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2.
Methods Mol Biol ; 2222: 363-379, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301102

RESUMO

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.


Assuntos
Bandeamento Cromossômico , Análise Citogenética , Genoma , Genômica , Hibridização in Situ Fluorescente , Filogenia , Cromomicinas/farmacologia , Bandeamento Cromossômico/métodos , Análise Citogenética/métodos , Código de Barras de DNA Taxonômico , DNA Ribossômico/genética , Genômica/métodos , Hibridização in Situ Fluorescente/métodos , Pinus/classificação , Pinus/genética
3.
Molecules ; 25(24)2020 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-33327391

RESUMO

A stable intense resistance called "nonhost resistance" generates a complete multiple-gene resistance against plant pathogenic species that are not pathogens of pea such as the bean pathogen, Fusarium solani f. sp. phaseoli (Fsph). Chitosan is a natural nonhost resistance response gene activator of defense responses in peas. Chitosan may share with cancer-treatment compounds, netropsin and some anti-cancer drugs, a DNA minor groove target in plant host tissue. The chitosan heptamer and netropsin have the appropriate size and charge to reside in the DNA minor groove. The localization of a percentage of administered radio-labeled chitosan in the nucleus of plant tissue in vivo indicates its potential to transport to site(s) within the nuclear chromatin (1,2). Other minor groove-localizing compounds administered to pea tissue activate the same secondary plant pathway that terminates in the production of the anti-fungal isoflavonoid, pisatin an indicator of the generated resistance response. Some DNA minor groove compounds also induce defense genes designated as "pathogenesis-related" (PR) genes. Hypothetically, DNA targeting components alter host DNA in a manner enabling the transcription of defense genes previously silenced or minimally expressed. Defense-response-elicitors can directly (a) target host DNA at the site of transcription or (b) act by a series of cascading events beginning at the cell membrane and indirectly influence transcription. A single defense response, pisatin induction, induced by chitosan and compounds with known DNA minor groove attachment potential was followed herein. A hypothesis is formulated suggesting that this DNA target may be accountable for a portion of the defense response generated in nonhost resistance.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Quitosana/farmacologia , Substâncias Intercalantes/farmacologia , Netropsina/farmacologia , Ervilhas/genética , Doenças das Plantas/genética , Pterocarpanos/farmacologia , Antineoplásicos Fitogênicos/química , Benzimidazóis/química , Benzimidazóis/farmacologia , Quitosana/química , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Cromomicinas/química , Cromomicinas/farmacologia , DNA de Plantas/genética , DNA de Plantas/metabolismo , Resistência à Doença/genética , Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Regulação da Expressão Gênica de Plantas , Proteínas HMGA/genética , Proteínas HMGA/metabolismo , Substâncias Intercalantes/química , Netropsina/química , Ervilhas/imunologia , Ervilhas/metabolismo , Ervilhas/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pterocarpanos/química , Transcrição Genética
4.
Mar Drugs ; 18(10)2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-33096696

RESUMO

A marine-derived actinomycete (Streptomyces sp. MBTI36) exhibiting antibacterial activities was investigated in the present study. The strain was identified using genetic techniques. The 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces microflavus. Furthermore, a new chromomycin A9 (1), along with chromomycin Ap (2), chromomycin A2 (3), and chromomycin A3 (4), were isolated from the ethyl acetate extract. Their structures were determined using extensive spectroscopic methods including 1D and 2D NMR, and HRMS, as well as comparisons with previously reported data. Compounds 1-4 showed potent antibacterial activities against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). During a passage experiment, minimum inhibitory concentration (MIC) values for compounds 1-4 showed no more than a 4-fold increase from the starting MIC value, indicating that no resistance was detected over the 21 passages.


