Tubulin–Na+ ,K +-ATPase interaction: Involvement in enzymatic regulation and cellular function

A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na⁺,K ⁺-ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin–NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na ⁺. Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na ⁺ and K ⁺, and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na ⁺/K ⁺ homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.     

Novel histamine H3 receptor antagonists based on the 4-[(1H-imidazol-4-yl)methyl]piperidine scaffold

We report the discovery of novel histamine H(3) receptor antagonists based on 4-[(1H-imidazol-4-yl)methyl]piperidine. The most potent compounds in the series (e.g., 7) result from the attachment of a substituted aniline amide to the main pharmacophore piperidine via a two-methylene linker.     

Prevention of tubulin/aldose reductase association delays the development of pathological complications in diabetic rats

Journal of Physiology and Biochemistry - In recent studies, we found that compounds derived from phenolic acids (CAFs) prevent the formation of the tubulin/aldose reductase complex and,...     

Accumulation of cadmium, zinc, and copper by Helianthus annuus L.: impact on plant growth and uptake of nutritional elements

We investigated the effects on physiological response, trace elements and nutrients accumulation of sunflower plants grown in soil contaminated with: 5 mg kg(-1) of Cd; 5 and 300 mg kg(-1) of Cd and Zn, respectively; 5, 300, and 400 mg kg(-1) of Cd, Zn, and Cu, respectively. Contaminants applied did not produce large effects on growth, except in Cd-Zn-Cu treatment in which leaf area and total dry matter were reduced, by 15%. The contamination with Cd alone did not affect neither growth nor physiological parameters, despite considerable amounts of Cd accumulated in roots and older leaves, with a high bioconcentration factor from soil to plant. By adding Zn and then Cu to Cd in soil, significant were the toxic effects on chlorophyll content and water relations due to greater accumulation of trace elements in tissues, with imbalances in nutrients uptake. Highly significant was the interaction between shoot elements concentration (Cd, Zn, Cu, Fe, Mg, K, Ca) and treatments. Heavy metals concentrations in roots always exceeded those in stem and leaves, with a lower translocation from roots to shoots, suggesting a strategy of sunflower to compartmentalise the potentially toxic elements in physiologically less active parts in order to preserve younger tissues.     

Proteasome inhibition and ROS generation by 4-nerolidylcatechol induces melanoma cell death

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half‐life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4‐Nerolidylcatechol (4‐NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4‐NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl‐1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4‐NC is time‐dependent upon the pro‐apoptotic protein Noxa, which is able to bind and neutralize Mcl‐1. We demonstrate the role of 4‐NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4‐NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.     

Aromatic acids are chemoattractants for Pseudomonas putida

A quantitative capillary assay was used to show that aromatic acids, compounds that are chemorepellents for Escherichia coli and Salmonella sp., are chemoattractants for Pseudomonas putida PRS2000. The most effective attractants were benzoate; p-hydroxybenzoate; the methylbenzoates; m-, p-, and o-toluate; salicylate; DL-mandelate; beta-phenylpyruvate; and benzoylformate. The chemotactic responses to these compounds were inducible. Taxis to benzoate and m-toluate was induced by beta-ketoadipate, a metabolic intermediate formed when benzoate is dissimilated via enzymes specified by chromosomal genes. Benzoylformate taxis was induced by benzoylformate and L(+)-mandelate. Taxis to mandelate, benzoylformate, and beta-phenylpyruvate was exhibited by cells grown on mandelate, but not by cells grown on benzoate. Cells grown on benzoate were chemotactic to benzoate, the toluates, p-hydroxybenzoate, and salicylate. These results show that P. putida synthesizes at least two distinct chemoreceptors for aromatic acids. Although DL-mandelate was an effective attractant in capillary assays, additional experiments indicated that the cells were actually responding to benzoylformate, a metabolite formed from mandelate. With the exception of mandelate taxis, chemotaxis to aromatic acids was not dependent on the expression of pathways for aromatic degradation. Therefore, the tactic responses exhibited by cells cannot be attributed to an effect of the oxidation of aromatic acids on the energy metabolism of cells.     

