A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca²⁺ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[¹²⁵I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([¹²⁵I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [¹²⁵I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca²⁺. This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca²⁺ and calmodulin decreases by approximately the same amount. However, incubation with Ca²⁺ alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [¹²⁵I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E₁ conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca²⁺ + C28 for SERCA and Ca²⁺ alone for PMCA), and one in which the enzyme is fully active (Ca²⁺ for SERCA and Ca²⁺-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.Although membrane proteins constitute more than 20% of the total proteins, the structure of only few of them is known in detail. An important group of integral membrane proteins are ion-motive ATPases. These proteins belong to the family of P-type ATPases, which share in common the formation of an acid-stable phosphorylated intermediate as part of its reaction cycle. Crystallographic information is available for a few members of this family. There are several crystal structures of the Ca²⁺ pump of sarcoplasmic reticulum (SERCA)² revealing different conformations (1–5), and recently, crystal structures of the H⁺-ATPase (6) and of the Na,K-ATPase were reported as well (7).We are interested in obtaining structural information about the plasma membrane calcium pump (PMCA). This pump is an integral part of the Ca²⁺ signaling mechanism (8). It is highly regulated by calmodulin, which activates this protein by binding to an auto-inhibitory region and changing the conformation of the pump from an inhibited state to an activated one (8, 9). Crystallization of PMCA is particularly challenging because there is no natural source from which this protein can be obtained in large quantities. Moreover, the presence of several isoforms in the same tissue further complicates efforts to obtain a homogeneous sample suitable for crystallization.Information about the structure and assembly of the transmembrane domain of an integral membrane protein can also be obtained from the analysis of the lipid-protein interactions. In this work, we have used a hydrophobic photolabeling method to study the noncovalent interactions between PMCA and the surrounding phospholipids under different experimental conditions that lead to known conformations. We employed the photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[¹²⁵I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([¹²⁵I]TID-PC/16) that has been previously used to analyze lipid-protein interfaces (10–12). This reagent is located in the phospholipidic milieu, and upon photolysis it reacts indiscriminately with its molecular neighbors. It is thus possible to directly analyze the interaction between a membrane protein and lipids belonging to its immediate environment (13–15). By measuring the amount of labeling of SERCA in conditions that promote conformations for which there are well resolved crystal structures, we were able to validate this photolabeling approach as a convenient tool for analyzing conformational changes within transmembrane regions. Furthermore, using this technique on PMCA and comparing the results obtained for SERCA, we were able to draw structural conclusions about these proteins under activated and inhibited states.     

Growth, lipid accumulation, and fatty acid composition in obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts

Obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts were cultured in a carbon-rich medium at different temperatures to investigate whether growth parameters, lipid accumulation, and fatty acid (FA) composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to a lower temperature decreased their specific growth rate, but did not affect their biomass yield (Y_X/S). Obligate and facultative psychrophiles exhibited the highest Y_X/S. Acclimation to lower temperature decreased the lipid yield (Y_L/X) in mesophilic yeasts, but did not affect Y_L/X in facultative psychrophilic ones. Similar Y_L/X were found in both groups of psychrophiles, suggesting that lipid accumulation is not a distinctive characteristic of adaptation to permanently cold environments. The unsaturation of FAs was one major adaptive feature of the yeasts colonizing permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expense of linoleic acid, whereas it was scarce or absent in all the other strains. Increased unsaturation of FAs was also a general acclimatory response of facultative psychrophiles to a lower temperature. These results improve the knowledge of the responses enabling psychrophilic yeasts to cope with the cold and may be of support for potential biotechnological exploitation of these strains.     

Enantioselective catalytic reduction of ketoimines with trichlorosilane promoted by readily available chiral Lewis bases

The straightforward synthesis of a series of enantiomerically pure Lewis basic amides by simple condensation of commercially available enantiopure diamines with picolinic acid is reported. These compounds were shown to promote the enantioselective reduction of ketoimines with trichlorosilane. Working with the model substrate N-phenyl benzophenone imine, the new organocatalysts led to the formation of the corresponding amine, with excellent chemical efficiency (up to 99% chemical yield) and good stereoselectivity (up to 73% ee). Up to 83% of enantioselectivity was reached in the reduction of differently substituted imines.     

