS: Mapping of the antigenic epitopes of Ro60 autoantigen to IAg7 of the NOD mice was performed using structural modeling and in-vitro stimulation. Tetraazatricyclo-dodecane (TATD) and 8-Azaguanine (8-Aza) were previously identified as potential binders to IAg7 of the NOD mice using in silico drug screening. Mice were treated with 20mgs/kg via IP every day five days/week for 23 weeks. Disease profiling was conducted. FINDINGS: Specific peptides of Ro60 autoantigen were identified to bind to IAg7 and stimulated splenocytes of the NOD mice. Treating NOD mice with TATD or 8-Azaguanine alleviated SjS symptoms by improving salivary and lacrimal gland secretory function, decreasing the levels of autoantibodies, and reducing the severity of lymphocytic infiltration in the salivary and lacrimal glands. INTERPRETATION: This study presents a novel therapeutic approach for SjS by identifying small molecules capable of inhibiting T cell response via antigen-specific presentation. FUNDING: CQN is supported financially in part by PHS grants AI130561, DE026450, and DE028544 from the National Institutes of Health.


Subject(s)
Alkanes/chemistry , Antigen Presentation/immunology , Azaguanine/pharmacology , Histocompatibility Antigens Class II/chemistry , Polycyclic Compounds/pharmacology , Sjogren's Syndrome/drug therapy , Animals , Antimetabolites, Antineoplastic/pharmacology , Female , Histocompatibility Antigens Class II/immunology , Mice , Mice, Inbred NOD , Sjogren's Syndrome/immunology , Sjogren's Syndrome/pathology
3.
J Appl Microbiol ; 130(6): 2018-2028, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33152175

ABSTRACT

AIM: Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. This study determines whether the E. amylovora guanine-hypoxanthine transporter (EaGhxP) is required for virulence and if it can import the E. amylovora produced toxic analogue 6-thioguanine (6TG) into cells. METHODS AND RESULTS: Characterization of EaGhxP in guanine transport deficient Escherichia coli reveals that it can transport guanine, hypoxanthine and the toxic analogues 8-azaguanine (8AG) and 6TG. Similarly, EaGhxP transports 8AG and 6TG into E. amylovora cells. EaGhxP has a high affinity for 6TG with a Ki of 3·7 µmol l-1 . An E. amylovora ⊿ghxP::Camr strain shows resistance to growth on 8AG and 6TG. Although EaGhxP is expressed during active disease propagation, it is not necessary for virulence as determined on immature apple and pear assays. CONCLUSIONS: EaGhxP is not required for virulence, but it does import 6TG into E. amylovora cells. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of the disease establishment process, E. amylovora synthesizes and exports a toxic guanine derivative 6TG. Our results are counter intuitive and show that EaGhxP, an influx transporter, can move 6TG into cells raising questions regarding the role of 6TG in disease establishment.


Subject(s)
Erwinia amylovora/metabolism , Guanine/metabolism , Hypoxanthine/metabolism , Nucleobase Transport Proteins/metabolism , Thioguanine/metabolism , Azaguanine/metabolism , Erwinia amylovora/enzymology , Erwinia amylovora/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Malus/microbiology , Nucleobase Transport Proteins/genetics , Plant Diseases/microbiology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
4.
Nat Commun ; 11(1): 1614, 2020 03 31.
Article in English | MEDLINE | ID: mdl-32235841

ABSTRACT

The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.


Subject(s)
Bacteria/metabolism , Nitric Oxide/metabolism , Triazoles/metabolism , Azaguanine/metabolism , Bacteria/enzymology , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Products , Biosynthetic Pathways/genetics , Genes, Bacterial/genetics , Nitric Oxide Synthase/metabolism , Nitrogen , Streptomyces/enzymology , Streptomyces/genetics , Streptomyces/metabolism , Synthetic Biology
5.
Chembiochem ; 20(17): 2262-2270, 2019 09 02.
Article in English | MEDLINE | ID: mdl-30983115

