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1.
ACS Omega ; 9(16): 18516-18525, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38680310

RESUMO

The fruit extract of Melaleuca quinquenervia yielded a total of 19 compounds, including two novel spiro-biflavonoid enantiomers (1a and 1b) and a chalcone derivative (3). Their structures were determined through spectroscopic analysis. The enantiomers of the racemic mixture of compound 1 were successfully resolved into (+)-1 and (-)-1 using chiral-phase HPLC. Single-crystal X-ray diffraction analysis was also used to confirm the structure of 1. The enantiomeric configurations of 1 and 2 were determined through a comparison of the calculated and experimental electronic circular dichroism spectra. Compounds 2 (melanervin), 14 (methyl betulinate), 15 (3-O-acetylbetulinic acid), and 16 (pyracrenic acid) were found to be highly cytotoxic, with compound 16 showing superior growth inhibition of nonsmall cell lung cancer cells (A549 cells) (IC50 2.8 ± 0.1 µM) compared to cisplatin (IC50 3.3 ± 0.0 µM), a positive control chemotherapeutic drug. Both compound 16 and cisplatin were significantly more cytotoxic toward A549 lung cancer cells compared to nontumorigenic Vero E6 cells.

2.
ACS Chem Biol ; 18(8): 1797-1807, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37487226

RESUMO

Cyanobacteria are tremendous producers of biologically active natural products, including the potent anti-inflammatory compound tolypodiol. However, linking biosynthetic gene clusters with compound production in cyanobacteria has lagged behind that in other bacterial genera. Tolypodiol is a meroterpenoid originally isolated from the cyanobacterium HT-58-2. Here we describe the identification of the tolypodiol biosynthetic gene cluster through heterologous expression in Anabaena and in vitro protein assays of a methyltransferase found in the tolypodiol biosynthetic gene cluster. We have also identified similar biosynthetic gene clusters in cyanobacterial and actinobacterial genomes, suggesting that meroterpenoids with structural similarity to the tolypodiols may be synthesized by other microbes. We also report the identification of two new analogs of tolypodiol that we have identified in both the original and heterologous producer. This work further illustrates the usefulness of Anabaena as a heterologous expression host for cyanobacterial compounds and how integrated approaches can help to link natural product compounds with their producing biosynthetic gene clusters.


Assuntos
Produtos Biológicos , Diterpenos , Metiltransferases , Família Multigênica
3.
J Nat Prod ; 86(2): 276-289, 2023 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-36746775

RESUMO

Sixteen new quinoline alkaloids (1a-7, 8a, 9, 10, 13-15, 17, and 21) and 10 known analogs (8b, 11, 12, 16, 18-20, and 22-24), along with three known cyclopeptide alkaloids (25-27), were isolated from the roots of Waltheria indica. The structures of the new compounds were elucidated by detailed NMR and circular dichroism with computational support and mass spectrometry data interpretation. Anti-inflammatory potential of isolates was evaluated based on inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production and tumor necrosis factor-alpha (TNF-α)-induced nuclear factor kappa B (NF-κB) activity with cell culture models. In the absence of cell growth inhibition, compounds 6, 8a, 9-11, 13, 21, and 24 reduced TNF-α-induced NF-κB activity with IC50 values ranging from 7.1 to 12.1 µM, comparable to the positive control (BAY 11-7082, IC50 = 9.7 µM). Compounds 6, 8a, 8b, and 11 showed significant NO-inhibitory activity with IC50 values ranging from 11.0 to 12.8 µM, being more active than the positive control (l-NMMA, IC50 = 22.7 µM). Structure-activity relationships indicated that NO inhibitory activity was significantly affected by C-8 substitution. Inhibition of LPS-induced nitric oxide synthase (iNOS) by 8b [(5S)-waltherione M, IC50 11.7 ± 0.8 µM] correlated with inhibition of iNOS mRNA expression. The biological potential of W. indica metabolites supports the traditional use of this plant for the treatment of inflammatory-related disorders.


