Evaluation of curcumin and cisplatin-induced DNA damage in PC12 cells by the alkaline comet assay

A very appropriate method for antigenotoxicity evaluation of antioxidants is the comet assay, since this analytical method detects initial DNA lesions that are still subject to repair; in other words, lesions that are very associated to damages resulting from the generation and subsequent action of reactive species. However, a solid evaluation should be developed in order to avoid inexact interpretations. In our study, besides the association of curcumin with cisplatin, curcumin and cisplatin agents were also tested separately. Classical genotoxic compounds, when tested by the comet assay, present an increase in the nucleoid tail; however, the cisplatin treatment has resulted in a decrease of DNA migration. This was an expected effect, as the cross-links between cisplatin and DNA decrease the DNA electrophoretic mobility. A similar effect was observed with the curcumin treatment, which decreased the nucleoid tail. Such effect was not expected and reinforced the necessity of including in the study, separate treatment groups with potentially antigenotoxic substances. The comet assay results have been analyzed using specific software for image analysis, as well as the classical visual analysis, and we have observed that the effect of decrease in DNA electrophoretic mobility was more easily observed when the data were analyzed by the software.

Effect of Mn deficiency and legume inoculation on rhizosphere pH in highly alkaline soils

Although plant growth is often limited at high pH, little is known about root-induced changes in the rhizospheres of plants growing in alkaline soils. The effect of Mn deficiency in Rhodes grass (Chloris gayana cv. Pioneer) and of legume inoculation in lucerne (Medicago sativa L. cv. Hunter River), on the rhizosphere pH of plants grown in highly alkaline bauxite residue was investigated. Rhizosphere pH was measured quantitatively, with a micro pH electrode, and qualitatively, with an agar/pH indicator solution. Manganese deficiency in Rhodes grass increased root-induced acidification of the rhizosphere in a soil profile in which N was supplied entirely as NO3-. Rhizosphere pH in the Mn deficient plants was up to 1.22 pH units lower than that of the bulk soil, while only 0.90 to 0.62 pH units lower in plants supplied with adequate Mn. When soil N was supplied entirely as NO3-, rhizosphere acidification was more efficient in inoculated lucerne (1.75 pH unit decrease) than in non-inoculated lucerne (1.16 pH unit decrease). This difference in capacity to lower rhizosphere pH is attributable to the ability of the inoculated lucerne to fix atmospheric N2 rather than relying on the soil N (NO3 ) reserves as the non-inoculated plants. Rhizosphere acidification in both Rhodes grass and lucerne was greatest in the meristematic root zone and least in the maturation root zone.

Mg induced Ca deficiency under alkaline conditions

Little is known about Mg induced Ca deficiency in alkaline conditions, and the relationship between Mg induced Ca deficiency and Na induced Ca deficiency. Dilute nutrient solutions (dominated by Mg) were used to investigate the effect of Ca activity ratio (CAR) on the growth of mungbeans (Vigna radiata (L.) Wilczek cv. Emerald). At pH 9.0, root growth was reduced below a critical CAR of 0.050 (corresponding to 90 % relative root length). Root growth was found to be limited more in Mg solutions than had been previously observed for Na solutions. Using a CAR equation modified with plasma membrane binding constants (to incorporate the differing antagonistic effects of Mg and Na), new critical CAR values were calculated for both Na (0.56) and Mg (0.44) dominated solutions. This modified CAR equation permits the calculation of CAR irrespective of the dominant salt present.

Dragmacidins: New protein phosphatase inhibitors from a southern Australian deep-water marine sponge, Spongosorites sp.

A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

Murine DEP-1, a receptor protein tyrosine phosphatase, is expressed in macrophages and is regulated by CSF-1 and LPS

The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A valiant mRNA Tvas identified that encodes an alternate carboxyl-terminus and 3' UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount irt situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation.

Crystal structure of mammalian purple acid phosphatase

Background: Mammalian purple acid phosphatases are highly conserved binuclear metal-containing enzymes produced by osteoclasts, the cells that resorb bone. The enzyme is a target for drug design because there is strong evidence that it is involved in bone resorption. Results: The 1.55 Angstrom resolution structure of pig purple acid phosphatase has been solved by multiple isomorphous replacement. The enzyme comprises two sandwiched beta sheets flanked by or-helical segments. The molecule shows internal symmetry, with the metal ions bound at the interface between the two halves. Conclusions: Despite less than 15% sequence identity, the protein fold resembles that of the catalytic domain of plant purple acid phosphatase and some serine/threonine protein phosphatases. The active-site regions of the mammalian and plant purple acid phosphatases differ significantly, however. The internal symmetry suggests that the binuclear centre evolved as a result of the combination of mononuclear ancestors. The structure of the mammalian enzyme provides a basis for antiosteoporotic drug design.