Assuntos
Antibacterianos/farmacologia , Cromomicinas/farmacologia , Streptomyces/química , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/isolamento & purificação , Organismos Aquáticos/química , Organismos Aquáticos/classificação , Organismos Aquáticos/genética , Organismos Aquáticos/metabolismo , Cromomicinas/química , Cromomicinas/isolamento & purificação , Farmacorresistência Bacteriana , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , Streptomyces/classificação , Streptomyces/genética
5.
J Genet ; 982019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31819022

RESUMO

A wide range of diploid number of chromosomes and the body size of Channa congeners are useful combination of characters for studying the factors controlling the body size. In this study, the karyological information was superimposed on the evolutionary tree generated by 16S rRNA mitochondrial gene sequences. Here, the metaphase chromosome complements stained with Giemsa, AgNO3 and CMA3 were prepared from six snakehead murrel fish species collected from northeast India. The diploid chromosome numbers and the fundamental arms of C. aurantimaculata (2n = 52, NF = 98), C. gachua (2n = 56, NF = 84), C. marulius (2n = 44, NF = 58), C. orientalis (2n = 52, NF = 74), C. punctata (2n = 32, NF = 60) and C. striata (2n = 40, NF = 48) were calculated by the analysis of metaphase chromosome complements. Both methods of nucleolar organizer region (NOR) localization, silver nitrate and chromomycin A3, revealed NOR pairs of 1, 2, 3, 1, 4 and 3 in C. aurantimaculata, C. gachua, C. marulius, C. orientalis, C. punctata and C. striata, respectively. The subject species showed primitive type of asymmetrical chromosomes, except the C. punctata. The variation in 2n for C. orientalis (2n = 52, 78) and C. gachua (2n = 52, 78, 104) of a complete haploid set indicates the possibility of either ploidy change in C. orientalis and C. gachua, if we consider 2n = 52 or the Robertsonian rearrangements in different populations of these two species. The chromosome evolution tree was constructed on 16S rRNA ML-phylogenetic tree using ChromEvol 1.3. The analysis of chromosome evolution explained the loss or gain of chromosome, duplications or semiduplications mechanism. For time scaling the chromosomeevolution, the node age of available 16S rRNA gene of Channa species were estimated, which was also used for estimating the time when chromosomal changes occurred in context of geological time-scale.


Assuntos
Evolução Molecular , Peixes/genética , Cariótipo , RNA Ribossômico 16S/genética , Animais , Cromomicinas , Cromossomos , Diploide , Peixes/classificação , Índia , Metáfase , Filogenia
6.
Environ Microbiol ; 21(2): 814-826, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30585380

RESUMO

The well-known role of antibiotics in killing sensitive organisms has been challenged by the effects they exert at subinhibitory concentrations. Unfortunately, there are very few published reports on the advantages these molecules may confer to their producers. This study describes the construction of a genetically verified deletion mutant of Streptomyces flaviscleroticus unable to synthesize chromomycin. This mutant was characterized by a rapid loss of viability in stationary phase that was correlated with high oxidative stress and altered antioxidant defences. Altered levels of key metabolites in the mutant signalled a redistribution of the glycolytic flux toward the PPP to generate NADPH to fight oxidative stress as well as reduction of ATP-phosphofructokinase and Krebs cycle enzymes activities. These changes were correlated with a shift in the preference for carbon utilization from glucose to amino acids. Remarkably, chromomycin at subinhibitory concentration increased longevity of the non-producer and restored most of the phenotypic features' characteristic of the wild type strain. Altogether these observations suggest that chromomycin may have antioxidant properties that would explain, at least in part, some of the phenotypes of the mutant. Our observations warrant reconsideration of the secondary metabolite definition and raise the possibility of crucial roles for their producers.