Assuntos
Alcaloides , Malvaceae , Quinolinas , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Lipopolissacarídeos/farmacologia , Alcaloides/farmacologia , Anti-Inflamatórios/farmacologia , Malvaceae/química , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico
4.
Mar Drugs ; 21(2)2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36827142

RESUMO

Six new nostocyclophanes and four known compounds have been isolated from Nostoc linckia (Nostocaceae) cyanobacterial strain UTEX B1932. The new compounds, nostocyclophanes E-J (1-6), were characterized by NMR and MS techniques. The known compounds were nostocyclophanes B-D, previously isolated from this strain, and dedichloronostocyclophane D. Structural modifications on the new [7.7]paracyclophane analogs 1-5, isolated from the 80% methanol fraction, range from simple changes such as the lack of methylation or halogenation to more unusual modifications such as those seen in nostocyclophane H (4), in which the exocyclic alkyl chains are of different length; this is the first time this modification has been observed in this family of natural products. In addition, nostocyclophane J (6) is a linear analog in which C-20 is chlorinated in preparation for the presumed enzymatic Friedel-Craft cyclization needed to form the final ring structure, analogous to the biosynthesis of the related cylindrocyclophanes. Nostocyclophane D, dedichloronostocyclophane D, and nostocyclophanes E-J demonstrated moderate to weak growth inhibition against MDA-MB-231 breast cancer cells.


Assuntos
Nostoc , Nostoc/química , Espectroscopia de Ressonância Magnética
5.
Molecules ; 27(10)2022 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-35630746

RESUMO

Seven new coumarinolignans, walthindicins A-F (1a, 1b, 2-5, 7), along with five known analogs (6, 8-11), were isolated from the roots of Waltheria indica. The structures of the new compounds are determined by detailed nuclear magnetic resonance (NMR), circular dichroism (CD) with extensive computational support, and mass spectroscopic data interpretation. Compounds were tested for their antioxidant activity in Human Cervical Cancer cells (HeLa cells). Compounds 1a and 6 showed higher reactive oxygen species (ROS) inhibitory activity at 20 µg/mL when compared with other natural compound-based antioxidants such as ascorbic acid. Considering the role of ROS in nuclear-factor kappa B (NF-κB) activation, compounds 1a and 6 were evaluated for NF-κB inhibitory activity and showed a concentration-dependent inhibition in Human Embryonic Kidney 293 cells (Luc-HEK-293).


Assuntos
Cumarínicos , Lignanas , Malvaceae , NF-kappa B , Espécies Reativas de Oxigênio , Cumarínicos/química , Cumarínicos/farmacologia , Células HEK293 , Células HeLa , Humanos , Lignanas/química , Lignanas/farmacologia , Malvaceae/química , NF-kappa B/antagonistas & inibidores , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Raízes de Plantas/química , Espécies Reativas de Oxigênio/antagonistas & inibidores
6.
Toxins (Basel) ; 14(4)2022 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-35448876

RESUMO

Glucocorticoid-resistant asthma, which predominates with neutrophils instead of eosinophils, is an increasing health concern. One potential source for the induction of neutrophil-predominant asthma is aerosolized lipopolysaccharide (LPS). Cyanobacteria have recently caused significant tidal blooms, and aerosolized cyanobacterial LPS has been detected near the cyanobacterial overgrowth. We hypothesized that cyanobacterial LPS contributes to lung inflammation by increasing factors that promote lung inflammation and neutrophil recruitment. To test this hypothesis, c57Bl/6 mice were exposed intranasally to LPS from the cyanobacterium member, Geitlerinema sp., in vivo to assess neutrophil infiltration and the production of pro-inflammatory cytokines and chemokines from the bronchoalveolar fluid by ELISA. Additionally, we exposed the airway epithelial cell line, A549, to Geitlerinema sp. LPS in vitro to confirm that airway epithelial cells were stimulated by this LPS to increase cytokine production and the expression of the adhesion molecule, ICAM-1. Our data demonstrate that Geitlerinema sp. LPS induces lung neutrophil infiltration, the production of pro-inflammatory cytokines such as Interleukin (IL)-6, Tumor necrosis factor-alpha, and Interferongamma as well as the chemokines IL-8 and RANTES. Additionally, we demonstrate that Geitlerinema sp. LPS directly activates airway epithelial cells to produce pro-inflammatory cytokines and the adhesion molecule, Intercellular Adhesion Molecule-1 (ICAM-1), in vitro using the airway epithelial cell line, A549. Based on our findings that use Geitlerinema sp. LPS as a model system, the data indicate that cyanobacteria LPS may contribute to the development of glucocorticoid-resistant asthma seen near water sources that contain high levels of cyanobacteria.


