NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer Part I. Discovering the guidelines

The development of “molecular rulers” would allow one to quantitatively locate intercalants within the liposomal bilayer. To this end, we have attempted to correlate the ¹³C NMR chemical shift of a polarizable “reporter” carbon (e.g., carbonyl) of the intercalant—with the E_T(30) polarity it experiences, and with its Angstrom distance from the interface. This requires families of molecules with the same two “reporter carbons” separated by a fixed distance, residing at various depths/polarities within the bilayer. The families studied included 4,4-dialkylcyclohexa-2,5-dienones 1, benzenediacetic esters 15, benzenedipropionic esters 17, 4-alkoxybenzaldehydes 19 and methyl 4-alkoxybenzoates 22. These compounds possessed the following characteristics: (1) a planar backbone; (2) polar/hydrophilic “head” groups; (3) modular hydrophobic tails; (4) large changes in the ¹³C NMR chemical shift (Δδ) of the reporter atoms with solvent polarity. These studies revealed a fifth requirement, namely: (5) the reporter carbons must not be strongly conjugated, lest it reflect the charge build-up at another site within the conjugated system.     

Improving the clinical efficiency of T2 mapping of ligament integrity

Current MR methods use T₂^? relaxation time as a surrogate measure of ligament strength. Currently, a multi-echo voxel-wise least squares fit is the gold standard to create T₂^? maps; however, the post-processing is time-intensive and serves as a stopgap for clinical use. The study objective was to determine if an alternative method could improve post-processing time without sacrificing fidelity of T₂^? values for eventual translational use in the clinic. Using a 6 echo FLASH sequence, three different methods were used to determine intact posterior cruciate ligament (PCL) median T₂^? Two of these methods utilized a voxel-wise method to establish T₂^? maps: (1) a current “gold standard” method using a voxel-wise 6 echo least-squares fit (6LS) and (2) a voxel-wise 2 echo point T₂^? determination (2MM). The third method used median ligament signal intensity and a single nonlinear least-squares fit (6LS_ROI) instead of a voxel-wise basis. The resulting median T₂^? values of the PCL and computational time were compared. The median T₂^? values were 42% higher using the 2MM compared to the 6LS method (p<0.0001). However, a strong correlation was found for the median T₂^? values between the 2MM and 6LS methods (R²=0.80). The median T₂^? values were not significantly different between the 6LS and 6LS_ROI methods (p=0.519). Using the 2MM (which provides a regional map) and the 6LS_ROI (which efficiently provides the median T₂^? value) methods in tandem would take only minutes of post-processing computational time compared to the 6LS method (~540 min), and hence would facilitate clinical application of T₂^? maps to predict ligament structural properties as a patient outcome measure.     

LAS测算森林冠层上方温度结构参数的可行性

利用位于河南省济源市的华北低丘山地30年生栓皮栎-侧柏-刺槐人工混交林2010年4月至8月每月连续7d,LAS直接测算的森林冠层上方湍流结构参数,与经过湍流谱方法计算处理的三维超声风速/温度仪的观测数据比较,分析LAS测算低丘山地森林冠层温度湍流结构的可行性。结果表明:水平风速和温度湍流谱都有明显的惯性区出现(斜率-2/3);LAS直接测算的湍流温度结构参数与利用该惯性区的数据计算的结果具有较好的一致性,说明在起伏非均匀下垫面上,采用LAS观测湍流结构的变化情况具有较好的可行性。     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Probing the steric barrier of nonionic surfactant vesicles with melittin

The role of the surface polymer brush of nonionic surfactant vesicles (NSV) in inhibiting interactions with small membrane-perturbing molecules was investigated using the bee venom peptide melittin as a probe. The interaction between melittin and NSV was compared with that of distearoylphosphatidylcholine (DSPC) vesicles and sterically stabilised liposomes (SSL) containing 5 mol% pegylated distearoylphosphatidylethanolamine (DSPE.E₄₄). The degree of melittin interaction with the various vesicles was determined by measuring peptide binding and folding, using intrinsic tryptophan fluorescence and circular dichroism respectively, in addition to monitoring the release of encapsulated carboxyfluorescein dye. NSV composed of 1,2-di-O-octadecyl-rac-glyceryl-3-(ω-dodecaethylene glycol) (2C₁₈E₁₂) showed a strong affinity for melittin, whilst exhibiting ~ 50% less bound peptide than SSL. 2C₁₈E₁₂:Chol vesicles showed reduced melittin interaction, in a manner consistent with Chol incorporation into DSPC vesicles. These results are discussed with respect to the effect of Chol on the in-plane order of 2C₁₈E₁₂ bilayers and consequent attenuation of hydrophobic interactions with the peptide. NSV formed from equimolar mixtures of polyoxyethylene-n-stearoyl ethers C₁₈E₂ and C₁₈E₂₀ showed a greater interaction with melittin than 2C₁₈E₁₂. However, replacing C₁₈E₂₀ with C₁₈E₁₀ was sufficient to achieve an attenuation of melittin interaction similar to that observed in 2C₁₈E₁₂:Chol vesicles. This indicates that the presence of surface polymer brush alone may confer resistance to melittin, provided hydrophobic interactions between the peptide and the vesicles can be minimised, through improved in-plane bilayer order.     

