Properties of unsaturated phospholipid bilayers: Effect of cholesterol

Properties of hydrated unsaturated phosphatidylcholine (PC) lipid bilayers containing 40 mol % cholesterol and of pure PC bilayers have been studied. Various methods were applied, including molecular dynamics simulations, self-consistent field calculations, and the pulsed field gradient nuclear magnetic resonance technique. Lipid bilayers were composed of 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules. Lateral self-diffusion coefficients of the lipids in all these bilayers, mass density distributions of atoms and atom groups with respect to the bilayer normal, the C-H and C-C bond order parameter profiles of each phospholipid hydrocarbon chain with respect to the bilayer normal were calculated. It was shown that the lateral self-diffusion coefficient of PC molecules of the lipid bilayer containing 40 mol % cholesterol is smaller than that for a corresponding pure PC bilayer; the diffusion coefficients increase with increasing the degree of unsaturation of one of the PC chains in bilayers of both types (i.e., in pure bilayers or in bilayers with cholesterol). The presence of cholesterol in a bilayer promoted the extension of saturated and polyunsaturated lipid chains. The condensing effect of cholesterol on the order parameters was more pronounced for the double C=C bonds of polyunsaturated chains than for single C-C bonds of saturated chains.     

Shedding light on the immunomodulatory properties of galectins: novel regulators of innate and adaptive immune responses

Galectins are a large family of structurally related beta-galactoside-binding proteins that play a pivotal role in the control of cell differentiation, proliferation, activation and apoptosis of many different cell types including immune cells. By crosslinking specific glycoconjugates, different members of the galectin family behave as pro-inflammatory or anti-inflammatory "cytokine-like" mediators, acting at different levels of innate and adaptive immune responses. Here we will review recent advances on the role of galectins in key events of the immune and inflammatory response, such as tolerance induction, cell cycle progression, cell adhesion, chemotaxis, antigen presentation and apoptosis. In particular we will examine the influence of individual members of the galectin family in the physiology of different immune cell types involved in innate and adaptive immune responses. Moreover, we will discuss the importance of these sugar-binding proteins as therapeutic targets in Th1- and Th2-mediated immune disorders, an exciting area for future research.     

Degradation of the herbicide atrazine by the soil mycelial fungus INBI 2-26(-)--a producer of cellobiose dehydrogenase

Nonsporulating mycelial fungi producing cellobiose dehydrogenase (CDH) and isolated from soils of South Vietnam with high residual content of dioxins are capable of growing on a solid medium in the presence of high atrazine concentrations (to 500 mg/l). At 20 and 50 mg/l atrazine, the area of fungal colonies was 1.5-1.2-fold larger, respectively, compared with control colonies of the same age, whereas development of the colonies at 500 mg/l atrazine was delayed by 5 days, compared with controls grown in the absence of atrazine. Surface cultivation of the fungus on a minimal medium with glucose as a sole source of carbon and energy decreased the initial concentration of atrazine (20 mg/l) 50 times in 40 days; in addition, no pronounced sorption of atrazine by mycelium was detected. This was paralleled by accumulation in the culture medium of extracellular CDH; atrazine increased the synthesis of this enzyme two- to threefold. Accumulation of beta-glucosidase (a mycelium-associated enzyme) and cellulases preceded the formation of CDH.     

Re-infestation of houses by Triatoma dimidiata after intra-domicile insecticide application in the Yucatán peninsula, Mexico

In most countries, Chagas disease transmission control remains based on domestic insecticide application. We thus evaluated the efficacy of intra-domicile cyfluthrin spraying for the control of Triatoma dimidiata, the only Chagas disease vector in the Yucatán peninsula, Mexico, and monitored potential re-infestation every 15 days for up to 9 months. We found that there was a re-infestation of houses by adult bugs starting 4 months after insecticide application, possibly from sylvatic/peridomicile areas. This points out the need to take into account the potential dispersal of sylvatic/peridomestic adult bugs into the domiciles as well as continuity action for an effective vector control.     

Isolation and Primary Identification of Methylotrophic Yeast Hansenula polymorpha Mutants for Peroxisome Biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorphaHF246leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45°C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45°C but could grow at optimal temperature (37°C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10pex10 mutants (4ts mutants among them); group 2 included 19 mutants that failed to complement otherpex testers: 1 pex1; 2 pex4(1ts); 6 pex5(5ts); 3 pex8; 1 pex13; 6 (3ts) pex19; group 3 contained 22 multiple mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30°C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. Analysis of the spore population from the hybrids of remaining 14 mutants with the pex tester demonstrated the presence of methanol-utilizing segregants, which indicates mutation localization in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed us to localize this mutation in the only PEX gene (PEX1 or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

Isolation and Characterization of a Cellobiose Dehydrogenase Formed by a Nonsporulating Mycelial Fungus INBI 2-26(-)

