Viviana Rivarola

In vitro protein synthesis is affected by the herbicide 2,4-dichlorophenoxyacetic acid in Azospirillum brasilense.

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 Cds 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.

DNA and protein synthesis inhibition in chinese hamster ovary cells by dichlorophenoxyacetic acid

The effects of the herbicide dichlorophenoxyacetic acid (2,4-D) on DNA and protein synthesis were investigated in chinese hamster ovary cells (CHO) employing two different methods. The results showed that the herbicide affects DNA and protein synthesis depending on the stage of growth and method of treatment. 2,4-D action appears to concentrate the cells mainly in the G1/S boundary of the cell cycle. The effect is expressed as an inhibition of DNA and protein synthesis. This effect was revealed not only by the chemical determination of DNA and protein synthesis but also by experiments using autoradiography, using the labelling index to detect the incorporation of [3H]thymidine into the cells. Labelling of the cell nucleus was reduced markedly when cells treated with 2,4-D were in confluency for 4 days after reaching plateau growth.

Response of Azospirillum brasilense Cd to Sodium Chloride Stress

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.

EFFECTS OF 2,4-DICHLOROPHENOXYACETIC ACID ON POLYAMINE SYNTHESIS IN CHINESE HAMSTER OVARY CELLS

It was found that 1 mM 2,4-dichlorophenoxyacetate (2,4-D) inhibited DNA and protein synthesis in Chinese hamster ovary (CHO) cells. When the possible relationship of this phenomenon to the presence of polyamines in the culture medium was investigated, it was found that: (a) the pesticide inhibited ornithine decarboxylase activity; (b) when the concentration of polyamines present in cells treated with the pesticide was determined, the putrescine concentration did not change, and the spermine and spermidine concentration decreased; (c) the addition of spermidine and spermine to CHO cells grown in the presence of 2,4-D normalized DNA and protein synthesis. Putrescine did not have any effect.

Colonization of wheat by Azospirillum brasilense Cd is impaired by saline stress.

The effect of saline stress on the colonization of wheat was analyzed by using Azospirillum brasilense Cd carrying the fusion of the reporter gene lacZ (β-galactosidase) with the N2 fixation gene promoter nifA. Colonization was also studied by inducing para-nodules on wheat roots using 2,4-D, establishing that these structures acted as bacterium protected niches. Bacteria grown under standard conditions were distributed along the whole root system, except the elongation zone, and colonized the para-nodules. Bacteria experiencing saline stress were mainly localized at the root tips and the lateral roots. In 2,4-D treated plants, most of the bacteria were present around the basal surface of the modified lateral root structures. Using the MPN method, there were not statistical differences between the numbers of control and stressed bacteria. As this method estimates endophytic colonization in contrast with the one using X-gal, which emphasizes colonization on the root surface, both procedures demonstrated to be necessary, concluding that salt treatment reduced surface colonization (X-gal) but not colonization inside the root. The bacterial counts made on inoculated wheat roots indicated higher numbers of both control and stressed bacteria in roots treated with 2,4-D compared with untreated roots.

The interaction of 2,4-dichlorophenoxyacetic acid, ribosomes and polyamines in Azospirillum brasilense

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.

2,4-Dichlorophenoxyacetic acid affects the attachment of Azospirillum brasilense Cd to maize roots

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.

2,4-Dichlorophenoxyacetic Acid Action on in Vitro Protein Synthesis and its Relation to Polyamines

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the in vitro synthesis of proteins was studied in Chinese hamster ovary cells. A remarkable inhibition of the synthesis of proteins was observed when cells grew for 24 h in presence of 1 mM 2,4-D. This effect was reversed by adding 0.1 mM of the three polyamines (putrescine, spermidine and spermine) to the cultured cells. The mRNA is not altered, indicating that the 2,4-D action is located at a different locus, which may be the ribosomes. From these studies, one can speculate that the alterations in the protein synthesis may be a consequence of the effect of 2,4-D on the polyamine metabolism.

Characterization of 2,4-dichlorophenoxyacetic acid transport and its relationship with polyamines in Azospirillum brasilense

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.

Problem-based learning in veterinary medicine: protein metabolism.

A problem-based approach to teaching biochemistry to veterinary students is described which aims to encourage the finding and learning of biochemical information that is relevant to real-life veterinary problems.