Calorimetric and molecular mechanics studies of the thermotropic phase behavior of membrane phospholipids

In this review, we summarize the results of recent studies on the main phase transition behavior of phospholipid bilayers using the combined approaches of molecular mechanics simulations and high-resolution differential scanning calorimetry. Following a brief overview of the phase transition phenomenon exhibited by the lipid bilayer, we begin with the review by showing how several structural parameters underlying various phospholipids including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol are defined and determined. Specifically, these structural parameters are obtained with saturated lipids packed in the gel-state bilayer using computer-based molecular mechanics calculations. Then we proceed to present the calorimetric data obtained with the lipid bilayer composed of saturated phospholipids as it undergoes the gel-to-liquid-crystalline phase transition in excess water. The general equations that can correlate the gel-to-liquid-crystalline phase transition temperature (T_m) of the lipid bilayer with the structural parameters of the lipid molecule constituting the lipid bilayer are subsequently presented. From these equations, two tables of predicated T_m values for well over 400 molecular species of saturated phosphatidylcholine and saturated phosphatidylethanolamine are generated. We further review the structure and chain-melting behavior of a large number of sn-1 saturated/sn-2 unsaturated phospholipids. Two T_m-diagrams are shown, from which the effects of the number and the position of one to five cis carbon–carbon double bonds on T_m can be viewed simultaneously. Finally, in the last part of this review, simple molecular models that have been invoked to interpret the characteristic T_m trends exhibited by lipid bilayers composed of unsaturated lipids with different numbers and positions of cis carbon–carbon double bonds as seen in the T_m-diagram are presented.     

Two distinct plant respiratory physiotypes might exist which correspond to fast-growing and slow-growing species

The origin of the carbon atoms in CO₂ respired by leaves in the dark of several plant species has been studied using ¹³C/¹²C stable isotopes. This study was conducted using an open gas exchange system for isotope labeling that was coupled to an elemental analyzer and further linked to an isotope ratio mass spectrometer (EA–IRMS) or coupled to a gas chromatography–combustion-isotope ratio mass spectrometer (GC–C-IRMS). We demonstrate here that the carbon, which is recently assimilated during photosynthesis, accounts for nearly ca. 50% of the carbon in the CO₂ lost through dark respiration (R_d) after illumination in fast-growing and cultivated plants and trees and, accounts for only ca. 10% in slow-growing plants. Moreover, our study shows that fast-growing plants, which had the largest percentages of newly fixed carbon of leaf-respired CO₂, were also those with the largest shoot/root ratios, whereas slow-growing plants showed the lowest shoot/root values.     

Dissociation constants of phenothiazine drugs incorporated in phosphatidylcholine bilayer of small unilamellar vesicles as determined by carbon-13 nuclear magnetic resonance spectrometric titration

The dissociation constants (pK_ms) of the phenothiazine drugs promazine, chlorpromazine, and triflupromazine, incorporated in the phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUV), were investigated by a ¹³C nuclear magnetic resonance (NMR) titration method employing their N-¹³CH₃ (ionizable group) labelled derivatives. Use of the labelled drugs enabled direct observations of the ionization equilibrium of the N-dimethyl group. A second derivative spectrophotometric study proved that 95-98% of the phenothiazine species in the sample solutions (200 μM phenothiazine in the presence of 27 mM PC SUV) were incorporated into the PC bilayer, which simplified the calculation of pK_m values by allowing that the phenothiazines in the aqueous phase could be neglected. The pK_m values were calculated from the chemical shift dependence of the N-dimethyl ¹³C NMR signal on the pH value of sample solutions. The pK_m values obtained were smaller than those measured in aqueous solutions by about one unit. The existence of cholesterol (30 mol%) in the PC bilayer showed little effect on the pK_m values, suggesting that cholesterol in the bilayer does not largely affect the interfacial region where the N-dimethyl group of the incorporated phenothiazines is located. The results offered clear evidence for the pK_m decrease and provided their precise values.     

Deprotonation of β-cyclodextrin in alkaline solutions

Variable pH ¹³C NMR and ¹H NMR spectroscopic studies of the β-cyclodextrin (β-CD) in alkaline aqueous solutions revealed that β-CD does not deprotonate at pH < 12.0. Further increase in solution pH results in the deprotonation of OH-groups adjacent to C-2 and C-3 carbon atoms of β-CD glucopyranose units, whereas the deprotonation of OH-groups adjacent to C-6 carbon atoms is expressed less markedly. The pK_a values for β-CD OH-groups adjacent to C-2 and C-3 carbon atoms are rather close, pK_a1,2 being 13.5 ± 0.2 (22.5 °C).     

