Time-resolved methods in biophysics. 4. Broadband pump-probe spectroscopy system with sub-20 fs temporal resolution for the study of energy transfer processes in photosynthesis.

In this paper we discuss how to push the temporal resolution limits of transient absorption spectroscopy in order to detect very fast processes (energy relaxation, energy or charge transfer, vibrational coherence) taking place in molecules of biological relevance. After reviewing the main principles of femtosecond pump-probe spectroscopy, we describe an experimental setup based on two synchronized non-collinear optical parametric amplifiers (NOPAs). Each NOPA can be independently configured to generate ultra-broadband sub-10 fs visible pulses, tunable 10-15 fs visible pulses, tunable 15-40 fs near-infrared pulses (900-1500 nm). This system enables to perform pump-probe experiments over nearly two octaves of spectrum with sub-20 fs temporal resolution. We then present an application example highlighting the capability of this instrument to track excited state dynamics in biomolecules on the sub-100 fs timescale: the study of carotenoid-bacteriochlorophyll energy transfer processes in peripheral light-harvesting complexes (LH2) from purple bacteria. We show that, by comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it is possible to visualize in the time domain the primary events in photosynthesis.     

Helicobacter pylori EstV: identification, cloning, and characterization of the first lipase isolated from an epsilon-proteobacterium

Bacterial lipases are attracting an enormous amount of attention due to their wide biotechnological applications and due to their roles as virulence factors in some bacteria. Helicobacter pylori is a significant and widespread pathogen which produces a lipase(s) and phospholipases that seem to play a role in mucus degradation and the release of proinflammatory and cytotoxic compounds. However, no H. pylori lipase(s) has been isolated and described previously. Therefore, a search for putative lipase-encoding genes was performed by comparing the amino acid sequences of 53 known lipolytic enzymes with the deduced proteome of H. pylori. As a result, we isolated, cloned, purified, and characterized EstV, a novel lipolytic enzyme encoded by open reading frame HP0739 of H. pylori 26695, and classified it in family V of the bacterial lipases. This enzyme has the properties of a small, cell-bound carboxylesterase (EC 3.1.1.1) that is active mostly with short-chain substrates and does not exhibit interfacial activation. EstV is stable and does not require additional cofactors, and the maximum activity occurs at 50 degrees C and pH 10. This unique enzyme is the first lipase isolated from H. pylori that has been described, and it might contribute to ulcer development, as inhibition by two antiulcer substances (beta-aescin and glycyrrhizic acid) suggests. EstV is also the first lipase from an epsilon-proteobacterium to be described. Furthermore, this enzyme is a new member of family V, probably the least-known family of bacterial lipases, and the first lipase of this family for which kinetic behavior, inhibition by natural substances, and other key biochemical features are reported.     

Mass transfer enhancement in moving biofilm structures

Biofilms are layers of microbial cells growing on an interface and they can form highly complex structures adapted to a wide variety of environmental conditions. Biofilm streamers have a small immobile base attached to the support and a flexible tail elongated in the flow direction, which can vibrate in fast flows. Herein we report numerical results for the role of the periodical movement of biofilm streamers on the nutrient uptake and in general on the solute mass transfer enhancement due to flow-induced oscillations. We developed what to our knowledge is a novel two-dimensional fluid-structure interaction model coupled to unsteady solute mass transport and solved the model using the finite element method with a moving mesh. Results demonstrate that the oscillatory movement of the biofilm tail significantly increases the substrate uptake. The mass transfer coefficient is the highest in regions close to the streamer tip. The reason for substrate transfer enhancement is the increase in speed of tip movement relative to the surrounding liquid, thereby reducing the thickness of the mass transfer boundary layer. In addition, we show that the relative mass transfer enhancement in unsteady conditions compared with the rigid static structure is larger at higher flow velocities, and this relative increase favors a more flexible structure.     