Assuntos
Antibacterianos/biossíntese , Cromomicinas/biossíntese , Estresse Oxidativo , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Deleção de Genes , Glicólise , NADP/metabolismo , Streptomyces/genética
7.
Sci Rep ; 7(1): 15122, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29123209

RESUMO

Identification of parameters predicting assisted reproductive technologies (ARTs) success is a major goal of research in reproduction. Quality of gametes is essential to achieve good quality embryos and increase the success of ARTs. We evaluated two sperm parameters, chromatin maturity and expression of the sperm specific calcium channel CATSPER, in relation to ART outcomes in 206 couples undergoing ARTs. Chromatin maturity was evaluated by Chromomycin A3 (CMA3) for protamination and Aniline Blue (AB) for histone persistence and CATSPER expression by a flow cytometric method. CMA3 positivity and CATSPER expression significantly predicted the attainment of good quality embryos with an OR of 6.6 and 14.3 respectively, whereas AB staining was correlated with fertilization rate. In the subgroup of couples with women ≤35 years, CATSPER also predicted achievement of clinical pregnancy (OR = 4.4). Including CMA3, CATSPER and other parameters affecting ART outcomes (female age, female factor and number of MII oocytes), a model that resulted able to predict good embryo quality with high accuracy was developed. CMA3 staining and CATSPER expression may be considered two applicable tools to predict ART success and useful for couple counseling. This is the first study demonstrating a role of CATSPER expression in embryo development after ARTs programs.


Assuntos
Canais de Cálcio/análise , Cromatina/química , Fertilização In Vitro , Expressão Gênica , Reprodução , Técnicas de Reprodução Assistida , Espermatozoides/fisiologia , Adulto , Compostos de Anilina/metabolismo , Cromomicinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Espermatozoides/química , Coloração e Rotulagem , Resultado do Tratamento , Adulto Jovem
8.
Angew Chem Int Ed Engl ; 56(30): 8761-8765, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28544401

RESUMO

Small-molecule compounds targeting trinucleotide repeats in DNA have considerable potential as therapeutic or diagnostic agents against many neurological diseases. NiII (Chro)2 (Chro=chromomycin A3) binds specifically to the minor groove of (CCG)n repeats in duplex DNA, with unique fluorescence features that may serve as a probe for disease detection. Crystallographic studies revealed that the specificity originates from the large-scale spatial rearrangement of the DNA structure, including extrusion of consecutive bases and backbone distortions, with a sharp bending of the duplex accompanied by conformational changes in the NiII chelate itself. The DNA deformation of CCG repeats upon binding forms a GGCC tetranucleotide tract, which is recognized by NiII (Chro)2 . The extruded cytosine and last guanine nucleotides form water-mediated hydrogen bonds, which aid in ligand recognition. The recognition can be accounted for by the classic induced-fit paradigm.


Assuntos
Cromomicinas/farmacologia , DNA/efeitos dos fármacos , Níquel/farmacologia , Compostos Organometálicos/farmacologia , Cromomicinas/química , DNA/química , Humanos , Modelos Moleculares , Níquel/química , Compostos Organometálicos/química , Repetições de Trinucleotídeos/efeitos dos fármacos
9.
Nat Prod Commun ; 12(4): 571-577, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30520599

RESUMO

A new antibiotic complex of six aureolic acids was isolated from the marine sediment-associated strain Streptomyces sp. KMM 9048. Four of the compounds (3-6) were found to be similar but not identical to the known chromomycins A2, A3, demethyl chromomycin A3 and A4. The two remaining.compounds; A2₋1 (1) and A3₋1 (2), were established as novel chromomycin analogs, which did not contain sugar B. Spectroscopic methods including ID and 2D NMR, and HRMS and MS/MS were applied for structure elucidation. Compounds 1-5 showed strong antimicrobial activity against Gram-positive indicatory bacteria Enterococcusfaecium, Staphylococcus aureus, S. epidernzidis, and Bacillus subtilis. Antitumor assay indicated that all tested compounds, in different manners, inhibited colony formation of RPMI-7951 and SK-Mel-28 cancer cells. This is the first study reporting the inhibitory effects of chromomycin analogs 1-5 on the colony formation of the investigated cancer cell lines. Compound 3, in a concentration of 5 nM, inhibited colony formation of RPMI-7951 and SK-Mel-28 cells by 82 % and 72 %, respectively. Our finding indicated that, of the compounds tested, 3 and 4 are promising anticancer and antimicrobial agents.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacocinética , Sedimentos Geológicos/microbiologia , Plicamicina/farmacologia , Streptomyces/química , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Cromomicinas/química , Cromomicinas/isolamento & purificação , Cromomicinas/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade Microbiana , Plicamicina/química , Plicamicina/isolamento & purificação , Streptomyces/genética , Streptomyces/isolamento & purificação , Streptomyces/metabolismo , Espectrometria de Massas em Tandem
10.
Biol Res ; 48: 58, 2015 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-26466995