ABSTRACT

The impact of 7-deaza-8-azaguanine (DAG) and 7-deaza-8-azaisoguanine (DAiG) modifications on the geometry and stability of the G:C Watson-Crick (cWW) base pair and the G:iC and iG:C reverse Watson-Crick (tWW) base pairs has been characterized theoretically. In addition, the effect on the same base pairs of seven C7-substituted DAG and DAiG derivatives, some of which have been previously experimentally characterized, has been investigated. Calculations indicate that all of these modifications have a negligible impact on the geometry of the above base pairs, and that modification of the heterocycle skeleton has a small impact on the base-pair interaction energies. Instead, base-pair interaction energies are dependent on the nature of the C7 substituent. For the 7-substituted DAG-C cWW systems, a linear correlation between the base-pair interaction energy and the Hammett constant of the 7-substituent is found, with higher interaction energies corresponding to more electron-withdrawing substituents. Therefore, the explored modifications are expected to be accommodated in both parallel and antiparallel nucleic acid duplexes without perturbing their geometry, while the strength of a base pair (and duplex) featuring a DAG modification can, in principle, be tuned by incorporating different substituents at the C7 position.


Subject(s)
Azaguanine/pharmacology , Base Pairing/drug effects , Cytosine/analogs & derivatives , Cytosine/chemistry , Hydrogen Bonding/drug effects , Azaguanine/analogs & derivatives , Azaguanine/chemistry , Molecular Structure , Thermodynamics
6.
Int Immunopharmacol ; 67: 152-159, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30551032

ABSTRACT

This study identified 8-azaguanine (8-AG) as a novel immunomodulatory drug (IMiD) through a high-throughput screen of the Preswick Chemical Library in a model of human NK cell cytotoxicity against blood cancer cells. 8-AG, originally developed as an antineoplastic agent, significantly increased the cytotoxicity of NK cells and was superior in this activity to previously known IMiDs, such as fluoxetine and amphotericin B, identified from the same library. IFN-γ expression was also slightly increased by 8-AG. Mechanistically, 8-AG increased conjugate formation between NK and target cells and subsequent cytolytic granule polarization, but not calcium mobilization, regulation of activating receptors, or expression of perforin or granzyme B. Thus, the antineoplastic activity of 8-AG should be re-evaluated in light of this novel potentiating effect on NK cells.


Subject(s)
Antineoplastic Agents/therapeutic use , Azaguanine/therapeutic use , Hematologic Neoplasms/drug therapy , Immunologic Factors/therapeutic use , Killer Cells, Natural/immunology , Amphotericin B/therapeutic use , Cells, Cultured , Cytoplasmic Granules/metabolism , Cytotoxicity, Immunologic , Drug Screening Assays, Antitumor , Fluoxetine/therapeutic use , Granzymes/metabolism , Humans , Lymphocyte Activation , Perforin/genetics , Perforin/metabolism , Small Molecule Libraries
7.
Bioconjug Chem ; 29(7): 2265-2277, 2018 07 18.
Article in English | MEDLINE | ID: mdl-29771499

ABSTRACT

The α-anomers of 8-aza-2'-deoxyguanosine (αGd*) and 2'-deoxyguanosine (αGd) were site-specifically incorporated in 12-mer duplexes opposite to the four canonical DNA constituents dA, dG, dT, and dC. Oligodeoxyribonucleotides containing αGd* display significant fluorescence at slightly elevated pH (8.0). Oligodeoxyribonucleotides incorporating ß-anomeric 8-aza-2'-deoxyguanosine (Gd*) and canonical dG were studied for comparison. For αGd* synthesis, an efficient purification of anomeric 8-azaguanine nucleosides was developed on the basis of protected intermediates, and a new αGd* phosphoramidite was prepared. Differences were observed for sugar conformations ( N vs S) and p Ka values of anomeric nucleosides. Duplex stability and mismatch discrimination were studied employing UV-dependent melting and fluorescence quenching. A gradual fluorescence change takes place in duplex DNA when the α-nucleoside αGd* was positioned opposite to the four canonical ß-nucleosides. The strongest fluorescence decrease appeared in duplexes incorporating αGd*-Cd base pair matches. Decreasing fluorescence corresponds to increasing Tm values. For mismatch discrimination, the α-anomers αGd* and αGd are more efficient than the corresponding ß-nucleosides. Duplexes with single "purine-purine" αGd*-αGd* or αGd-αGd base pairs are significantly more stable than those displaying ß-d configuration. CD spectra indicate that single mutations by α-anomeric nucleosides do not affect the global structure of B-DNA.