Persistence and degrading activity of free and immobilised allochthonous bacteria during bioremediation of hydrocarbon-contaminated soils

Rhodococcus sp. and Pseudomonas sp. bioremediation experiments were carried out using free and immobilized cells on natural carrier material (corncob powder) in order to evaluate the feasibility of its use in the bioremediation of hydrocarbon-contaminated soils. Terminal restriction fragment length polymorphism analysis was performed on the 16S rRNA gene as molecular fingerprinting method in order to assess the persistence of inoculated strains in the soil over time. Immobilized Pseudomonas cells degraded hydrocarbons more efficiently in the short term compared to the free ones. Immobilization seemed also to increase cell growth and stability in the soil. Free and immobilized Rhodococcus cells showed comparable degradation percentages, probably due to the peculiarity of Rhodococcus cells to aggregate into irregular clusters in the presence of hydrocarbons as sole carbon source. It is likely that the cells were not properly adsorbed on the porous matrix as a result of the small size of its pores. When Rhodococcus and Pseudomonas cells were co-immobilized on the matrix, a competition established between the two strains, that probably ended in the exclusion of Pseudomonas cells from the pores. The organic matrix might act as protective agent, but it also possibly limited cell density. Nevertheless, when the cells were properly adsorbed on the porous matrix, the immobilization became a suitable bioremediation strategy.     

High glucose levels induce inhibition of Na,K-ATPase via stimulation of aldose reductase, formation of microtubules and formation of an acetylated tubulin/Na,K-ATPase complex

Our previous studies demonstrated that acetylated tubulin forms a complex with Na(+),K(+)-ATPase and thereby inhibits its enzyme activity in cultured COS and CAD cells. The enzyme activity was restored by treatment of cells with l-glutamate, which caused dissociation of the acetylated tubulin/Na(+),K(+)-ATPase complex. Addition of glucose, but not elimination of glutamate, led to re-formation of the complex and inhibition of the Na(+),K(+)-ATPase activity. The purpose of the present study was to elucidate the mechanism underlying this effect of glucose. We found that exposure of cells to high glucose concentrations induced: (a) microtubule formation; (b) activation of aldose reductase by the microtubules; (c) association of tubulin with membrane; (d) formation of the acetylated tubulin/Na(+),K(+)-ATPase complex and consequent inhibition of enzyme activity. Exposure of cells to sorbitol caused similar effects. Studies on erythrocytes from diabetic patients and on tissues containing insulin-insensitive glucose transporters gave similar results. Na(+),K(+)-ATPase activity was >50% lower and membrane-associated tubulin content was >200% higher in erythrocyte membranes from diabetic patients as compared with normal subjects. Immunoprecipitation analysis showed that acetylated tubulin was a constituent of a complex with Na(+),K(+)-ATPase in erythrocyte membranes from diabetic patients. Based on these findings, we propose a mechanism whereby glucose triggers a synergistic effect of tubulin and sorbitol, leading to activation of aldose reductase, microtubule formation, and consequent Na(+),K(+)-ATPase inhibition.     

Avenues for increasing salt tolerance of crops,and the role of physiologically based selection traits

Increased salt tolerance is needed for crops grown in areas at risk of salinisation. This requires new genetic sources of salt tolerance, and more efficient techniques for identifying salt-tolerant germplasm, so that new genes for tolerance can be introduced into crop cultivars. Screening a large number of genotypes for salt tolerance is not easy. Salt tolerance is achieved through the control of salt movement into and through the plant, and salt-specific effects on growth are seen only after long periods of time. Early effects on growth and metabolism are likely due to osmotic effects of the salt, that is to the salt in the soil solution. To avoid the necessity of growing plants for long periods of time to measure biomass or yield, practical selection techniques can be based on physiological traits. We illustrate this with current work on durum wheat, on selection for the trait of sodium exclusion. We have explored a wide range of genetic diversity, identified a new source of sodium exclusion, confirmed that the trait has a high heritability, checked for possible penalties associated with the trait, and are currently developing molecular markers. This illustrates the potential for marker-assisted selection based on sound physiological principles in producing salt-tolerant crop cultivars.