Assuntos
Asma , Cianobactérias , Pneumonia , Animais , Asma/patologia , Quimiocinas/metabolismo , Cianobactérias/metabolismo , Citocinas/metabolismo , Glucocorticoides/metabolismo , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Pulmão/patologia , Camundongos , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/patologia
7.
J Nat Prod ; 85(2): 415-425, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35142496

RESUMO

As part of a study examining polar metabolites produced by cyanobacterial strains, we examined media extracts of a Calothrix sp. (strain R-3-1) and a Scytonema sp. (strain U-3-3). The cell mass of each was separated from the media, and HP20 resin was added for adsorption of secreted metabolites, a relatively unexplored area of cyanobacterial chemistry. HPLC-UV-LCMS-guided isolation led to the discovery of seven sesquiterpenoid compounds with five new, one known, and one previously isolated as the methyl ester. Through a complement of 1D and 2D NMR spectroscopic techniques, the planar structures and relative configurations of the seven compounds were elucidated. Spironostoic acid (1), 11,12-didehydrospironostoic acid (2), and 12-hydroxy-2-oxo-11-epi-hinesol (4) are spirovetivane-type compounds from R-3-1, while stigolone (5), 11R,12-dihydroxystigolone (6), and 11S,12-dihydroxystigolone (7) are three eudesmane-type compounds from U-3-3. Circular dichroism was utilized to decipher the absolute configurations of new compounds 1, 2, 4, 5, 6, and 7. Due to the structural variety observed among the spirovetivane- and eudesmane-type compounds in the literature and often a lack of clarity in how determinations were made, computational spectra and model compounds were used to support the interpretation of ECD and NMR spectra. A straightforward process to determine the configuration of these systems is presented.


Assuntos
Sesquiterpenos de Eudesmano , Sesquiterpenos , Meios de Cultura , Estrutura Molecular , Sesquiterpenos/química , Sesquiterpenos de Eudesmano/química
8.
PLoS Pathog ; 17(5): e1009570, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33989345

RESUMO

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ sequestration is part of "nutritional immunity", where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.


Assuntos
Granuloma/microbiologia , Lipidômica , Mycobacterium tuberculosis/fisiologia , Proteoma , Transcriptoma , Zinco/deficiência , Adaptação Fisiológica , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Granuloma/imunologia , Homeostase , Interações Hospedeiro-Patógeno , Humanos , Pulmão/microbiologia , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Oxirredução , Estresse Oxidativo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
9.
Appl Microbiol Biotechnol ; 104(16): 6977-6989, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32601736

RESUMO

This study aimed to develop a bioprocess using plant oil as the carbon source for lipid-assimilating yeast to produce high-value astaxanthin. Using high-oleic safflower oil as a model, efficient cell growth and astaxanthin production by the engineered Yarrowia lipolytica strain ST7403 was demonstrated, and a considerable portion of astaxanthin was found excreted into the spent oil. Astaxanthin was the predominant carotenoid in the extracellular oil phase that allowed facile in situ recovery of astaxanthin without cell lysis. Autoclaving the safflower oil medium elevated the peroxide level but it declined quickly during fermentation (reduced by 84% by day 3) and did not inhibit cell growth or astaxanthin production. In a 1.5-L fed-batch bioreactor culture with a YnB-based medium containing 20% safflower oil, and with the feeding of casamino acids, astaxanthin production reached 54 mg/L (53% excreted) in 28 days. Further improvement in astaxanthin titer and productivity was achieved by restoring leucine biosynthesis in the host, and running fed-batch fermentation using a high carbon-to-nitrogen ratio yeast extract/peptone medium containing 70% safflower oil, with feeding of additional yeast extract/peptone, to attain 167 mg/L astaxanthin (48% excreted) in 9.5 days of culture. These findings facilitate industrial microbial biorefinery development that utilizes renewable lipids as feedstocks to not only produce high-value products but also effectively extract and recover the products, including non-native ones.Key Points• Yarrowia lipolytica can use plant oil as a C-source for astaxanthin production.• Astaxanthin is excreted and accumulated in the extracellular oil phase.• Astaxanthin is the predominant carotenoid in the extracellular oil phase.• Plant oil serves as a biocompatible solvent for in situ astaxanthin extraction. Graphical abstract.


