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AIMS: This work was conducted to identify the antifungal compounds produced by two previously isolated Bacillus sp. strains: ARP(2) 3 and MEP(2) 18. Both strains were subjected to further analysis to determine their taxonomic position and to identify the compounds responsible for their antifungal activity as well as to evaluate the efficiency of these strains to control sclerotinia stem rot in soybean. METHODS AND RESULTS: The antifungal compounds were isolated by acid precipitation of cell-free supernatants, purified by RP-HPLC and then tested for antagonistic activity against Sclerotinia sclerotiorum. Mass spectra from RP-HPLC eluted fractions showed the presence of surfactin C(15) , fengycins A (C(16) -C(17)) and B (C(16)) isoforms in supernatants from strain ARP(2) 3 cultures, whereas the major lipopeptide produced by strain MEP(2) 18 was iturin A C(15) . Alterations in mycelial morphology and sclerotial germination were observed in the presence of lipopeptides-containing supernatants from Bacillus strains cultures. Foliar application of Bacillus amyloliquefaciens strains on soybean plants prior to S. sclerotiorum infection resulted in significant protection against sclerotinia stem rot compared with noninoculated plants or plants inoculated with a nonlipopeptide-producing B. subtilis strain. CONCLUSIONS: Both strains, renamed as B. amyloliquefaciens ARP(2) 3 and MEP(2) 18, were able to produce antifungal compounds belonging to the cyclic lipopeptide family. Our data suggest that the foliar application of lipopeptide-producing B. amyloliquefaciens strains could be a promising strategy for the management of sclerotinia stem rot in soybean. SIGNIFICANCE AND IMPACT OF THE STUDY: Sclerotinia stem rot was ranked as one of the most severe soybean disease in Argentina and worldwide. The results of this study showed the potential of B. amyloliquefaciens strains ARP(2) 3 and MEP(2) 18 to control plant diseases caused by S. sclerotiorum.

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Glass ionomer cements are important options in restorative and preventive dentistry due to their adhesion to the tooth surface and to fluoride release, which can decrease the risk of recurrent caries. The topical use of acidulated and neutral fluoride gels has been frequent in dentistry. However, this procedure can adversely affect the surface of restorative materials, increasing their roughness and the retention of dental plaque. Thus, this study evaluated the period in which Vitremer glass ionomer cement maintains its antimicrobial activity over Streptococcus mutans ATCC 25175, as well as the effects of topical application of acidulated and neutral fluoride gels on these microbiological parameters and on the superficial characteristics of the restorative material. It was verified that the antimicrobial activity of Vitremer is very transient, decreasing to an undetectable level after four days, and the topical application of fluoride gel did not restore this activity. It was observed that S. mutans ATCC 25175 adheres to this restorative material, and the topical fluorides did not affect this event. The surface of Vitremer was not altered by the application of fluoride gels.

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DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis, and has been shown to induce apoptosis. In this paper, the relation between the effects of DFMO on the polyamine content, apoptotic index and Fas expression in HEP-2 cells was determined. Fas is a type I membrane protein with a molecular mass of 45 kDa, which mediates apoptosis. The results suggest that the treatment with the polyamine inhibitor DFMO induced the expression of the surface antigen Fas, which could be responsible for trigger apoptosis in these cells.(AU)

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DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis, and has been shown to induce apoptosis. In this paper, the relation between the effects of DFMO on the polyamine content, apoptotic index and Fas expression in HEP-2 cells was determined. Fas is a type I membrane protein with a molecular mass of 45 kDa, which mediates apoptosis. The results suggest that the treatment with the polyamine inhibitor DFMO induced the expression of the surface antigen Fas, which could be responsible for trigger apoptosis in these cells.

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The photodynamic effects of 5,10,15, 20-tetra(4-methoxyphenyl)porphyrin (TMP) on a Hep-2 cell line were investigated. TMP toxicity in the dark and in relation to illumination with visible light was examined. Hep-2 cells were treated with different TMP concentrations (1, 5 and 10 microM). The uptake of TMP by Hep-2 cells increased with TMP concentration and an increase of the initial uptake rate was observed with increasing TMP concentrations. However, after 24 h of incubation, a similar value of intracellular TMP concentration was reached at all three concentrations of TMP added. Cell toxicity induced by TMP was analyzed in the dark at different concentrations of the photosensitizer and at several incubation periods. The cell mortality obtained after exposure of the cell cultures to visible light was exclusively due to the photosensitization effect of TMP produced by light irradiation. Staining with the hematoxylin-eosin method demonstrated that treatment with TMP, followed by exposure to visible light, notably increased the apoptotic figures. Fas antigen was only expressed in these conditions. The results contribute to the understanding of the photodynamic therapy (PDT) mechanism produced by TMP on Hep-2 carcinoma cell line.

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DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis. The goal of this study was to determine the effects of DFMO on the expression of cyclin A at different stages of the cell cycle of Hep-2 cells. The cell cycle analysis, done by measuring the incorporation of thymidine in the cell DNA, revealed that DFMO produced a lower and constant level of that incorporation; this effect is probably due to the incapacity of the cells to culminate the phase S of the cell cycle. The expression of cyclin A increased in the phases S and G2 in control cells, almost disappearing in phase M. However, in DFMO treated cultures, the expression of cyclin A was increased in M and this effect remained still after 48 h treatment. We conclude that polyamines could exert an effect on the cyclin destruction mechanism, and the depletion caused by DFMO would alter this mechanism.