Crystallization and preliminary X-ray diffraction studies of mammalian purple acid phosphatase

The oxidized form of purple acid phosphatase from pig allantoic fluid has been crystallized in the presence of phosphate using the hanging-drop technique. The crystals belong to the space group P2(1)2(1)2(1) and have unit-cell parameters a = 66.8, b = 70.3, c = 78.7 Angstrom. Diffraction data collected from a cryocooled crystal using a conventional X-ray source extend to 1.55 Angstrom resolution. A knowledge of the three-dimensional structure of mammalian purple acid phosphatase will aid in understanding the substrate specificity of the enzyme and will be important in the rational design of inhibitors, with potential in the treatment of bone diseases.

'Tetol': a stereo-rigid four-strand motif for alkali and alkaline earth metal ion coordination

The tetraalcohol 2,3,5,6-endo,endo,endo,endo-tetrakis(hydroxymethyl]bicyclo[2.2.1]heptane (tetol, 1) has been prepared and crystallises readily as the lithium(I) complex [Li(1)(2)]Cl, forming an oligomeric multi-chain structure in which pairs of alcohols from two crystallographically independent tetol molecules bind lithium ions tetrahedrally. However, formation of monomeric structures in solution is inferred from electrospray mass spectroscopy, which has also shown evidence of exchange of lithium ion in the complexed species by added alkaline earth ions. (C) 2000 Elsevier Science S.A. All rights reserved.

Functional expression cloning reveals proapoptotic role for protein phosphatase 4

Functional expression cloning strategies are highly suitable for the analysis of the molecular control of apoptosis. This approach has two critical advantages. Firstly, it eliminates prior assumptions about the properties of the proteins involved, and, secondly, it selectively targets proteins that are causally involved in apoptosis control and which affect the crucial cellular decision between survival and death. The application of this strategy to the isolation of cDNAs conferring resistance to dexamethasone and gamma-irradiation resulted in the isolation of a partial cDNA for the catalytic subunit of protein phosphatase 4 (PP4). Cells transfected with this partial cDNA in an expression vector downregulated PP4 and were resistant to both dexamethasone and UV radiation, as demonstrated by both membrane integrity and colony-forming assays. These observations suggest that PP4 plays an important proapoptotic role in T lymphocytes.

Neoformation of micas in soils surrounding an alkaline-saline lake of Pantanal wetland, Brazil

The mineralogy and origin of micas were investigated in incipient soils surrounding a modem alkaline-saline lake of Nhecolandia, a sub-region of Pantanal wetland. Soils were sampled along a toposequence and analyzed by XRD, TEM-EDS, and ICP-MS. The studied micas, mainly concentrated in a green horizon, are dioctahedral, strongly associated with Fe(3+) and Al, and interstratified with smectite layers. Classification of individual crystals shows that glauconite and Fe-illite are the dominant micas, but one crystal of illite was recognized. Si-rich amorphous materials are associated with small crystallites in the mica-enriched horizon. A recent study shows that water samples from the studied lake and the surrounding water table have high pH, negative Eh, temperatures up to 40 C. high concentration of K. and low concentration of Si(OH)(4). Experimental studies of micas synthesis reported in the literature show that similar water conditions allow for dioctahedral mica crystallization from initial precipitation of amorphous hydroxides. Therefore, water characteristics combined with presence of Si-rich amorphous materials in the mica-enriched horizon suggest that the micas of the study area are neoformed. The alternated origin of illite, glauconite, and Fe-illite mixed-layer minerals probably occurs due to seasonal variations of pH. temperature, and chemical composition of waters in microenvironments, since the changes at this scale are possibly faster and more extreme. (C) 2010 Elsevier B.V. All rights reserved.

MINERALOGY AND GENESIS OF SMECTITES IN AN ALKALINE-SALINE ENVIRONMENT OF PANTANAL WETLAND, BRAZIL

Smectite formation in alkaline-saline environments has been attributed to direct precipitation from solution and/or transformation from precursor minerals, but these mechanisms are not universally agreed upon in the literature. The objective of this work was to investigate the mineralogy of smectites in the soils surrounding a representative alkaline-saline lake of Nhecolandia, a sub-region of the Pantanal wetland, Brazil, and then to identify the mechanisms of their formation. Soils were sampled along a toposequence and analyzed by X-ray diffraction, transmission electron microscopy-energy dispersive X-ray analysis, and inductively coupled plasma-mass spectrometry. Water was collected along a transect involving the studied toposequence and equilibrium diagrams were calculated using the databases PHREEQC and AQUA. The fine-clay fraction is dominated by smectite, mica, and kaolinite. Smectites are concentrated at two places in the toposequence: an upper zone, which includes the soil horizons rarely reached by the lake-level variation; and a lower zone, which includes the surface horizon within the area of seasonal lake-level variation. Within the upper zone, the smectite is dioctahedral, rich in Al and Fe, and is classified as ferribeidellite. This phase is interstratified with mica and vermiculite and has an Fe content similar to that of the mica identified. These characteristics suggest that the ferribeidellite originates from transformation of micas and that vermiculite is an intermediate phase in this transformation. Within the lower zone, smectites are dominantly trioctahedral, Mg-rich, and are saponitic and stevensitic minerals. In addition, samples enriched in these minerals have much smaller rare-earth element (REE) contents than other soil samples. The water chemistry shows a geochemical control of Mg and saturation with respect to Mg-smectites in the more saline waters. The REE contents, water chemistry, and the presence of Mg-smectite where maximum evaporation is expected, suggest that saponitic and stevensitic minerals originate by chemical precipitation from the water column of the alkaline-saline lake.

Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling

Trichophyton rubrum is a dermatophyte that infects human skin and nails. Its growth on keratin as its carbon source shifts the ambient pH from acidic to alkaline, which may be an efficient strategy for its successful infection and maintenance in the host. In this study, we used suppression subtractive hybridization to identify genes preferentially expressed in T rubrum incubated at either pH 5.0 or pH 8.0. The functional grouping of the 341 overexpressed unigenes indicated proteins putatively involved in diverse cellular processes, such as membrane remodeling, cellular transport, metabolism, cellular protection, fungal pathogenesis, gene regulation, interaction with the environment, and iron uptake. Although the basic metabolic machinery identified under both growth conditions seems to be functionally similar, distinct genes are upregulated at acidic or alkaline pHs. We also isolated a large number of genes of unknown function, probably unique to T rubrum or dermatophytes. Interestingly, the transcriptional profiling of several genes in a pacC mutant suggests that, in T rubrum, the transcription factor PacC has a diversity of metabolic functions, in response to either acidic or alkaline ambient pH. (C) 2009 Elsevier Ltd. All rights reserved.

Evaluation of DNA damage by the alkaline comet assay of the olfactory and respiratory epithelia of dogs from the city of Sao Paulo, Brazil

Animals kept as pets may be considered sentinels for environmental factors to which humans could be exposed. Olfactory and respiratory epithelia are directly subjected to airborne factors, which could cause DNA lesions, and the alkaline comet assay is considered a reliable tool for the assessment of DNA damage. The objective of this work is to evaluate the extent of DNA damage by the comet assay of the olfactory and respiratory epithelia of dogs from different regions of the city of sao Paulo, Brazil. Thirty-three clinically healthy dogs, aged 5 years or more, were used in the study, with 7 from the North region of Sao Paulo, 7 from the South region, 3 dogs from the East region, and 16 dogs from the West city region. Three dogs younger than 6 months were used as controls. DNA damage was analyzed by the alkaline comet assay. We observed no difference in histopathological analysis of olfactory and respiratory epithelia between dogs from different regions of Sao Paulo. Dogs older than 5 years presented significantly higher comet length in both olfactory and respiratory epithelia, when compared with controls, indicating DNA damage. When separated by regions, olfactory and respiratory epithelia presented similar DNA damage in dogs from different regions of Sao Paulo, corroborating with similar levels of particulate matter index (PM10) in all regions of the city. In this study, we report for the first time that the comet assay can be used to quantify the extent of DNA damage in dog olfactory and respiratory epithelia, and that comet length (DNA damage) increases with age, probably due to environmental factors. Air pollution, as measured by PM 10, can be responsible for this DNA damage. (C) 2009 Elsevier GmbH. All rights reserved.

Acid phosphatase and cathepsin D are active expressed enzymes in the placenta of the cat

Enzymes are crucial for the metabolism of macromolecular substrates. In the great majority of cells, most enzymes are constitutive. Nevertheless, inducible enzymes can predominate, determining specialized cell functions. Within this context, histochemistry/immunohistochemistry and biochemistry were used to investigate expression of peroxidase and reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-oxidase, as well as the expression and activity of cathepsin D and acid phosphatase, in trophoblast cells within the endotheliochorial labyrinth and marginal hematoma of the term cat placenta. In the marginal hematoma, elevated Cathepsin D expression and activity was accompanied by erythrophagocytosis. In contrast, acid phosphatase activity was much more intense in the labyrinth, where metabolic exchanges occur. Peroxidase and NAD(P)H-oxidase were predominantly active in trophoblast cells within endosomal vesicles of different placental compartments, indicating that, although reactive oxygen species might participate in endosomal/lysosomal processes, they are not territorially specific or functional markers. These findings highlight differential characteristics of cathepsin D and acid phosphatase activity within each placental compartment, thereby contributing to the comprehension of the territorial role played by the placenta and facilitating future metabolic studies. (C) 2007 Elsevier Ltd. All rights reserved.