RESUMO

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemia salina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells. Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.


Assuntos
Antineoplásicos/farmacologia , Microbiologia do Solo , Streptomyces/química , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antineoplásicos/isolamento & purificação , Artemia/classificação , Artemia/efeitos dos fármacos , Bovinos , Linhagem Celular , Chlorocebus aethiops , Cromatografia/métodos , Cromomicinas/classificação , Cromomicinas/farmacologia , Formazans , Glicerol/análogos & derivados , Glicerol/farmacologia , Células HeLa , Humanos , Concentração Inibidora 50 , Larva/efeitos dos fármacos , Células MCF-7 , Microscopia Eletrônica de Varredura , Mineração , Paquistão , Filogenia , RNA Ribossômico 16S/genética , Sais , Análise de Sequência de RNA , Solo/química , Streptomyces/isolamento & purificação , Streptomyces/ultraestrutura , Streptomyces griseus/classificação , Sais de Tetrazólio , Células Vero
11.
Biol. Res ; 48: 1-10, 2015. ilus, graf, tab
Artigo em Inglês | LILACS | ID: biblio-950822

RESUMO

BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.


Assuntos
Humanos , Animais , Bovinos , Microbiologia do Solo , Streptomyces/química , Antineoplásicos/farmacologia , Paquistão , Filogenia , Artemia/classificação , Artemia/efeitos dos fármacos , Sais , Solo/química , Streptomyces/isolamento & purificação , Streptomyces/ultraestrutura , Streptomyces griseus/classificação , Sais de Tetrazólio , Células Vero , RNA Ribossômico 16S/genética , Cromomicinas/classificação , Cromomicinas/farmacologia , Células HeLa , Microscopia Eletrônica de Varredura , Linhagem Celular , Chlorocebus aethiops , Cromatografia/métodos , Análise de Sequência de RNA , Concentração Inibidora 50 , Células MCF-7 , Formazans , Glicerol/análogos & derivados , Glicerol/farmacologia , Larva/efeitos dos fármacos , Mineração , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antineoplásicos/isolamento & purificação
12.
Mar Drugs ; 12(12): 5839-55, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25486109

RESUMO

The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.


Assuntos
Autofagia/efeitos dos fármacos , Melanoma/tratamento farmacológico , Plicamicina/análogos & derivados , Brasil , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromomicina A3/metabolismo , Cromomicinas/farmacologia , Células HCT116 , Células HL-60 , Humanos , Melanoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Plicamicina/farmacologia , Streptomyces/química
13.
Methods Mol Biol ; 1115: 309-23, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24415481

RESUMO

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.