Genetic diversity and geographic differentiation of disjunct Atlantic and Amazonian populations of <Emphasis Type="Italic">Psychotria ipecacuanha</Emphasis> (Rubiaceae)

Ipecac (Psychotria ipecacuanha) is a perennial, medicinal herb that grows in the understory of semi-deciduous tropical forests in the Neotropics. Ipecacs present a widely disjunct distribution, with two of its three ranges occurring in Brazil. The Amazonian populations are at least 1600 km from the nearest Atlantic populations. This work used ISSR markers to compare the genetic diversity and structure of populations from the two Brazilian ranges. Lower genetic diversity in Amazon populations (P = 60.11%, Hs = 0.18) and higher genetic diversity in Atlantic populations (P = 73.94%, Hs = 0.20) were detected. Differentiation between ranges were high (θ ^B = 0.6838, G_ST-B = 0.6665). AMOVA revealed that 65.3% of the total molecular variance can be attributed to regional differences between the two ranges. Principal coordinate analyses and cluster analyses organized ipecacs at either individual or population level into two exclusive groups that correspond each to one of the two disjunct ranges, without exception. The results do not support a scenario that postulates human-mediated, long-distance dispersal events as a plausible origin for the distribution of the Brazilian ipecacs, but indicate geographic isolation as a long-standing barrier to genetic exchange and connectivity among populations from different ranges. Conservation implications are discussed.     

Availability of and access to critical habitats in regulated rivers: effects of low-head barriers on threatened lampreys

1.  Conservation of freshwater animal populations requires their access to, as well as sufficient availability of, critical habitats, such as those for reproduction. Abundant small-scale barriers may cause extensive fragmentation of freshwater habitat but, by comparison to larger structures their effects are rarely considered by catchment managers. The relationship between the distribution of, and access to, spawning habitat in a regulated river, characterized by abundant small barriers, was examined for river lamprey Lampetra fluviatilis , a threatened migratory fish.
2.  Telemetry of adult lamprey in the River Derwent, North East England was used to quantify upriver migration and access to spawning habitat, together with surveys of spawning habitat availability and spawning activity between 2002 and 2007.
3.  Access in to the Derwent appeared severely restricted by a tidal barrage, beyond which lamprey migrated rapidly in unobstructed reaches. Of all lamprey tagged in the lower 4 km of river, or ascending the barrage, 64% and 17% passed the first and second weirs respectively, with high flows crucial for this. Although over 98% of lamprey spawning habitat occurred more than 51 km upstream, on average just 1.8% of river lamprey spawners were recorded there.
4.  In order to protect or rehabilitate species or species assemblages, greater attention needs to be paid to the relative spatial distribution of low-head barriers and the resultant availability of key habitats within individual catchments. This is particularly important given the renewed emphasis internationally on low-head hydropower solutions as a source of renewable energy, and the rapid growth in numbers of low-head barriers in many catchments.     

Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium

Cadmium (Cd) is a toxic heavy metal, which can cause severe damage to plant development. The aim of this work was to characterize ultrastructural changes induced by Cd in miniature tomato cultivar Micro-Tom (MT) mutants and their wild-type counterpart. Leaves of diageotropica (dgt) and Never ripe (Nr) tomato hormonal mutants and wild-type MT were analysed by light, scanning and transmission electron microscopy in order to characterize the structural changes caused by the exposure to 1 mM CdCl₂. The effect of Cd on leaf ultrastructure was observed most noticeably in the chloroplasts, which exhibited changes in organelle shape and internal organization, of the thylakoid membranes and stroma. Cd caused an increase in the intercellular spaces in Nr leaves, but a decrease in the intercellular spaces in dgt leaves, as well as a decrease in the size of mesophyll cells in the mutants. Roots of the tomato hormonal mutants, when analysed by light microscopy, exhibited alterations in root diameter and disintegration of the epidermis and the external layers of the cortex. A comparative analysis has allowed the identification of specific Cd-induced ultrastructural changes in wild-type tomato, the pattern of which was not always exhibited by the mutants.     

Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

Mitochondria Are Linked to Calcium Stores in Striated Muscle by Developmentally Regulated Tethering Structures

Bi-directional calcium (Ca²⁺) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca²⁺ stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca²⁺ release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca²⁺ stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR–mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.     

Anaesthesia of free-ranging Northern chamois (<Emphasis Type="Italic">Rupicapra rupicapra</Emphasis>) with xylazine/ketamine and reversal with atipamezole

One-hundred and fifty-five free-ranging Northern chamois (Rupicapra rupicapra) were anaesthetised in the course of a restocking programme using xylazine plus ketamine. Mean ± SD dosages for xylazine and ketamine were 1.9 ± 0.5 and 2.2 ± 0.7 mg/kg, respectively. In 57 chamois, sedation was reversed using 0.3 ± 0.1 mg/kg atipamezole. Although all the anaesthetic dosages tested immobilised free-ranging Northern chamois, shorter induction times (4.8 ± 2.6 min), deeper sedation with no reaction to handling in >90% of the animals and quick reversal (4.0 ± 2.7 min) were obtained using 2.5 mg/kg xylazine plus 3.0 mg/kg ketamine reversed with 0.25 mg/kg atipamezole. Under the conditions of this study, suggested standard doses are 63 mg/animal xylazine plus 76 mg/animal ketamine reversed by 6.3 mg/animal atipamezole. This anaesthetic protocol improves the results from the previous study of Dematteis et al. (Vet Rec 163:184–189, 2008) using xylazine alone.