Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.     

Role of the cyclooxygenase pathway in the protection against postischemic stunning in conscious sheep

Objective: There are controversial reports in conscious animals regarding the role of cyclooxygenase-2 in late preconditioning (LP). This study analyzed the effect of COX-2 involvement in non-preconditioned hearts (NP) and in mediation of LP protection against stunning in conscious sheep submitted to a prolonged reversible ischemia. Methods: Six groups were considered: NP: 12 min ischemia and 120 min reperfusion; LP consisting of six periods of 5 min-ischemia-5 min reperfusion 24 h before the 12 min ischemia; NP and LP with either the non-selective COX-1 and COX-2 inhibitor, aspirin (20 mg/kg), or the specific COX-2 inhibitor, celecoxib (3 mg/kg) before the 12 min ischemic period. Results: Mean postischemic wall thickening fraction (as % of preischemic values) improved from 49.6 ± 4.0% in NP to 72.5 ± 3.5% in LP (p < 0.01) and a similar protection was obtained with aspirin and celecoxib in NP hearts (p < 0.01). Neither aspirin nor celecoxib administration prior to the prolonged ischemia on day 2 abrogated LP improvement of postischemic dysfunction. Moreover, LP with aspirin improved the protective response (80.7 ± 2.6%) over that obtained with aspirin in NP hearts (66.6 ± 4.7%, p < 0.05). This effect was not obtained with celecoxib. Conclusions: Aspirin and celecoxib showed that COX-2 has a detrimental effect on mechanical cardioprotection in NP hearts of conscious sheep submitted to a prolonged reversible ischemia, and does not seem to participate as mediator of LP. Aspirin revealed a similar COX-1 deleterious action, since only when both COX-1 and COX-2 were inhibited, LP was put in evidence adding functional improvement over that obtained in NP hearts treated with aspirin.     

Kinetics of bimolecular reactions in model bilayers and biological membranes. A critical review

The quantitative study of the probability of molecular encounters giving rise to a reaction in membranes is a challenging discipline. Model systems, model in the sense that they use model bilayers and model reactants, have been widely used for this purpose, but the methodologies employed for the analysis of the results obtained in experiments, and for experimental design, are so disparate that a concerned experimentalist has difficulty in deciding about the value of each approach. This review intends to examine the several approaches that can be found in the literature showing, when feasible, the weakness, strengths and limits of application of each of them. There is not, so far, a full experimental validation of the most promising theories for the analysis of reactions in two dimensions, what leaves open a large field for new research. The major challenge resides in the time range in which the processes take place, but the possibilities of the existing techniques for these studies are far from exhausted. We review also the attempts of several authors to quantitatively analyze the kinetics of reactions in biological membranes. Especially in this field, the recently developed microspectroscopies enclose a still unexplored potential.     

Biomass and primary production of a 8–11 m depth meadow versus <3 m depth meadows of the seagrass Cymodocea nodosa (Ucria) Ascherson

Current knowledge about the abundance, growth, and primary production of the seagrass Cymodocea nodosa (Ucria) Ascherson is biased towards shallow (depth <3 m) meadows although this species also forms extensive meadows at larger depths along the coastlines. The biomass and primary production of a C. nodosa meadow located at a depth of 8–11 m was estimated at the time of maximum annual vegetative development (summer) using reconstruction techniques, and compared with those available from shallow meadows of this species. A depth-referenced data base of values at the time of maximum annual development was compiled to that end. The vegetative development of C. nodosa at 8–11 m depth was not different from that achieved by shallow (depth <3 m) meadows of this species. Only shoot density, which decreased from 1637 to 605 shoots m⁻², and the annual rate of elongation of the horizontal rhizome, which increased from 23 to 71 cm apex⁻¹ year⁻¹, were different as depth increased from <3 to 8–11 m. Depth was a poor predictor of the vegetative development and primary production of C. nodosa. The biomass of rhizomes and roots decreased with depth (g DW m⁻² = 480 (±53, S.E.) − 32 (±15, S.E.) depth (in m); R² = 0.12, F = 4.65, d.f. = 35, P = 0.0381) which made total biomass of the meadow to show a trend of decrease with depth but the variance of biomass data explained by depth was low. The annual rate of elongation of the horizontal rhizome showed a significant positive relationship with depth (cm apex⁻¹ year⁻¹ = 18 (±5.1, S.E.) + 5.0 (±1.33, S.E.) depth (in m); R² = 0.50, F = 14.07, d.f. = 14, P = 0.0021). As shoot size and growth did not change significantly with depth, the reduction of shoot density should drive any changes of biomass and productivity of C. nodosa as depth increases. The processes by which this reduction of C. nodosa abundance with depth occur remain to be elucidated.     