Subject(s)
Azaguanine/chemistry , Base Pairing , Guanine/chemistry , Oligodeoxyribonucleotides/chemistry , Base Pair Mismatch , DNA, B-Form/chemistry , Fluorescence , Nucleic Acid Conformation , Transition Temperature
8.
Article in English | MEDLINE | ID: mdl-29437626

ABSTRACT

The high acquisition rate of drug resistance by Mycobacterium tuberculosis necessitates the ongoing search for new drugs to be incorporated in the tuberculosis (TB) regimen. Compounds used for the treatment of other diseases have the potential to be repurposed for the treatment of TB. In this study, a high-throughput screening of compounds against thiol-deficient Mycobacterium smegmatis strains and subsequent validation with thiol-deficient M. tuberculosis strains revealed that ΔegtA and ΔmshA mutants had increased susceptibility to azaguanine (Aza) and sulfaguanidine (Su); ΔegtB and ΔegtE mutants had increased susceptibility to bacitracin (Ba); and ΔegtA, ΔmshA, and ΔegtB mutants had increased susceptibility to fusaric acid (Fu). Further analyses revealed that some of these compounds were able to modulate the levels of thiols and oxidative stress in M. tuberculosis This study reports the activities of Aza, Su, Fu, and Ba against M. tuberculosis and provides a rationale for further investigations.


Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Azaguanine/pharmacology , Mutation/genetics , Mycobacterium tuberculosis/genetics , Oxidative Stress/drug effects , Oxidative Stress/genetics , Sulfaguanidine/pharmacology , Sulfhydryl Compounds/metabolism
9.
Chembiochem ; 18(24): 2408-2415, 2017 12 14.
Article in English | MEDLINE | ID: mdl-29024251

ABSTRACT

A series of nucleotide analogues, with a hypoxanthine base moiety (8-aminohypoxanthine, 1-methyl-8-aminohypoxanthine, and 8-oxohypoxanthine), together with 5-methylisocytosine were tested as potential pairing partners of N8 -glycosylated nucleotides with an 8-azaguanine or 8-aza-9-deazaguanine base moiety by using DNA polymerases (incorporation studies). The best results were obtained with the 5-methylisocytosine nucleotide followed by the 1-methyl-8-aminohypoxanthine nucleotide. The experiments demonstrated that small differences in the structure (8-azaguanine versus 8-aza-9-deazaguanine) might lead to significant differences in recognition efficiency and selectivity, base pairing by Hoogsteen recognition at the polymerase level is possible, 8-aza-9-deazaguanine represents a self-complementary base pair, and a correlation exists between in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions were observed).


Subject(s)
DNA-Directed DNA Polymerase/metabolism , DNA/metabolism , Purine Nucleotides/metabolism , 5-Methylcytosine , Azaguanine , Base Pairing , Escherichia coli/enzymology , Escherichia coli/genetics , Hypoxanthine
10.
PLoS Negl Trop Dis ; 10(12): e0005174, 2016 12.
Article in English | MEDLINE | ID: mdl-27935961

ABSTRACT

The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira.


Subject(s)
Communicable Diseases, Emerging/microbiology , Leptospira/isolation & purification , Leptospirosis/microbiology , Shrews/microbiology , Animals , Azaguanine/pharmacology , Bacterial Outer Membrane Proteins/genetics , Bacterial Typing Techniques , China/epidemiology , Communicable Diseases, Emerging/epidemiology , Communicable Diseases, Emerging/transmission , Cricetinae , Disease Vectors , Humans , Introduced Species , Ireland/epidemiology , Japan/epidemiology , Leptospira/classification , Leptospira/drug effects , Leptospira/pathogenicity , Leptospirosis/epidemiology , Leptospirosis/transmission , Lipoproteins/genetics , Malaysia/epidemiology , Polymerase Chain Reaction , Prohibitins , RNA, Ribosomal, 16S , Serogroup , Zoonoses/epidemiology , Zoonoses/microbiology , Zoonoses/transmission
11.
Bioorg Med Chem Lett ; 25(20): 4593-6, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26320620

ABSTRACT

A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue to green high quantum yield fluorescence in a variety of organic and aqueous solutions, high extinction coefficients, and large Stokes shifts often above 100 nm. The probes are highly cell permeable, and exhibit stable bright fluorescence once intracellular; therefore are suited to the design of biosensors.