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DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis, and has been shown to induce apoptosis. In this paper, the relation between the effects of DFMO on the polyamine content, apoptotic index and Fas expression in HEP-2 cells was determined. Fas is a type I membrane protein with a molecular mass of 45 kDa, which mediates apoptosis. The results suggest that the treatment with the polyamine inhibitor DFMO induced the expression of the surface antigen Fas, which could be responsible for trigger apoptosis in these cells.

Assuntos

RESUMO

DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis. The goal of this study was to determine the effects of DFMO on the expression of cyclin A at different stages of the cell cycle of Hep-2 cells. The cell cycle analysis, done by measuring the incorporation of thymidine in the cell DNA, revealed that DFMO produced a lower and constant level of that incorporation; this effect is probably due to the incapacity of the cells to culminate the phase S of the cell cycle. The expression of cyclin A increased in the phases S and G2 in control cells, almost disappearing in phase M. However, in DFMO treated cultures, the expression of cyclin A was increased in M and this effect remained still after 48 h treatment. We conclude that polyamines could exert an effect on the cyclin destruction mechanism, and the depletion caused by DFMO would alter this mechanism.

RESUMO

DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis, and has been shown to induce apoptosis. In this paper, the relation between the effects of DFMO on the polyamine content, apoptotic index and Fas expression in HEP-2 cells was determined. Fas is a type I membrane protein with a molecular mass of 45 kDa, which mediates apoptosis. The results suggest that the treatment with the polyamine inhibitor DFMO induced the expression of the surface antigen Fas, which could be responsible for trigger apoptosis in these cells.

RESUMO

This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis

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This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis

Assuntos

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This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis.

Assuntos

RESUMO

This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis.

RESUMO

Growth of Azospirillum brasilense Cd in the presence of different NaCl concentrations showed that it tolerates up to 200 mM NaCl in the medium, without appreciable decline in growth rate. At 300 mM NaCl, a decrease of 66% in growth was observed at 24 h of culture. At 48 h of culture, bacteria in the presence of 300 mM NaCl reached the maximum optical density value that was attained at 12 h by control cultures. This investigation was designed to elucidate the effect of saline stress on Azospirillum brasilense Cd and the physiologic mechanism involved in its possible salinity tolerance. For this reason, studies of other osmolytes, as well as of putrescine metabolism and protein patterns were done with bacteria grown with this NaCl concentration in the medium, at 24 and at 48 hours. A. brasilense responded to saline stress elevating the intracellular concentration of glutamate at 24 h, and of K+ at 48 h. Glucan pattern, putrescine metabolism, and total and periplasmic protein patterns of the treated group showed several changes with respect to the control. In spite of the several cellular functions affected by saline stress, the results imply that A. brasilense Cd shows salinity tolerance in these experimental conditions.

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2.4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide widely applied to forage, grain and cereals. We previously determined that 1 mM 2,4-D diminished cell growth and cellular activity of Azospirillum brasilense Cd. The present work was designed to determine the possible effect of this herbicide--at concentrations used on crops--on the attachment of the bacteria to maize roots, since this step is of prime importance for the growth stimulation of the plant obtained with Azospirillum brasilense. In this paper we demonstrate that 2,4-D alters the bacterial adhesion to maize roots.

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We have previously shown that 2,4-dichlorophenoxyacetic acid (2,4-D) inhibits Azospirillum brasilense growth, the synthesis of DNA, RNA and proteins. These toxic effects are prevented when polyamines are added to the culture medium. The purposes of our research were to determine the effects of the herbicide on the number of viable Azospirillum brasilense cells, characterize the 2,4-D transport system and to study the effects of polyamines upon the latter in this microorganism. We found that 2,4-D reduced the number of viable cells and that 2,4-D transport is energy-independent, since it was not affected by metabolic inhibitors. Polyamines did not alter 2,4-D uptake, further supporting the hypothesis that the herbicide most likely produces its toxic effects by interfering with the polyamine metabolism.

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We had previously demonstrated that 1 mM 2,4-D inhibited cell growth, nucleic acid synthesis and protein synthesis (at the ribosomal level) of Azospirillum brasilense. These alterations were prevented by the presence of polyamines in the culture medium. On the other hand, polyamines did not affect the 2,4-D uptake. In this paper we demonstrate that 2,4-D alters the metabolism of polyamines and increases their uptake.

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2,4-Dichlorophenoxyacetic acid (2,4-D) is an herbicide used extensively in agriculture. We had previously determined that 1 mM 2,4-D could inhibit cell growth, DNA and protein synthesis of Azospirillum brasilense. The present work was designed to determine if these alterations are a consequence of 2,4-D action on polyamine biosynthesis and if the protein synthesis inhibition is a result of ribosomal impairment. In this paper we demonstrate that 2,4-D alters the metabolism of polyamines and, thus, affects protein synthesis at the ribosomal level.

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The effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on growth and protein, DNA and RNA synthesis of Azospirillum brasilense Cd were studied. At a concentration of 1 mM, 2,4-D inhibited cell growth, an effect that was reversed either by transferring bacteria to a control (2,4-D-free) medium or to a 2,4-D-treated medium supplemented with polyamines. The herbicide also affected in vitro protein synthesis, either when Azospirillum brasilense Cd's own cellular mRNA or an artificial mRNA was used. This effect was also reversed by the addition of polyamines to the 2,4-D-treated medium. Similar results were observed when DNA synthesis was studied in synchronous cultures. Taking into account the effects of this herbicide on animal cells (V.A. Rivarola and H.F. Balegno, Toxicology, 68 (1991) 109) we postulate that the mechanism of action of 2,4-D is similar on both procaryotic and eucaryotic cells, probably acting through the polyamine metabolism.

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