A nonsporulating fungus isolated from dioxin-containing tropical soils forms cellobiose dehydrogenase when grown in media supplemented by a source of cellulose. The enzyme purified to homogeneity by SDS-PAGE (yield, 43%) had an M_r of 95 kDa; its pH optimum was in the range 5.5–7.0; more than 50% activity was retained at pH 4.0–8.0 (citrate–phosphate buffer). The absorption spectrum of the enzyme in the visible range had the characteristic appearance of flavocytochrome proteins. Cellobiose dehydrogenase oxidized cellobiose and lactose (the respective K _M values at pH 6.0 equaled 4.5 ± 1.5 and 56 M) in the presence of dichlorophenolindophenol (K _M,_app = 15 ± 3 M at pH 6.0) taken as an electron acceptor. Other sugars were barely if at all oxidized by the enzyme. Neither ethyl--D-cellobioside, heptobiose, nor chitotriose inhibited the enzymatic oxidation of lactose, even under the conditions of 100-fold molar excess. The enzyme was weakly inhibited by sodium azide dichlorophenolindophenol reduction and exhibited an affinity for amorphous cellulose. At 55°C and pH 6.0 (optimum stability), time to half-maximum inactivation equaled 99 min. The enzyme reduced by cellobiose was more stable than the nonreduced form. Conversely, the presence of an oxidizer (dichlorophenolindophenol) decreased the stability eight times at pH 6.0. In addition, the enzyme acted as a potent reducer of the one-electron acceptor cytochrome c ³⁺ (K _{M app} = 15 M at pH 6.0).     

The antimetastatic effect of a single low dose of cyclophosphamide involves modulation of galectin-1 and Bcl-2 expression

We have demonstrated that a single low dose of cyclophosphamide has an antimetastatic effect on lymphoma (L-TACB)-bearing rats by modulating the host immune response. Galectin-1, a member of the galectin family with specificity for beta-galactosides, has potent immunomodulatory properties by regulating cell-matrix interactions and T-cell apoptosis. Since galectin-1 is expressed by highly metastatic tumors, in the present study we investigated the participation of this beta-galactoside-binding protein in cyclophosphamide-induced immunomodulation. Inbred " e" rats were s.c. challenged with L-TACB. After 14 days, half of the animals received an i.p. injection of cyclophosphamide (10 mg/kg), and on day 21 tumors and spleens were excised. Cell extracts were prepared and galectin-1 expression was determined by Western blot analysis and correlated with Bcl-2 expression levels and the DNA fragmentation profile. Expression of galectin-1 was significantly decreased in tumors from cyclophosphamide-treated rats compared to non-treated rats. The same effect was observed regarding expression of Bcl-2 by tumors. In contrast, expression of Bcl-2 was significantly higher in spleens from treated animals than in non-treated rats. This effect correlated with a decreased intensity in the pattern of DNA fragmentation of spleen cells from cyclophosphamide-treated animals. Our results suggest that a single low dose of cyclophosphamide modulates the expression of galectin-1 and Bcl-2 by tumors, which could in turn influence the apoptotic threshold of spleen mononuclear cells. This mechanism could explain, at least in part, the antimetastatic effect evidenced in our tumor experimental model.     

Fungal decomposition of oat straw during liquid and solid state fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei 6/16) were grown on oat straw-based liquid and solid media, as well as in a bench-scale reactor, either individually or as co-cultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mould Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely, Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the co-culture of Coriolus hirsutus and Cerrena maxima caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw-based liquid medium, the basidiomycetes consumed up to 40% polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid state straw fermentation, white rot fungi consumed up to 75% cellulose and 55% lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for co-cultures of Mycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide-monitored solid-state fermentation of oat straw by the co-culture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

Opposite effects of galectin-1 on alternative metabolic pathways of L-arginine in resident,inflammatory, and activated macrophages

Recent evidence has implicated galectins and their carbohydrate ligands as master regulators of the inflammatory response. Galectin-1, a member of this family, has shown specific anti-inflammatory and immunoregulatory effects. To gain insight into the potential mechanisms involved in these effects, we investigated the effects of galectin-1 in L-arginine metabolism of peritoneal rat macrophages. Pretreatment of macrophages with galectin-1 resulted in a dose- and time-dependent inhibition of lipopolysaccharide-induced nitric oxide (NO) production, accompanied by a decrease in inducible nitric oxide synthase (iNOS) expression (the classic pathway of L-arginine). On the other hand, galectin-1 favored the balance toward activation of L-arginase, the alternative metabolic pathway of L-arginine. Inhibition of NO production was not the result of increased macrophage apoptosis because addition of this beta-galactoside-binding protein to macrophages under the same experimental conditions did not affect the apoptotic threshold of these cells. To understand how endogenous galectin-1 is regulated in macrophages under inflammatory stress, we finally explored the ultrastructural distribution, expression, and secretion of galectin-1 in resident, inflammatory, and activated macrophages. This study provides an alternative cellular mechanism based on the modulation of L-arginine metabolism to understand the molecular basis of the anti-inflammatory properties displayed by this carbohydrate-binding protein.     

Dynamics of two electrically coupled chaotic neurons: Experimental observations and model analysis

Conductance-based models of neurons from the lobster stomatogastric ganglion (STG) have been developed to understand the observed chaotic behavior of individual STG neurons. These models identify an additional slow dynamical process – calcium exchange and storage in the endoplasmic reticulum – as a biologically plausible source for the observed chaos in the oscillations of these cells. In this paper we test these ideas further by exploring the dynamical behavior when two model neurons are coupled by electrical or gap junction connections. We compare in detail the model results to the laboratory measurements of electrically-coupled neurons that we reported earlier. The experiments on the biological neurons varied the strength of the effective coupling by applying a parallel, artificial synapse, which changed both the magnitude and polarity of the conductance between the neurons. We observed a sequence of bifurcations that took the neurons from strongly synchronized in-phase behavior, through uncorrelated chaotic oscillations to strongly synchronized – and now regular – out-of-phase behavior. The model calculations reproduce these observations quantitatively, indicating that slow subcellular processes could account for the mechanisms involved in the synchronization and regularization of the otherwise individual chaotic activities. Received: 28 June 1999 / Accepted in revised form: 30 June 2000