Subject(s)
Azaguanine/chemistry , Azaguanine/metabolism , Cell Membrane Permeability , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Azaguanine/analogs & derivatives , Azaguanine/chemical synthesis , Cell Line, Tumor , Fluorescence , Fluorescent Dyes/chemical synthesis , Humans , KB Cells , Microscopy, Confocal , Molecular Structure
12.
Molecules ; 21(1): E44, 2015 Dec 28.
Article in English | MEDLINE | ID: mdl-26729076

ABSTRACT

Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-ß-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-ß-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.


Subject(s)
Azaguanine/analogs & derivatives , Point Mutation , Purine-Nucleoside Phosphorylase/genetics , Azaguanine/chemistry , Catalysis , Catalytic Domain , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Humans , Molecular Structure , Purine-Nucleoside Phosphorylase/chemistry , Purine-Nucleoside Phosphorylase/metabolism
13.
J Biomol Struct Dyn ; 32(1): 27-35, 2014.
Article in English | MEDLINE | ID: mdl-23384120

ABSTRACT

Analogues of purine bases are highly relevant in the biological context and have been implicated as drug molecules for therapy against a number of diseases. Additionally, these molecules have been implicated to have a role in the prebiotic RNA world. However, experimental data on the structures of these molecules in aqueous solution is lacking. In this work, we report the ultraviolet resonance Raman spectra of 6-chloroguanine, 8-azaguanine and allopurinol, obtained with 260 nm excitation. The reported spectra have been assigned to normal modes computed from density functional theory (B3LYP/6-31G (d,p)) calculations. This work has been useful in identifying the solution-state structures of these molecules at neutral pH. We find that the guanine analogues 6-chloroguanine and 8-azaguanine exist as keto-N9H and keto-N7H tautomers in solution, respectively. On the other hand, the hypoxanthine analogue allopurinol exists as a mixture of keto-N9H and keto-N8H tautomers in solution. We predict that this work would be particularly useful in future vibrational studies where these molecules are present in complexes with their target proteins.


Subject(s)
Allopurinol/chemistry , Azaguanine/chemistry , Guanine/analogs & derivatives , Guanine/chemistry , Hydrogen-Ion Concentration , Molecular Structure , Spectrum Analysis, Raman
14.
Molecules ; 18(10): 12587-98, 2013 Oct 11.
Article in English | MEDLINE | ID: mdl-24126376

ABSTRACT

Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the recombinant calf PNP catalyzes ribosylation of 2,6-diamino-8-azapurine in a phosphate-free medium, with ribose-1-phosphate as ribose donor, but the ribosylation site is predominantly N7 and N8, with the proportion of N8/N7 ribosylated products markedly dependent on the reaction conditions. Both products are fluorescent. Application of the E. coli PNP gave a mixture of N8 and N9-substituted ribosides. Fluorescence of the ribosylated 2,6-diamino-8-azapurine has been briefly characterized. The highest quantum yield, ~0.9, was obtained for N9-ß-d-riboside (λmax 365 nm), while for N8-ß-d-riboside, emitting at ~430 nm, the fluorescence quantum yield was found to be close to 0.4. Ribosylation of 8-azaguanine with calf PNP as a catalyst goes exclusively to N9. By contrast, the E. coli PNP ribosylates 8-azaGua predominantly at N9, with minor, but highly fluorescent products ribosylated at N8/N7.


Subject(s)
Azaguanine/analogs & derivatives , Azaguanine/chemical synthesis , Escherichia coli Proteins/chemistry , Fluorescent Dyes/chemical synthesis , Purine-Nucleoside Phosphorylase/chemistry , Animals , Biocatalysis , Cattle , Glycosylation , Kinetics , Recombinant Proteins/chemistry , Ribosemonophosphates/chemistry
15.
Nucleic Acids Res ; 41(20): 9471-83, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23945945

ABSTRACT

Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.


