Study of the mitotic and meiotic chromosomes of sotol (<i>Dasylirion cedrosanum</i> Trel.)

The genus Dasylirion is a group of plants typically present in the Chihuahuan Desert, perennial, with a dioecious sexual behavior and commonly called sotoles. This genus has been little studied from the biological point of view, and the bases of its reproductive response remain unknown. In this work we studied the chromosome number and meiotic response of Dasylirion cedrosanum in the county of Saltillo, Coahuila, located at the North East of Mexico. For the preparation of mitotic chromosomes, we used a technique based on enzymatic treatment with pectolyase and cellulase, as well as staining with acetocarmin dye. For the study of meiosis, male flower buds were collected, fixed and stained for analysis with the same dye. As a result, the gametic (n = x = 19) and somatic chromosome (2n = 38) numbers of D. cedrosanum are reported for the first time, being consistent with previous findings in other Dasylirion species, which points to a constant ploidy level across the genus. Variation was observed in the morphology and size of the somatic chromosomes, with types ranging from submetacentric to subtelocentric, and sizes oscillating in a range of 4.43 µm, with an average total length of 112.38 µm for the diploid chromosome complement. This shows that the chromosome complement of D. cedrosanom would belong to a 3B classification of Stebins, with a medium variation between chromosome lengths and low chromosome asymmetry. This variation indicates the feasibility of constructing a chromosome ideotype for this species. The meiotic chromosome pairing showed a chromosome behavior consistent with a disomic inheritance characteristic of a diploid species, with prevalence of ring and chain bivalents, typically without pairing abnormalities. Bivalent configurations in all cases were symmetrical.The normal and symmetrical meiotic pairing indicates a balanced production of gametes, and suggests the absence of heteromorphic sex determination.     

Heterologous expression of a position 2-substituted (1-->3)-beta-D-glucan in Lactococcus lactis

Exopolysaccharides play an important role in the rheology and texture of fermented foods, and among these beta-glucans have immunomodulating properties. We show that the overproduction of the Pediococcus parvulus GTF glycosyltransferase in an uncapsulated Lactococcus lactis strain results in synthesis and secretion (300 mg liter(-1)) of a position 2-substituted (1-->3)-beta-D-glucan that has potential use as a food additive.     

Potential role of a new anti-beta3 integrin antibody in the development of intimal hyperplasia after vascular surgery: an in vitro smooth muscle cell model

The occurrence of intimal hyperplasia after vascular surgery is an ongoing concern in current clinical practice. Among the many factors involved in the development of this pathology, platelet adhesion and myointimal proliferation play a major role. Both these processes are mediated by integrins (mainly alphavbeta3 integrins). Over the past years, several substances have been designed to delay or inhibit the cell proliferation that leads to hyperplasia and mainly include monoclonal antibodies directed against integrins. The aim of the present study was to evaluate the effects of an antibody denoted P37 (anti beta3 integrin) on human smooth muscle cells (SMC) and its role in blocking the B3 subunit. To this end, SMC from human umbilical artery were cultured in the presence or absence of the cell substrate vitronectin (VN) and incubated with P37. After 4 days of treatment, determination was made of cell proliferation and migration. Smooth muscle cells grown on VN showed increased proliferation and migration compared to control VN-free cultures. However, the presence of P37 in the culture medium inhibited proliferation and reduced migration. Combined treatment with VN and P37 led to improved proliferation but VN was unable to reverse the effects on migration observed in the former cultures. Results suggest that in vitro, P37 is capable of blocking human SMC beta3 integrins and thus impedes cell proliferation and migration These findings may have clinical implications related to modulation of the development of hyperplasia.     

Different doses of adenoviral vector expressing IL-12 enhance or depress the immune response to a coadministered antigen: the role of nitric oxide

Joint immunization with two recombinant adenoviruses, one expressing hepatitis C virus (HCV) core and E1 proteins and another expressing IL-12 (RAdIL-12), strongly potentiates cellular immune response against HCV Ags in BALB/c mice when RAdIL-12 was used at doses of 1 x 105-1 x 107 plaque-forming units. However, cellular immunity against HCV Ags was abolished when higher doses (1 x 108 plaque-forming units) of RAdIL-12 were used. This immunosuppressive effect was associated with marked elevation of IFN-gamma and nitric oxide in the serum and increased cell apoptosis in the spleen. Administration of N-nitro-L -arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, to mice that received high doses of RAdIL-12 was lethal, whereas no apparent systemic toxicity by L -NAME was observed in those immunized with lower doses of the adenovirus. Interestingly, in mice immunized with recombinant adenovirus expressing core and E1 proteins of HCV in combination with RAdIL-12 at low doses (1 x 107 plaque-forming units), L -NAME inhibited T cell proliferation and CTL activity in response to HCV Ags and also production of Abs against adenoviral proteins. In conclusion, gene transfer of IL-12 can increase or abolish cell immunity against an Ag depending of the dose of the vector expressing the cytokine. IL-12 stimulates the synthesis of NO which is needed for the immunostimulating effects of IL-12, but apoptosis of T cells and immunosuppression ensues when IFN-gamma and NO are generated at very high concentrations.     