Assuntos
Bandeamento Cromossômico/métodos , Hibridização in Situ Fluorescente/métodos , Filogenia , Cromomicinas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Fixação de Tecidos
14.
Cytogenet Genome Res ; 140(1): 62-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23635472

RESUMO

Sites of 5S and 45S rDNA are more commonly located on different chromosomes of most angiosperms. Previous investigations have shown that in the subfamily Aurantioideae these sites may appear closely linked (adjacent sites), as in Poncirustrifoliata, or completely isolated, as in some species of Citrus. In the present work, the distribution of rDNA sites was investigated in representatives of 9 genera of Aurantioideae by FISH and CMA banding, aiming to understand the evolution of adjacent sites in the subfamily. A total of 57 rDNA sites were observed, 40 of them being adjacent to each other. All adjacent sites displayed the 45S rDNA array more terminally located. Assuming that the linked 5S-45S rDNA arrangement was the ancestral condition in Aurantioideae, the isolated rDNA sites observed in Clausena excavata,Bergera koenigii, and Fortunella obovata, as well as the complete linkage loss in Citrus maxima and C. medica indicates that unlinked sites arose independently several times in the evolution of the group. The linkage loss may be due to independent dispersion of one or both rDNA sequence families followed by deletion of the corresponding array in the adjacent site. The possible mechanisms behind these events and their occurrence in other groups are discussed.


Assuntos
Cromossomos de Plantas/genética , Sequência Conservada , DNA de Plantas/análise , RNA Ribossômico 5S/genética , RNA Ribossômico/genética , Rutaceae/genética , Sequência de Bases , Cromomicinas/metabolismo , Cromossomos de Plantas/metabolismo , DNA de Plantas/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Evolução Molecular , Ligação Genética , Variação Genética , Hibridização in Situ Fluorescente , Indóis/metabolismo , Cariótipo , Cariotipagem , Metáfase , RNA de Plantas/análise , RNA de Plantas/genética , Rutaceae/metabolismo , Especificidade da Espécie
15.
Microbiol Res ; 167(10): 590-5, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22789867

RESUMO

A marine-derived actinomycete (Streptomyces sp. WBF16) exhibiting antitumor activities was investigated. The strain was identified using morphological, biochemical and genetic techniques. 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces coelicolor A3 (2). Furthermore, a new aureolic acid (Chromomycin B, 1), along with Chromomycin A(2) (2) and Chromomycin A(3) (3) were isolated from its secondary metabolites. Their structures were determined by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. Compounds 1-3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1 and HUVEC.


Assuntos
Cromomicinas/química , Cromomicinas/farmacologia , Plicamicina/química , Plicamicina/farmacologia , Streptomyces/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cromomicinas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células HCT116 , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Espectroscopia de Ressonância Magnética/métodos , Biologia Marinha , Plicamicina/metabolismo , Streptomyces/química , Streptomyces/classificação
16.
Bioorg Med Chem ; 19(17): 5183-9, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21807523

RESUMO

Two chromomycin SA analogs, chromomycin SA(3) and chromomycin SA(2), along with deacetylchromomycin A(3) and five previously reported chromomycin analogs were isolated from a marine-derived Streptomyces sp. The structures of the new compounds were determined by spectroscopic methods including 1D and 2D NMR techniques, HRMS and chemical methods. Chromomycin SA(3) and chromomycin SA(2) are the first naturally occuring chromomycin analogs with truncated side-chains. Biological evaluation of chromomycin analogs for cytotoxicity against two non-small cell lung cancer (NSCLC) cell-lines, A549 and HCC44, demonstrated a decrease in cytotoxicity for the truncated sides chain chromomycin analogs.


Assuntos
Cromomicinas/química , Streptomyces/química , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/toxicidade , Cromomicinas/isolamento & purificação , Cromomicinas/toxicidade , Ensaios de Seleção de Medicamentos Antitumorais , Espectroscopia de Ressonância Magnética , Conformação Molecular
17.
Microb Biotechnol ; 4(2): 226-38, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21342468

RESUMO

Mithramycin and chromomycin A(3) are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.