Coexistence and divergence of tropical dry forests and savannas in southern Mexico

Aim The purpose of the study was to assess the degree of floristic differentiation between tropical dry forest (TDF) and savanna occurring in a single landscape. This comparison provides information on the responses of vegetation to the prevailing environmental conditions, while it also allows us to make inferences about large‐scale events and processes, both biogeographical and evolutionary. Our approach included three levels of analysis: (1) taxonomic, (2) morphological and (3) vegetational. Location The seasonal dry tropical landscape in the Nizanda region, Oaxaca State, southern Mexico. The landscape comprises a complex vegetation mosaic in which tropical dry forest and savannas are the most conspicuous components. Methods Comparisons between TDF and savanna were based on inventories for these communities produced after 8 years of botanical survey. At the taxonomic level, the relative representation of taxa of different hierarchical levels in each community was examined. Morphological analyses required the classification of species on each of three criteria: (1) growth form, (2) life form and (3) growth habit. Vegetation level analysis was based on the frequencies of taxa in one hundred 100‐m² composition plots with which matrices of binary data were constructed for species, genera and families. These were subjected to classification analysis with Ward's method and using Euclidean distances as the dissimilarity algorithm. Results The combined flora for both communities comprised 600 species, 375 genera and 94 families; between them they shared 31, 40 and 34 taxa, respectively. The corresponding Sørensen similarity values were 10%, 21% and 72%, respectively. Ranking genera and families according to their species richness displayed large differences between savanna and TDF. Large differences between these communities were observed for Acanthaceae, Cactaceae, Euphorbiaceae and Mimosaceae, whereas Fabaceae and Asteraceae had similar high ranks according to the species richness in the two systems. The growth form spectrum diverged between the two communities, with TDF having more trees, shrubs and climbers. Savanna was characterised by forbs and graminoid herbs. Growth habit spectra revealed a clear dominance of herbaceous and suffruticose plants in savanna, and of woody elements and epiphytes in TDF. Regarding Raunkiaer's life forms, savanna had relatively more hemicryptophytes, and TDF more phanerophytes. Classification analyses showed that savanna and TDF forest samples kept their identities, regardless of taxonomic level (species, genera and families) at which the analyses were performed. Main conclusions The TDF and savanna of Nizanda represent two floristic systems with a large degree of differentiation at all taxonomic levels and patterns of morphological attributes. This suggest that the two floristic sets have evolved independently for extended periods of time, despite their close proximity. One important implication of this floristic differentiation is the large joint contribution made by these communities to the regional flora.     

Diversity of intersegmental auditory neurons in a bush cricket

Various auditory interneurons of the duetting bush cricket Ancistrura nigrovittata with axons ascending to the brain are presented. In this species, more intersegmental sound-activated neurons have been identified than in any other bush cricket so far, among them a new type of ascending neuron with posterior soma in the prothoracic ganglion (AN4). These interneurons show not only morphological differences in the prothoracic ganglion and the brain, but also respond differently to carrier frequencies, intensity and direction. As a set of neurons, they show graded differences for all of these parameters. A response type not described among intersegmental neurons of crickets and other bush crickets so far is found in the AN3 neuron with a tonic response, broad frequency tuning and little directional dependence. All neurons, with the exception of AN3, respond in a relatively similar manner to the temporal patterns of the male song: phasically to high syllable repetitions and rhythmically to low syllable repetitions. The strongest coupling to the temporal pattern is found in TN1. In contrast to behavior the neuronal responses depend little on syllable duration. AN4, AN5 and TN1 respond well to the female song. AN4 (at higher intensities) and TN1 respond well to a complete duet.     