PHYTOEXTRACTION OF METAL POLLUTED SOILS AROUND A Pb-Zn MINE BY CROP PLANTS

In order to assess their practical capability for the absorption and accumulation of Pb, Zn, and Cu, five common crop plants, i.e. maize (Zea mays), sunflower (Helianthus annuus), canola (Brassica napus), barley (Hordeum vulgare) and White lupine (Lupinus albus) were tested in pot experiments using six soil samples taken from mine tailings, pasture and arable soils around an old Pb-Zn mine in Spain. Metal concentration ranges of the soils were 76.2–785 mg kg^?1, 127–1652 mg kg^?1, and 12.4–82.6 mg kg^?1 for Zn, Pb, and Cu, respectively. With the exception of the highest polluted sample, soil total metal concentration did not influence significantly biomass yields of each crop for the different growth substrates. The order found for the total metal accumulation rate (TMAR) in the crops was Zn>>Pb > Cu, with maize reaching the highest metal concentrations. Pb root concentrations were markedly higher than those of shoots for all the crops, while Zn and Cu were translocated to shoots more efficiently. Concentrations of metals extracted by EDTA and BCR sequential extraction were well correlated, in general, with both root metal content and TMAR. CaCl₂-extracted Zn was well correlated with root concentrations, TMAR and, in some cases, with shoot contents. Our study showed that the test crops were not feasible to remediate the heavily or moderately contaminated soils studied here in order to achieve the total metal soil concentrations required by the current European laws.     

Antimicrobial activity of the acidophilic eukaryotic microalga Coccomyxa onubensis

Extremophilic microalgae are unexplored as a source of pharmaceuticals despite the fact that its biomass can be produced at large scale with low risk of contamination. A significant amount of antimicrobial activity was produced by extracts obtained from the eukaryotic acidophilic microalgae Coccomyxa onubensis in non‐polar solvents, such as hexane, diethyl ether, and chloroform or in weakly polar solvents, such as dichloromethane, against Gram‐negative and Gram‐positive bacteria, and also the yeast Candida albicans. The most effective activity was shown by chloroform extract against Escherichia coli S, Salmonella enterica, and Proteus mirabilis; hexane extract against P. mirabilis, Sa. enterica, and Ca. albicans; dichloromethane extract against Sa. enterica or diethyl ether extract against E. coli S and the Gram‐positive Staphylococcus aureus MB. The lowest minimum inhibitory concentration values were recorded against E. coli S (305 μg mL ^?1) and P. mirabilis (153 μg mL ^?1) (using chloroform extract) and against P. mirabilis (106 μg mL^?1) (using hexane extract). Fatty acids, but not carotenoids, seem to be involved in the antimicrobial activity of this microalga. However, further biochemical and biotechnological studies must be conducted in order to characterize and purify the bioactive principles from Co. onubensis for assessing its potential as a pharmaceutical source and feasibility of production.     

Improvement of DNA transfection with cationic liposomes

The increasing use of cationic liposomes as vectors for DNA transfection of eukaryotic cells is due to its high efficiency and reproducibility. After the interaction of the DNA cationic-liposome complexes (DNA-CLC) with the plasma membrane, the entry into the cells delivers the DNA-CLC to the endosome-lysosome pathway where some of the DNA-CLC are degraded. The non-degraded DNA that escapes to the cytoplasm, still has to transverse the nuclear membrane to be transcribed and then translated. To improve the efficiency of the whole process, we can manipulate the DNA (sequences, promoters, enhancers, nuclear localisation signals, etc), the DNA-CLC (lipids) or the plasmatic, endosomal and/or nuclear cellular membranes (ultrasound, electroporation, Ca++, pH of the endosomes, mitosis, fusogenic peptides, nuclear localisation signals, etc). Most of these methods have been generally used individually but in combination, may greatly improve the efficiency and reproducibility of in vitro transfection. While much of this work remains yet to be done and present results further explored, the application of these efforts is essential to the future development of new gene therapy strategies.     