Assuntos
Carbono/metabolismo , Óleo de Cártamo/química , Yarrowia/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Biomassa , Reatores Biológicos/microbiologia , Meios de Cultura/química , Fermentação , Nitrogênio/química , Xantofilas/metabolismo , Yarrowia/genética
10.
J Nat Prod ; 83(5): 1691-1695, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32282204

RESUMO

Chemical investigation of cyanobacterial strain HT-58-2, which most closely aligns with the genus Brasilomena, has led to the isolation of two compounds related to tolypodiol. The structures and absolute configuration of 6-deoxytolypodiol (1) and 11-hydroxytolypodiol (2) were elucidated by spectroscopic and spectrometric analysis. While tolypodiol previously showed anti-inflammatory activity in a mouse ear edema assay, only 2 reduced in vitro thromboxane B2 and superoxide anion (O2-) generation from Escherichia coli lipopolysaccharide-activated rat neonatal microglia to any appreciable degree.


Assuntos
Anti-Inflamatórios/farmacologia , Cianobactérias/química , Diterpenos/química , Otopatias/tratamento farmacológico , Escherichia coli/química , Lipopolissacarídeos/química , Superóxidos/química , Tromboxano B2/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Edema , Camundongos , Ratos
11.
J Org Chem ; 85(2): 318-326, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31815480

RESUMO

Tolyporphins L-R (2-8) have been isolated from a mixed cyanobacterium-microbial culture. The structures of tolyporphins L and M have been revised to four constitutional isomers, isolated as two mixtures of dioxobacteriochlorins (2/3 and 4/5). In contrast, tolyporphin P (6) is a fully oxidized tetrapyrrole, while tolyporphins Q and R (7 and 8) are oxochlorins. X-ray structures are reported for the first time for tolyporphins A (1), R (8), and E (9), revealing unexpected stereochemical variation within the series.


Assuntos
Cianobactérias/química , Porfirinas/química , Tetrapirróis/química , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Estrutura Molecular , Porfirinas/isolamento & purificação , Análise Espectral/métodos , Tetrapirróis/isolamento & purificação
12.
Org Lett ; 21(20): 8449-8453, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31591889

RESUMO

The bioactivity-guided examination of a Leptolyngbya sp. led to the isolation of leptazolines A-D (1-4), from the culture media, along with two degradation products (5 and 6). Density functional theory nuclear magnetic resonance calculations established the relative configurations of 1 and 2 and revealed that the calculated shifts depended on the operating system when using the "Willoughby-Hoye" Python scripts to streamline the processing of the output files, a previously unrecognized flaw that could lead to incorrect conclusions.


Assuntos
Cianobactérias/química , Oxazóis/síntese química , Espectroscopia de Ressonância Magnética/normas , Estrutura Molecular , Oxazóis/química , Padrões de Referência , Estereoisomerismo
13.
J Nat Prod ; 82(9): 2668-2671, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461285

RESUMO

Four compounds (1-4) were isolated from a Hawaiian sponge of the genus Myrmekioderma. Myrmenaphthol A (1) incorporates two unusual elements into an oxidized steroidal core: a naphthyl AB-ring system and a hydroxy group at C-2. A comparison of the experimental and predicted electronic circular dichroism (ECD) spectra of 1 assigned an S configuration to the lone stereocenter (ΔESI = 0.75; similarity factor 0.8137). Known compounds, cinanthrenol A (2), 3,4-dihydroxypregna-5,17-diene-10,2-carbolactone (3), and 3,4-dihydroxypregna-5,20-diene-10,2-carbolactone (4), were also isolated. Despite literature reports of competitive inhibition at nanomolar levels for 2, neither 2 nor the structurally related 1 showed any activity against estrogen receptors at the concentrations tested.


Assuntos
Poríferos/química , Animais , Dicroísmo Circular , Havaí , Estrutura Molecular , Análise Espectral/métodos
14.
Mar Drugs ; 17(7)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31331110