Subject(s)
Anti-HIV Agents/pharmacology , Azaguanine/pharmacology , HIV-1/drug effects , Quinolines/pharmacology , RNA Splicing/drug effects , RNA, Viral/metabolism , Thiophenes/pharmacology , rev Gene Products, Human Immunodeficiency Virus/antagonists & inhibitors , CD4-Positive T-Lymphocytes/virology , Cell Line , HIV-1/genetics , HIV-1/physiology , HeLa Cells , Humans , Viral Structural Proteins/genetics , Viral Structural Proteins/metabolism , Virus Replication/drug effects , rev Gene Products, Human Immunodeficiency Virus/analysis
16.
Int J Syst Evol Microbiol ; 63(Pt 7): 2457-2462, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23203626

ABSTRACT

Strain Eri-1(T) was isolated from a water sample on the campus of Kyushu University, Fukuoka, Japan. The motility and morphology of the isolate were similar to those of members of the genus Leptospira, but the spiral structure of the isolate was sharper under dark-field microscopy. Cells were 10.6 ± 1.3 µm long and 0.2 µm in diameter, with a wavelength of 0.9 µm and an amplitude of 0.4 µm. Strain Eri-1(T) grew in Korthof's medium at both 13 and 30 °C, and also in the presence of 8-azaguanine. 16S rRNA gene-based phylogenetic analysis placed strain Eri-1(T) within the radiation of the genus Leptospira where it formed a unique lineage within the clade of the known saprophytic species of the genus Leptospira. The strain was not pathogenic to hamsters. Strain Eri-1(T) exhibited low levels (11.2-12.6 %) of similarity by DNA-DNA hybridization to the three most closely related species of the genus Leptospira. The DNA G+C content of the genome of strain Eri-1(T) was 42.5 ± 0.1 mol%. These results suggest that strain Eri-1(T) represents a novel species of the genus Leptospira, for which the name Leptospira idonii sp. nov. is proposed. The type strain is Eri-1(T) ( = DSM 26084(T) = JCM 18486(T)).


Subject(s)
Leptospira/classification , Phylogeny , Water Microbiology , Animals , Azaguanine , Bacterial Typing Techniques , Base Composition , Cricetinae , DNA, Bacterial/genetics , Japan , Leptospira/genetics , Leptospira/isolation & purification , Male , Molecular Sequence Data , Nucleic Acid Hybridization , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
17.
Cancer Invest ; 30(5): 331-42, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22348536

ABSTRACT

The involvement of apoptosis in the cytotoxicity mediated by nucleoside analogues, namely azaguanine, and its implication in resistance are not well understood. Using human T-cell acute lymphoblastic leukemia cell lines, sensitive (CEM cells) and resistant to azaguanine (CM3 cells), we observe a decrease in the expression of proapoptotic proteins in CM3 cells, which may be related to the resistance to cell death induced by azaguanine. On the other hand, CM3 cells lack cross resistance with other anticarcinogenic drugs, suggesting that azaguanine may be used alternatively in the presence of chemoresistance. A better knowledge of the apoptotic pathways involved in leukemic cell death resistance may contribute to the development of therapeutic strategies, aimed to prevent chemotherapy resistance.


Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Apoptosis/drug effects , Azaguanine/therapeutic use , Leukemia/drug therapy , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , Immunophenotyping , Leukemia/immunology , Leukemia/parasitology
18.
J Mol Model ; 18(2): 493-500, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21541747

ABSTRACT

The interaction between 8-azaguanine (8-Azan) and bovine serum albumin (BSA) in Tris-HCl buffer solutions at pH 7.4 was investigated by means of fluorescence and ultraviolet-visible (UV-Vis) spectroscopy. At 298 K and 310 K, at a wavelength of excitation (λ (ex)) of 282 nm, the fluorescence intensity decreased significantly with increasing concentrations of 8-Azan. Fluorescence static quenching was observed for BSA, which was attributed to the formation of a complex between 8-Azan and BSA during the binding reaction. This was illuminated further by the UV-Vis absorption spectra and the decomposition of the fluorescence spectra. The thermodynamic parameters ∆G, ∆H, ∆S were calculated. The results showed that the forces acting between 8-Azan and BSA were typical hydrophobic forces, and that the interaction process was spontaneous. The interaction distance r between 8-Azan and BSA, evaluated according to fluorescence resonance energy transfer theory, suggested that there is a high possibility of energy transfer from BSA to 8-Azan. Theoretical investigations based on homology modeling and molecular docking suggested that binding between 8-Azan and BSA is dominated by hydrophilic forces and hydrogen bonding. The theoretical investigations provided a good structural basis to explain the phenomenon of fluorescence quenching between 8-Azan and BSA.


