Signal molecules in the peanut–bradyrhizobia interaction

Main nodulation signal molecules in the peanut–bradyrhizobia interaction were examined. Flavonoids exuded by Arachis hypogaea L. cultivar Tegua were genistein, daidzein and chrysin, the latest being released in lower quantities. Thin layer chromatography analysis from genistein-induced bacterial cultures of three peanut bradyrhizobia resulted in an identical Nod factor pattern, suggesting low variability in genes involved in the synthesis of these molecules. Structural study of Nod factor by mass spectrometry and NMR analysis revealed that it shares a variety of substituents with the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium spp. Nodulation assays in legumes nodulated by these rhizobia demonstrated differences between them and the three peanut bradyrhizobia. The three isolates were classified as Bradyrhizobium sp. Their fixation gene nifD and the common nodulation genes nodD and nodA were also analyzed. Accession numbers: AY427207, EF202193, EF158295 (16S rRNA gene of strains NLH25, NOD31 and NDEHE, respectively); DQ295199, DQ295200, DQ295201 (Partial nifD gene sequences of strains NLH25, NOD31 and NDEHE, respectively).     

Outdoor and indoor cultivation of Spirulina platensis in the extreme south of Brazil

Water supplemented with 10% or 20% (v/v) of Zarrouk medium was used to cultivate Spirulina platensis in closed and open bioreactors under controlled conditions (30 degrees C, 32.5 micromol m(-2) s(-1), 12 h light/dark photoperiod) and in a greenhouse (9.4 to 46 degrees C, up to 2800 micromol m(-2) s(-1), variable day length photoperiod) using different initial biomass concentrations (X0) in the extreme south of Brazil (32.05 degrees S, 52.11 degrees W). Under controlled conditions the maximum specific growth rate (micromax) was 0.102 d(-1), the biomass doubling time (t(d)) was 6.8 d, the maximum dry biomass concentration (Xmax) was 1.94 g L(-1) and the maximum productivity (Pmax) was 0.059 g L(-)1 d(-1), while the corresponding values in the greenhouse experiments were micromax = 0.322 d(-1), t(d) = 2.2 d, Xmax = 1.73 g L(-1) and Pmax = 0.112 g L(-1) d(-1). Under controlled conditions the highest values for these parameters occurred when X0 = 0.15 g L(-1), while in the greenhouse X0 = 0.4 g L(-1) produced the highest values. These results show that the cultivation of S. platensis in greenhouses in the extreme south of Brazil is technically viable and that the S. platensis inoculum and the concentration of Zarrouk medium can be combined in such a way as to obtain growth and productivity parameters comparable, or superior, to those occurring in bioreactors under controlled conditions of temperature, illuminance and photoperiod.     

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Ι) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at E _m = +197 mV (heme c) and −4.5 mV (heme b). Variable-temperature (4–120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe–4S]⁺ cluster and overlapping signals associated with at least three types of [4Fe–4S]⁺ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called “low-pH” and “high-pH,” changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Unraveling the mechanisms of tryptophan fluorescence quenching in the triosephosphate isomerase from Giardia lamblia

In the native state several proteins exhibit a quenching of fluorescence of their tryptophans. We studied triosephosphate isomerase from Giardia lamblia (GlTIM) to dissect the mechanisms that account for the quenching of fluorescence of its Trp. GlTIM contains four Trp per monomer (Trp75, Trp162, Trp173, and Trp196) distributed throughout the 3D structure. The fluorescence of the denatured enzyme is 3-fold higher than that of native GlTIM. To ascertain the origin of this phenomenon, single and triple mutants of Trp per Phe were made. The intrinsic fluorescence was determined, and the data were interpreted on the basis of the crystal structure of the enzyme. Our data show that the fluorescence of all Trp residues is quenched through two different mechanisms. In one, fluorescence is quenched by aromatic-aromatic interactions due to the proximity and orientation of the indole groups of Trp196 and Trp162. The magnitude of the quenching of fluorescence in Trp162 is higher than in the other three Trp. Fluorescence quenching is also due to energy transfer to the charged residues that surround Trp 75, 173 and 196. Further analysis of the fluorescence of GlTIM showed that, among TIMs from other parasites, Trp at position 12 exhibits rather unique properties.     

Latitudinal Variation in Leaf and Tree Traits of the Mangrove Avicennia germinans (Avicenniaceae) in the Central Region of the Gulf of Mexico

The variation in leaf mass per area, leaf nutrients (% carbon, nitrogen and phosphorus), and the allometric relation between tree height and diameter of the black mangrove, Avicennia germinans, were explored in nine mangrove forests in similar environments along a 5° latitudinal gradient in the central region of the Gulf of Mexico, as indicated by a southward increase in temperature and precipitation. There was no correlation between leaf nitrogen or phosphorus content and latitude. Leaf mass per area and leaf carbon content were positively correlated with latitude and negatively correlated with temperature and annual rainfall, whereas asymptotic tree height and maximum diameter showed the opposite trend. Such patterns suggest a trade-off between leaf traits and tree size which may be constrained by the same environmental factors along a dry-cold to humid-warm latitudinal gradient.     