Renal mitochondrial dysfunction in spontaneously hypertensive rats is attenuated by losartan but not by amlodipine

Mitochondrial dysfunction is associated with cardiovascular damage; however, data on a possible association with kidney damage are scarce. Here, we aimed at investigating whether 1) kidney impairment is related to mitochondrial dysfunction; and 2) ANG II blockade, compared with Ca2+ channel blockade, can reverse potential mitochondrial changes in hypertension. Eight-week-old male spontaneously hypertensive rats (SHR) received water containing losartan (40 mg.kg-1.day-1, SHR+Los), amlodipine (3 mg.kg-1.day-1, SHR+Amlo), or no additions (SHR) for 6 mo. Wistar-Kyoto rats (WKY) were normotensive controls. Glomerular and tubulointerstitial damage, systolic blood pressure, and proteinuria were higher, and creatinine clearance was lower in SHR vs. SHR+Los and WKY. In SHR+Amlo, blood pressure was similar to WKY, kidney function was similar to SHR, and renal lesions were lower than in SHR, but higher than in SHR+Los. In kidney mitochondria from SHR and SHR+Amlo, membrane potential, nitric oxide synthase, manganese-superoxide dismutase and cytochrome oxidase activities, and uncoupling protein-2 content were lower than in SHR+Los and WKY. In SHR and SHR+Amlo, mitochondrial H2O2 production was higher than in SHR+Los and WKY. Renal glutathione content was lower in SHR+Amlo relative to SHR, SHR+Los, and WKY. In SHR and SHR+Amlo, glutathione was relatively more oxidized than in SHR+Los and WKY. Tubulointerstitial alpha-smooth muscle actin labeling was inversely related to manganese-superoxide dismutase activity and uncoupling protein-2 content. These findings suggest that oxidant stress is associated with renal mitochondrial dysfunction in SHR. The mitochondrial-antioxidant actions of losartan may be an additional or alternative way to explain some of the beneficial effects of AT1-receptor antagonists.     

The role of foetal red blood cells in protecting cultured lymphocytes against diepoxybutane-induced chromosome breaks

Diepoxybutane (DEB) is an established mutagen that induces chromosome damage following in vitro treatment of peripheral blood lymphocytes. It is widely used to identify patients with Fanconi Anemia (FA), a clinical situation that is characterized, besides the hypersensitivity to DEB, by an elevated foetal haemoglobin (HbF) content in the peripheral blood. In a previous study, we showed that red blood cells (RBC) from normal individuals can protect cultured lymphocytes against chromosomal breaks induced by DEB and demonstrated the particular role of haemoglobin in the protective effect. In the present work, we studied the influence of RBC extracted from umbilical cord blood of neonates (F cells) on the frequency of DEB-induced chromosome breaks in lymphocyte cultures from normal individuals. Simultaneously, we determined individual GSTT1 and GSTM1 genotypes and the activity of Pi-class glutathione S-transferase (GSTP), catalase and superoxide dismutase (SOD) in adult and foetal RBC. Our results show that F cells, in comparison with adult RBC, elicit a better protection of cultured lymphocytes from normal individuals against chromosome breaks induced by DEB. Variability in the protective effect among RBC from different individuals was observed; we confirmed that the GSTT1 genotype modulates this inter-individual variability, but it is not sufficient to explain all of the protective effect of F cells. Our results suggest that the increased protective effect of F cells can be, at least in part, correlated with an increase in the activity of glutathione S-transferase, catalase and superoxide dismutase, in particular Cu/Zn SOD, in F cells compared with adult RBC.