RESUMO

Several known sesquiterpenoid quinones and quinols (1-9), and kauamide (10), a new polyketide-peptide containing an 11-membered heterocycle, were isolated from the extracts of the Hawaiian marine sponge Dactylospongia elegans. The planar structure of 10 was determined from spectroscopic analyses, and its relative and absolute configurations were established from density functional theory (DFT) calculations of the GIAO NMR shielding tensors, and advanced Marfey's analysis of the N-MeLeu residue, respectively. Compounds 1 and 3 showed moderate inhibition of ß-secretase 1 (BACE1), whereas 1-9 exhibited moderate to potent inhibition of growth of human glioma (U251) cells. Compounds 1-2 and 4-7 were also active against human pancreatic carcinoma (Panc-1) cells.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Poríferos/química , Sesquiterpenos/farmacologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Glioma/tratamento farmacológico , Glioma/patologia , Havaí , Compostos Heterocíclicos/química , Compostos Heterocíclicos/isolamento & purificação , Compostos Heterocíclicos/farmacologia , Humanos , Hidroquinonas/química , Hidroquinonas/isolamento & purificação , Hidroquinonas/farmacologia , Estrutura Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Quinonas/química , Quinonas/isolamento & purificação , Quinonas/farmacologia , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Neoplasias Pancreáticas
15.
Toxicol Sci ; 171(2): 421-430, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31271425

RESUMO

Cyanobacterial blooms are an increasing source of environmental toxins that affect both human and animals. After ingestion of cyanobacteria, such as Geitlerinema sp., toxins and lipopolysaccharide (LPS) from this organism induce fever, gastrointestinal illness, and even death. However, little is known regarding the effects of cyanobacterial LPS on human monocytes after exposure to LPS upon ingestion. Based on our previous data using Geitlerinema sp. LPS (which was previously named Oscillatoria sp., a genus belonging to the same order as Geitlerinema), we hypothesized that Geitlerinema sp. LPS would activate human monocytes to proliferate, phagocytose particles, and produce cytokines that are critical for promoting proinflammatory responses in the gut. Our data demonstrate that Geitlerinema sp. LPS induced monocyte proliferation and TNF-α, IL-1, and IL-6 production at high concentrations. In contrast, Geitlerinema sp. LPS is equally capable of inducing monocyte-mediated phagocytosis of FITC-latex beads when compared with Escherichia coli LPS, which was used as a positive control for our experiments. In order to understand the mechanism responsible for the difference in efficacy between Geitlerinema sp. LPS and E. coli LPS, we performed biochemical analysis and identified that Geitlerinema sp. LPS was composed of significantly different sugars and fatty acid side chains in comparison to E. coli LPS. The lipid A portion of Geitlerinema sp. LPS contained longer fatty acid side chains, such as C15:0, C16:0, and C18:0, instead of C12:0 found in E. coli LPS which may explain the decreased efficacy and toxicity of Geitlerinema sp. LPS in comparison to E. coli LPS.

16.
Microbiology (Reading) ; 164(10): 1229-1239, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30117798

RESUMO

Tolyporphins are structurally diverse tetrapyrrole macrocycles produced by the cyanobacterial culture HT-58-2. Although tolyporphins were discovered over 25 years ago, little was known about the microbiology of the culture. The studies reported herein expand the description of the community of predominantly alphaproteobacteria associated with the filamentous HT-58-2 cyanobacterium and isolate a dominant bacterium, Porphyrobacter sp. HT-58-2, for which the complete genome is established and growth properties are examined. Fluorescence in situ hybridization (FISH) analysis of the cyanobacterium-microbial community with a probe targeting the 16S rRNA of Porphyrobacter sp. HT-58-2 showed fluorescence emanating from the cyanobacterial sheath. Although genes for the biosynthesis of bacteriochlorophyll a (BChl a) are present in the Porphyrobacter sp. HT-58-2 genome, the pigment was not detected under the conditions examined, implying the absence of phototrophic growth. Comparative analysis of four Porphyrobacter spp. genomes from worldwide collection sites showed significant collinear gene blocks, with two inversions and three deletion regions. Taken together, the results enrich our understanding of the HT-58-2 cyanobacterium-microbial culture.


Assuntos
Alphaproteobacteria/fisiologia , Cianobactérias/metabolismo , Genoma Bacteriano/genética , Consórcios Microbianos , Alphaproteobacteria/classificação , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Mapeamento Cromossômico , DNA Bacteriano/genética , Hibridização in Situ Fluorescente , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
17.
Toxins (Basel) ; 10(4)2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29561785