Assuntos
Acetiltransferases/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Plicamicina/química , Plicamicina/metabolismo , Streptomyces griseus/enzimologia , Acetiltransferases/genética , Antineoplásicos/farmacologia , Proteínas de Bactérias/genética , Biotransformação , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Cromomicinas/metabolismo , Humanos , Plicamicina/farmacologia , Streptomyces griseus/química , Streptomyces griseus/genética , Streptomyces griseus/metabolismo
18.
Biochemistry ; 49(49): 10543-52, 2010 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-21067184

RESUMO

The antitumor antibiotics mithramycin A and chromomycin A(3) bind reversibly to the minor groove of G/C-rich regions in DNA in the presence of dications such as Mg(2+), and their antiproliferative activity has been associated with their ability to block the binding of certain transcription factors to gene promoters. Despite their biological activity, their use as anticancer agents is limited by severe side effects. Therefore, in our pursuit of new structurally related molecules showing both lower toxicity and higher biological activity, we have examined the binding to DNA of six analogues that we have obtained by combinatorial biosynthetic procedures in the producing organisms. All these molecules bear a variety of changes in the side chain attached to C-3 of the chromophore. The spectroscopic characterization of their binding to DNA followed by the evaluation of binding parameters and associated thermodynamics revealed differences in their binding affinity. DNA binding was entropically driven, dominated by the hydrophobic transfer of every compound from solution into the minor groove of DNA. Among the analogues, mithramycin SDK and chromomycin SDK possessed the higher DNA binding affinities.


Assuntos
Cromomicinas/química , Cromomicinas/metabolismo , Técnicas de Química Combinatória , DNA/metabolismo , Plicamicina/análogos & derivados , Plicamicina/metabolismo , Animais , Sítios de Ligação/fisiologia , Cromomicinas/biossíntese , Técnicas de Química Combinatória/métodos , DNA/química , Masculino , Modelos Moleculares , Conformação de Ácido Nucleico , Salmão , Testículo/química , Termodinâmica
19.
Biochem Pharmacol ; 79(10): 1418-27, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20093108

RESUMO

Differential cleavage at three restriction enzyme sites was used to determine the specific binding to DNA of the antitumour antibiotics mithramycin A (MTA), chromomycin A(3) (CRO) and six chromophore-modified analogues bearing shorter side chains attached at C-3, instead of the pentyl chain. All these antibiotics were obtained through combinatorial biosynthesis in the producer organisms. MTA, CRO and their six analogues showed differences in their capacity for inhibiting the rate of cleavage by restriction enzymes that recognize C/G-rich tracts. Changes in DNA melting temperature produced by these molecules were also analyzed, as well as their antiproliferative activities against a panel of colon, ovarian and prostate human carcinoma cell lines. Moreover, the cellular uptake of several analogues was examined to identify whether intracellular retention was related to cytotoxicity. These experimental approaches provided mutually consistent evidence of a seeming correlation between the strength of binding to DNA and the antiproliferative activity of the chromophore-modified molecules. Four of the analogues (mithramycin SK, mithramycin SDK, chromomycin SK and chromomycin SDK) showed promising biological profiles.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Cromomicinas/farmacologia , Enzimas de Restrição do DNA/antagonistas & inibidores , Plicamicina/análogos & derivados , Linhagem Celular Tumoral , Cromomicina A3/farmacologia , Neoplasias do Colo/tratamento farmacológico , Desoxirribonucleases de Sítio Específico do Tipo II/antagonistas & inibidores , Feminino , Citometria de Fluxo , Humanos , Masculino , Neoplasias Ovarianas/tratamento farmacológico , Plicamicina/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Relação Estrutura-Atividade
20.
Biochemistry (Mosc) ; 75(11): 1331-41, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21314600

RESUMO

Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A(3) (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this difference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and ~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV-stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.


Assuntos
Núcleo Celular/química , Cromomicinas/farmacologia , Distamicinas/farmacologia , Hepatócitos/química , Histonas/isolamento & purificação , Ácido Poliglutâmico , Raios Ultravioleta , Animais , Antibacterianos/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Cromatina/química , DNA/química , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/efeitos da radiação , Interfase , Conformação de Ácido Nucleico , Ratos
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