Subject(s)
Azaguanine/chemistry , Models, Molecular , Serum Albumin, Bovine/chemistry , Animals , Azaguanine/metabolism , Binding Sites , Cattle , Fluorescence Resonance Energy Transfer/methods , Humans , Protein Binding , Protein Structure, Secondary , Serum Albumin, Bovine/metabolism , Spectrometry, Fluorescence/methods , Spectrum Analysis/methods , Thermodynamics
19.
Trop Biomed ; 27(3): 632-8, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21399605

ABSTRACT

Leptospirosis is recognized as one of the important zoonotic diseases in the world including Malaysia. A total of 145 soil and water samples were collected from selected National Service Training Centres (NSTC) in Kelantan and Terengganu. The samples were inoculated into modified semisolid Ellinghausen McCullough Johnson Harris (EMJH) medium, incubated at room temperature for 1 month and examined under the dark-field microscope. Positive growth of the leptospiral isolates were then confirmed with 8-Azaguanine Test, Polymerase Chain Reaction (PCR) assay and Microscopic Agglutination Test (MAT). Fifteen cultures (10.34%) exhibited positive growths which were seen under dark field microscope whilst only 20% (3/15) were confirmed as pathogenic species. based on 8-Azaguanine Test and PCR. Serological identification of the isolates with MAT showed that hebdomadis was the dominant serovar in Terengganu. Pathogenic leptospires can be detected in Malaysian environment and this has the potential to cause an outbreak. Therefore, precautionary steps against leptospirosis should be taken by camp authorities to ensure the safety of trainees.


Subject(s)
Leptospira/isolation & purification , Soil Microbiology , Water Microbiology , Agglutination Tests , Animals , Antimetabolites/metabolism , Azaguanine/metabolism , Bacteriological Techniques , Culture Media/chemistry , Humans , Malaysia , Microscopy , Polymerase Chain Reaction , Serotyping
20.
Anticancer Res ; 29(11): 4489-96, 2009 Nov.
Article in English | MEDLINE | ID: mdl-20032396

ABSTRACT

BACKGROUND: Despite improvements in the treatment of patients with Ewing family tumours (EFT) during the past decades, the prognosis for patients with advanced disease is still unsatisfying. New treatment strategies have to be developed. MATERIALS AND METHODS: A hypoxanthine/aminopterin/thymidine (HAT)-sensitive EFT cell line was developed by repetitive treatment of the EFT cell line SK-N-MC with 8'-azaguanine (8AG). By using DNA microarrays, the gene expression profile of this cell line was characterized. Immunostimulatory activity was assessed by mixed lymphocyte/tumour cell culture (MLTC). Artificial fusion of tumour cells and dendritic cells was visualized by flow cytometry. RESULTS: After selection of 8AG-resistant cells, a cell line with high sensitivity for treatment with HAT was obtained. Expression of the X chromosome inactivation specific transcript XIST was higher in HAT-sensitive cells. Nevertheless, HAT-sensitive cells retained the EFT-associated gene expression profile. Moreover, in the presence of HAT, it was possible to use these cells without irradiation as stimulatory cells in MLTC or as fusion partner for dendritic cells. CONCLUSION: HAT-sensitive EFT cells might be an interesting tool for the development of new immunotherapeutic approaches for the treatment of EFT.


Subject(s)
Aminopterin/pharmacology , Hypoxanthine/pharmacology , Immunotherapy, Active/methods , Sarcoma, Ewing/immunology , Thymidine/pharmacology , Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/immunology , Azaguanine/pharmacology , Calmodulin-Binding Proteins/biosynthesis , Calmodulin-Binding Proteins/immunology , Cancer Vaccines/immunology , Cell Line, Tumor , Gene Expression Profiling , HLA-A2 Antigen/immunology , HLA-A2 Antigen/metabolism , Humans , Oligonucleotide Array Sequence Analysis , Peptides/immunology , Proto-Oncogene Protein c-fli-1/biosynthesis , Proto-Oncogene Protein c-fli-1/immunology , RNA-Binding Protein EWS , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/immunology , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathology
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