RESUMO

Cosmopolitan Gram-negative cyanobacteria may affect human and animal health by contaminating terrestrial, marine and freshwater environments with toxins, such as lipopolysaccharide (LPS). The cyanobacterial genus Scytonema (S) produces several toxins, but to our knowledge the bioactivity of genus Scytonema LPS has not been investigated. We recently reported that cyanobacterium Oscillatoria sp. LPS elicited classical and alternative activation of rat microglia in vitro. Thus, we hypothesized that treatment of brain microglia in vitro with either cyanobacteria S. javanicum or S. ocellatum LPS might stimulate classical and alternative activation with concomitant release of superoxide anion (O2-), matrix metalloproteinase-9 (MMP-9), cytokines and chemokines. Microglia were isolated from neonatal rats and treated in vitro with either S. javanicum LPS, S. ocellatum LPS, or E. coli LPS (positive control), in a concentration-dependent manner, for 18 h at 35.9 °C. We observed that treatment of microglia with either E. coli LPS, S. javanicum or S. ocellatum LPS generated statistically significant and concentration-dependent O2-, MMP-9 and pro-inflammatory cytokines IL-6 and TNF-α, pro-inflammatory chemokines MIP-2/CXCL-2, CINC-1/CXCL-1 and MIP-1α/CCL3, and the anti-inflammatory cytokine IL-10. Thus, our results provide experimental support for our working hypothesis because both S. javanicum and S. ocellatum LPS elicited classical and alternative activation of microglia and concomitant release of O2-, MMP-9, cytokines and chemokines in a concentration-dependent manner in vitro. To our knowledge this is the first report on the toxicity of cyanobacteria S. javanicum and S. ocellatum LPS to microglia, an immune cell type involved in neuroinflammation and neurotoxicity in the central nervous system.


Assuntos
Cianobactérias , Lipopolissacarídeos/toxicidade , Microglia/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Citocinas/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Microglia/metabolismo , Ratos Sprague-Dawley , Superóxidos/metabolismo
18.
Phytochem Anal ; 29(2): 205-216, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29110356

RESUMO

INTRODUCTION: Tolyporphins are unusual tetrapyrrole macrocycles produced by a non-axenic filamentous cyanobacterium (HT-58-2). Tolyporphins A-J, L, and M share a common dioxobacteriochlorin core, differ in peripheral substituents, and exhibit absorption spectra that overlap that of the dominant cyanobacterial pigment, chlorophyll a. Identification and accurate quantitation of the various tolyporphins in these chlorophyll-rich samples presents challenges. OBJECTIVE: To develop methods for the quantitative determination of tolyporphins produced under various growth conditions relative to that of chlorophyll a. METHODOLOGY: Chromatographic fractionation of large-scale (440 L) cultures afforded isolated individual tolyporphins. Lipophilic extraction of small-scale (25 mL) cultures, HPLC separation with an internal standard, and absorption detection enabled quantitation of tolyporphin A and chlorophyll a, and by inference the amounts of tolyporphins A-M. Absorption spectroscopy with multicomponent analysis of lipophilic extracts (2 mL cultures) afforded the ratio of all tolyporphins to chlorophyll a. The reported absorption spectral data for the various tolyporphins required re-evaluation for quantitative purposes. RESULTS AND DISCUSSION: The amount of tolyporphin A after 50 days of illumination ranged from 0.13 nmol/mg dry cells (media containing nitrate) to 1.12 nmol/mg (without nitrate), with maximum 0.23 times that of chlorophyll a. Under soluble-nitrogen deprivation after 35-50 days, tolyporphin A represents 1/3-1/2 of the total tolyporphins, and the total amount of tolyporphins is up to 1.8-fold that of chlorophyll a. CONCLUSIONS: The quantitative methods developed herein should facilitate investigation of the biosynthesis of tolyporphins (and other tetrapyrroles) as well as examination of other strains for production of tolyporphins. Copyright © 2017 John Wiley & Sons, Ltd.


Assuntos
Clorofila/química , Cromatografia Líquida de Alta Pressão/métodos , Cianobactérias/metabolismo , Porfirinas/análise , Análise Espectral/métodos , Tetrapirróis/análise , Clorofila A , Cianobactérias/crescimento & desenvolvimento , Porfirinas/química , Padrões de Referência , Reprodutibilidade dos Testes
19.
Appl Environ Microbiol ; 83(19)2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28754701