Ventilatory responses to acute and chronic hypoxia are altered in female but not male Paskin-deficient mice

Proteins harboring a Per-Arnt-Sim (PAS) domain are versatile and allow archaea, bacteria, and plants to sense oxygen partial pressure, as well as light intensity and redox potential. A PAS domain associated with a histidine kinase domain is found in FixL, the oxygen sensor molecule of Rhizobium species. PASKIN is the mammalian homolog of FixL, but its function is far from being understood. Using whole body plethysmography, we evaluated the ventilatory response to acute and chronic hypoxia of homozygous deficient male and female PASKIN mice (Paskin-/-). Although only slight ventilatory differences were found in males, female Paskin-/- mice increased ventilatory response to acute hypoxia. Unexpectedly, females had an impaired ability to reach ventilatory acclimatization in response to chronic hypoxia. Central control of ventilation occurs in the brain stem respiratory centers and is modulated by catecholamines via tyrosine hydroxylase (TH) activity. We observed that TH activity was altered in male and female Paskin-/- mice. Peripheral chemoreceptor effects on ventilation were evaluated by exposing animals to hyperoxia (Dejours test) and domperidone, a peripheral ventilatory stimulant drug directly affecting the carotid sinus nerve discharge. Male and female Paskin-/- had normal peripheral chemosensory (carotid bodies) responses. In summary, our observations suggest that PASKIN is involved in the central control of hypoxic ventilation, modulating ventilation in a gender-dependent manner.     

Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and beta2-integrin surface expression in human neutrophils

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH(i)) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na(+)/H(+) exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pH(i) was reduced while maintaining normal extracellular pH, by using an NH(4)Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH(4)Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, beta(2)-integrin (CD11b and CD18) was only slightly upregulated in the low-pH(i) condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pH(i). Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface.     

Removal of copper, lead, and zinc from contaminated water by saltbush biomass: analysis of the optimum binding, stripping, and binding mechanism

Experiments performed on the Cu(II), Pb(II), and Zn(II) binding by saltbush biomass (Atriplex canescens) showed that the metal binding increased as pH increased from 2.0 to 5.0. The highest amounts of Cu, Pb, and Zn bound by the native biomass varied from 48-89%, 89-94%, and 65-73%, respectively. The hydrolyzed biomass bound similar amount of Pb and 50% more Cu and Zn than the native. The esterified biomass had a lower binding capacity than native; however, esterified flowers bound 45% more Cu at pH 2.0 than native flowers. The optimum binding time was 10 min or less. More than 60% of the bound Cu was recovered using 0.1 mM HCl, while more than 90% of Pb was recovered with either HCl or sodium citrate at 0.1 mM. For Zn, 0.1 mM sodium citrate allowed the recovery of 75%. Results indicated that carboxyl groups participate in the Cu, Pb, and Zn binding.     

Alternative medium for production of Pleurotus ostreatus biomass and potential antitumor polysaccharides

Pleurotus species are recognized for producing beta-glucans with important medicinal properties as a constituent of the cellular wall of the fruiting body or of the mycelium. The aims of this work were to select a culture medium that maximized the production of biomass and polysaccharides produced by Pleurotus ostreatus DSM 1833 and to evaluate the selected medium in two values of initial oxygen transfer rate -K(L)a (10.2 and 19.3 h(-1)). A 2* *4 factorial design was constructed to evaluate the supplementation of wheat extract with corn steep liquor--CSL (10 or 20 g L(-1)), yeast extract--YE (2 or 5gL(-1)), ammonium sulfate--AS (0 or 5 g L(-1)) and glucose (20 or 40 g L(-1)). In terms of maximum productivity in biomass and global productivity in polysaccharides, the best values were obtained when 5 g L(-1) of YE and 40 g L(-1) of glucose were used. In terms of maximum concentration of biomass, the best results were obtained when 20 g L(-1) of CSL and 40 g L(-1) of glucose were used. The best results in terms of production of biomass and polysaccharides were achieved when lower initial K(L)a (10.2 h(-1)) was used.     

Design, synthesis and in vitro antiprotozoal activity of benzimidazole-pentamidine hybrids

A series of ten novel hybrids from benzimidazole and pentamidine were prepared using a short synthetic route. Each compound was tested in vitro against the protozoa Trichomonas vaginalis, Giardia lamblia, Entamoeba histolytica, Leishmania mexicana, and Plasmodium berghei, in comparison with pentamidine and metronidazole. Some analogues showed high bioactivity in the low micromolar range (IC(50)<1 microM) against the first four protozoa, which make them significantly more potent than either standard. 1,5-bis[4-(5-methoxy-1H-benzimidazole-2-yl)phenoxy]pentane (2) was 3- and 9-fold more potent againstG. lamblia than metronidazole and pentamidine, respectively. This compound was 23-, 108-, and 13-fold more active than pentamidine against T. vaginalis, E. histolytica and L. mexicana, respectively. Studying further structure-activity relationships through the use of bioisosteric substitution in these hybrids should provide new leads against protozoal diseases.