Synthesis and in vitro trypanocide activity of several polycyclic drimane-quinone derivatives

The Diels-Alder reaction between two polygodial-derived dienes and simple quinones to yield substituted naphtho- and anthraquinones, is described. The in vitro trypanocide activity for the series was determined. Two of the new compounds showed an activity ten and two times higher, respectively, than nifurtimox and benznidazole, the medicines of choice for the treatment of the acute Chagas' disease.     

Immunomodulation by Histoplasma capsulatum products; polyclonal activation and mitogenic effects

The presence of reactive spleen cells to sheep red blood cells (SRBC) in nonimmunized BALB/c mice injected with histoplasmin, the culture filtrate of Histoplasma capsulatum, was monitored for 21 days following inoculation. Polyclonal activation, as evidenced by a sharp increase in the number of anti-SRBC rosetteforming cells (RFC), as well as an enhanced response to heterologous non-cross-reactive erythrocytes from other species, was found in the spleens of these rodents on Days 11 to 13. Elimination of B-cell-derived RFC by the addition of complement indicated that the erythrocyte-binding cells consisted of both T- and B-lymphocytes. An immunosuppressive effect was detected if histoplasmin was injected 2 days before the antigen (SRBC), but could be reversed by injecting the filtrate 30 min prior to the antigen, as is found with polyclonal activators displaying immunosuppressive activity. Histoplasmin also had a mitogenic effect on lymphocyte obtained from the spleen, bone marrow, and thymus similar in magnitude to that produced by lipopolysaccharide (LPS) and concanaval in A. The biological significance of these findings is discussed.     

Late pregnancy suppresses relapses in experimental autoimmune encephalomyelitis: evidence for a suppressive pregnancy-related serum factor

Women with multiple sclerosis have significantly diminished disease activity during pregnancy. The purpose of our study was to identify the underlying mechanism for the diminished disease activity. We found that during the period of late pregnancy there is protection against paralysis, during both the induction and effector phases of relapsing experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. We did not find any changes in the cytokine secretion profiles or the proliferative activity of autoreactive T cells from mice induced during late pregnancy compared with virgin controls. In mice mated after disease onset, the inflammatory histologic lesions did not clear, despite marked clinical improvement during pregnancy. We found evidence for a serum factor present in late pregnancy that suppresses T cell activation. In the presence of sera taken from mice late in pregnancy, the proliferative response and IL-2 production of proteolipid protein p139-151-specific T cells were significantly diminished as compared with stimulation in the presence of normal mouse sera. In conclusion, serum from late pregnancy has the capacity to down-regulate T cell responses and might be associated with the amelioration of disease activity in experimental autoimmune encephalomyelitis.     

Dual effect of cyclic GMP on renal brush border Na-H antiporter.

The Na-H antiporter of renal-brush border membranes is inhibited by cyclic AMP and stimulated by protein kinase C. The proximal tubule contains guanylate cyclase and is capable of cyclic GMP production. The effect of cGMP on renal Na-H antiporter activity was analyzed in phosphorylated brush border membranes by 22Na uptake in the presence or absence of 1 mM amiloride. 8-Bromo cyclic GMP (1 microM) increased the amiloride-sensitive 22Na uptake in control from 1.26 +/- 0.13 to 1.54 +/- 0.12 nmol/mg/protein/10 sec, P less than 0.01, without altering the amiloride-insensitive component. In the absence of exogenous ATP, cGMP also stimulated the amiloride-sensitive 22Na uptake, which can be explained by the presence of endogenous ATP in concentrations of up to 50 microM in the membranes. In ATP-depleted membrane vesicles, however, cGMP inhibited the amiloride-sensitive 22Na uptake. These data indicate that cGMP acts on the Na-H antiporter by at least two different mechanisms, one of which is ATP dependent. It is likely that cGMP-dependent protein kinase mediates the stimulatory effects seen in the presence of ATP, and the inhibition seen in ATP-depleted membranes results from cGMP direct action on the Na-H antiporter.