RESUMO

The cyanobacterial culture HT-58-2 was originally described as a strain of Tolypothrix nodosa with the ability to produce tolyporphins, which comprise a family of distinct tetrapyrrole macrocycles with reported efflux pump inhibition properties. Upon reviving the culture from what was thought to be a nonextant collection, studies of culture conditions, strain characterization, phylogeny, and genomics have been undertaken. Here, HT-58-2 was shown by 16S rRNA analysis to closely align with Brasilonema strains and not with Tolypothrix isolates. Light, fluorescence, and scanning electron microscopy revealed cyanobacterium filaments that are decorated with attached bacteria and associated with free bacteria. Metagenomic surveys of HT-58-2 cultures revealed a diversity of bacteria dominated by Erythrobacteraceae, 97% of which are Porphyrobacter species. A dimethyl sulfoxide washing procedure was found to yield enriched cyanobacterial DNA (presumably by removing community bacteria) and sequence data sufficient for genome assembly. The finished, closed HT-58-2Cyano genome consists of 7.85 Mbp (42.6% G+C) and contains 6,581 genes. All genes for biosynthesis of tetrapyrroles (e.g., heme, chlorophyll a, and phycocyanobilin) and almost all for cobalamin were identified dispersed throughout the chromosome. Among the 6,177 protein-encoding genes, coding sequences (CDSs) for all but two of the eight enzymes for conversion of glutamic acid to protoporphyrinogen IX also were found within one major gene cluster. The cluster also includes 10 putative genes (and one hypothetical gene) encoding proteins with domains for a glycosyltransferase, two cytochrome P450 enzymes, and a flavin adenine dinucleotide (FAD)-binding protein. The composition of the gene cluster suggests a possible role in tolyporphin biosynthesis.IMPORTANCE A worldwide search more than 25 years ago for cyanobacterial natural products with anticancer activity identified a culture (HT-58-2) from Micronesia that produces tolyporphins. Tolyporphins are tetrapyrroles, like chlorophylls, but have several profound structural differences that reside outside the bounds of known biosynthetic pathways. To begin probing the biosynthetic origin and biological function of tolyporphins, our research has focused on studying the cyanobacterial strain, about which almost nothing has been previously reported. We find that the HT-58-2 culture is composed of the cyanobacterium and a community of associated bacteria, complicating the question of which organisms make tolyporphins. Elucidation of the cyanobacterial genome revealed an intriguing gene cluster that contains tetrapyrrole biosynthesis genes and a collection of unknown genes, suggesting that the cluster may be responsible for tolyporphin production. Knowledge of the genome and the gene cluster sharply focuses research to identify related cyanobacterial producers of tolyporphins and delineate the tolyporphin biosynthetic pathway.


Assuntos
Cianobactérias/metabolismo , Genoma Bacteriano , Porfirinas/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Cianobactérias/química , Cianobactérias/genética , Cianobactérias/crescimento & desenvolvimento , Metagenômica , Família Multigênica , Filogenia , Porfirinas/química
20.
Photochem Photobiol ; 93(5): 1204-1215, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28439932

RESUMO

Tolyporphins are tetrapyrrole macrocycles produced by a cyanobacterium-containing culture known as HT-58-2. Tolyporphins A-J are free base dioxobacteriochlorins, whereas tolyporphin K is an oxochlorin. Here, the photophysical characterization is reported of tolyporphin A and two synthetic analogues, an oxobacteriochlorin and a dioxobacteriochlorin. The characterization (in toluene, diethyl ether, ethyl acetate, dichloromethane, 1-pentanol, 2-butanone, ethanol, methanol, N,N-dimethylformamide and dimethylsulfoxide) includes static absorption and fluorescence spectra, fluorescence quantum yields and time-resolved data. The data afford the lifetime of the lowest singlet excited state and the yields of the nonradiative decay pathways (intersystem crossing and internal conversion). The three macrocycles exhibit only modest variation in spectroscopic and excited-state photophysical parameters across the solvents. The long-wavelength (Qy ) absorption band of tolyporphin A appears at ~680 nm and is remarkably narrow (full-width-at-half-maximum ~7 nm). The position of the long-wavelength (Qy ) absorption band of tolyporphin A (~680 nm) more closely resembles that of chlorophyll a (662 nm) than bacteriochlorophyll a (772 nm). The absorption spectra of tolyporphins B-I, K (which were available in minute quantities) are also reported in methanol; the spectra of B-I closely resemble that of tolyporphin A. Taken together, tolyporphin A generally exhibits spectral and photophysical features resembling those of chlorophyll a.


Assuntos
Cianobactérias/química , Fotoquímica , Porfirinas/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
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