Change in Apoplastic Aluminum during the Initial Growth Response to Aluminum by Roots of a Tolerant Maize Variety

Root elongation, hematoxylin staining, and changes in the ultrastructure of root-tip cells of an Al-tolerant maize variety (Zea mays L. C 525 M) exposed to nutrient solutions with 20 μm Al (2.1 μm Al³⁺ activity) for 0, 4, and 24 h were investigated in relation to the subcellular distribution of Al using scanning transmission electron microscopy and energy-dispersive x-ray microanalysis on samples fixed by different methods. Inhibition of root-elongation rates, hematoxylin staining, cell wall thickening, and disturbance of the distribution of pyroantimoniate-stainable cations, mainly Ca, was observed only after 4 and not after 24 h of exposure to Al. The occurrence of these transient, toxic Al effects on root elongation and in cell walls was accompanied by the presence of solid Al-P deposits in the walls. Whereas no Al was detectable in cell walls after 24 h, an increase of vacuolar Al was observed after 4 h of exposure. After 24 h, a higher amount of electron-dense deposits containing Al and P or Si was observed in the vacuoles. These results indicate that in this tropical maize variety, tolerance mechanisms that cause a change in apoplastic Al must be active. Our data support the hypothesis that in Al-tolerant plants, Al can rapidly cross the plasma membrane; these data clearly contradict the former conclusions that Al mainly accumulates in the apoplast and enters the symplast only after severe cell damage has occurred.It is largely recognized that root tips are the primary site of Al-induced injury in plants (Ryan et al., 1993). The accumulation of Al in root tips has been found to be significantly correlated with root-growth inhibition in maize (Zea mays L.) varieties differing in Al tolerance (Llugany, 1994; Llugany et al., 1994). In Al-sensitive maize plants an inhibition of root elongation has been observed after only 30 min of exposure to Al (Llugany et al., 1995). Such a short response time, in addition to the common belief (Kochian, 1995) that Al accumulates mainly in the apoplast and crosses the plasma membrane slowly, has led to the hypothesis that Al-induced inhibition of root elongation may be caused by toxicity mechanisms that occur in the apoplast (Rengel, 1990, 1996; Horst, 1995) and that there is no need for Al to enter the symplast to cause primary toxicity effects (Rengel, 1992). However, investigations using the highly Al-sensitive technique of secondary ion MS have shown that significant Al concentrations accumulate in the symplast of root-tip cells of soybean plants after only 30 min of exposure to Al (Lazof et al., 1994, 1996). Recent experiments on giant algae (Chara corallina) cells, where cell walls were separated from the cells by microsurgery, have also shown that Al uptake across the plasmalemma may be linear and occurs without delay (Rengel and Reid, 1997). These investigations support the view that symplastic phytotoxicity mechanisms may also be responsible for Al-induced inhibition of root elongation after short exposure times (Kochian, 1995).More information on the subcellular distribution of Al in root tips would help to establish both the relative importance of apoplastic and symplastic sites in the Al-toxicity syndrome and the role of Al compartmentation in Al resistance or tolerance. Unfortunately, ultrastructural investigations under environmentally realistic growth conditions that relate the subcellular localization of Al in root tips to root growth in Al-tolerant varieties are scarce (Delhaize et al., 1993). Major difficulties for such an approach are the low sensitivity of electron probe x-ray microanalysis for Al determination (Lazof et al., 1994, 1997) and the poor visual distinction of subcellular structures in freeze-dried samples, in combination with the extremely low Al tissue concentrations, which have been shown to cause inhibition of root elongation (Lazof et al., 1994, 1996).Using a highly sensitive monitoring technique for root growth, we have previously shown that 20 μm Al (2.1 μm Al³⁺ activity) causes a significant decrease in the relative root-elongation rate in the Al-tolerant maize var C 525 M after 112 min of exposure, whereas after 24 h the relative elongation rate did not differ from that of the controls (Llugany et al., 1995). In this paper we report results on the changes in the subcellular distribution of Al in root tips during the initial root-growth response (0–24 h) of var C 525 M exposed to 20 μm Al (2.1 μm Al³⁺ activity). Hematoxylin staining, ultrastructural observations, and EDXMA were performed on root tips after 0, 4, and 24 h of exposure of plants to control or Al-containing nutrient solutions to detect a possible relationship between changes in subcellular Al compartmentation and ultrastructural alterations, which may explain why, after a transient inhibition, the root-elongation rate recovers during the initial 24 h of exposure to Al. EDXMA with scanning TEM on glutaraldehyde-fixed, PA-stained, and freeze-substituted samples were performed. Although these techniques only allow a semiquantitative estimation of mineral contents, the better visual resolution obtained results in more reliable data on the subcellular localization than EDXMA with SEM on freeze-dried or frozen-hydrated bulk specimens (Van Steveninck and Van Steveninck, 1991).     

Effect of arthropod extracts on the multiplication of Toxoplasma gondii in mouse peritoneal macrophages

Treatment of toxoplasmosis usually causes secondary effects. It is important to find active substances extracted from natural organisms. In this work we studied some arthropod extracts that have effect against Toxoplasma multiplication inside mouse macrophages. After studying 382 extracts, 23 were selected on the basis of the activity and we found that 13 extracts from orders Polydesmida, Lepidoptera, Orthoptera and Hymenoptera exerted an important inhibition of Toxoplasma multiplication.     

Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates

Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III.