Trichinella spiralis Infection Suppressed Gut Inflammation with CD4+CD25+Foxp3+ T Cell Recruitment

In order to know the effect of pre-existing Trichinella spiralis infection on experimentally induced intestinal inflammation and immune responses, we induced colitis in T. spiralis-infected mice and observed the severity of colitis and the levels of Th1, Th2, and regulatory cytokines and recruitment of CD4⁺CD25⁺Foxp3⁺ T (regulatory T; T_reg) cells. Female C57BL/6 mice were infected with 250 muscle larvae; after 4 weeks, induction of experimental colitis was performed using 3% dextran sulfate sodium (DSS). During the induction period, we observed severity of colitis, including weight loss and status of stool, and evaluated the disease activity index (DAI). A significantly low DAI and degree of weight loss were observed in infected mice, compared with uninfected mice. In addition, colon length in infected mice was not contracted, compared with uninfected mice. We also observed a significant increase in production of pro-inflammatory cytokines, IL-6 and IFN-γ, in spleen lymphocytes treated with DSS; however, such an increase was not observed in infected mice treated with DSS. Of particular interest, production of regulatory cytokines, IL-10 and transforming growth factor (TGF)-β, in spleen lymphocytes showed a significant increase in mice infected with T. spiralis. A similar result was observed in mesenteric lymph nodes (MLN). Subsets of the population of T_reg cells in MLN and spleen showed significant increases in mice infected with T. spiralis. In conclusion, T. spiralis infection can inhibit the DSS-induced colitis in mice by enhancing the regulatory cytokine and T_reg cells recruitment.     

Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16_meso) and hyperthermophilic (S16_thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16_thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔC_p⁰) of 6.4 and 3.3 kJ mol^− 1 K^− 1, respectively. A strong linear correlation between ΔC_p⁰ and melting temperature (T_m) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16_meso folds through an apparent two-state model, whereas S16_thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16_thermo is more compact relative to S16_meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16_thermo that appears to account for the large difference in quantified ΔC_p⁰ values and, in turn, parts of the observed extreme thermal stability of S16_thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.     

Dynorphin induced magnetic ordering in lipid bilayers as studied by P NMR spectroscopy

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T_m=24°C), as examined by ³¹P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T_m but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T_m. These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T_m in parallel with the bilayer surface, because a single ³¹P NMR signal appeared at the perpendicular position of the ³¹P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T_m, because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated ³¹P NMR lineshape.     

Activation and Recruitment of Regulatory T Cells via Chemokine Receptor Activation in Trichinella spiralis-Infected Mice

As most infections by the helminth parasite elicit the recruitment of CD4⁺CD25⁺Foxp3⁺ T (T_reg) cells, many scientists have suggested that these cells could be used for the treatment of immune-mediated inflammation and associated diseases. In order to investigate the distribution and alteration of activated T_reg cells, we compared the expression levels of T_reg cell activation markers in the ileum and gastrocnemius tissues 1, 2, and 4 weeks after infection. The number of T_reg cells was monitored using GFP-coded Foxp3 transgenic mice. In mice at 1 week after Trichinella spiralis infection, the number of activated T_reg cells was higher than in the control group. In mice at 2 weeks after infection, there was a significant increase in the number of cells expressing Foxp3 and CTLA-4 when compared to the control group and mice at 1 week after infection. At 4 weeks after infection, T. spiralis was easily identifiable in nurse cells in mouse muscles. In the intestine, the expression of Gzmb and Klrg1 decreased over time and that of Capg remained unchanged for the first and second week, then decreased in the 4th week. However, in the muscles, the expression of most chemokine genes was increased due to T. spiralis infection, in particular the expression levels of Gzmb, OX40, and CTLA-4 increased until week 4. In addition, increased gene expression of all chemokine receptors in muscle, CXCR3, CCR4, CCR5, CCR9, and CCR10, was observed up until the 4th week. In conclusion, various chemokine receptors showed increased expressions combined with recruitment of T_reg cells in the muscle tissue.     

Molecular dynamics simulation of d-Benzedrine transmitting through molecular channels within D3R

Dex-Benzedrine (known as d-Benzedrine or SAT) acts in dopamine receptors of central nerve cell system. In clinic, SAT is used to treat a variety of diseases; meanwhile, it has dependence and addiction. In order to investigate the pharmacology and addiction mechanisms of SAT as a medicine, in this paper, we have studied the structure of D₃R complex protein with SAT, and based on which, using potential mean force with umbrella samplings and the simulated phospholipid bilayer membrane (or POPC bilayer membrane), the molecular dynamics simulation was performed to obtain free energy changes upon the trajectories for SAT moving along the molecular channels within D₃R. The free energy change for SAT transmitting toward the outside of cell along the functional molecular channel within D₃R is 83.5 kJ mol^?1. The change of free energy for SAT to permeate into the POPC bilayer membrane along the protective molecular channel within D₃R is 87.7 kJ mol^?1. Our previous work gave that the free energy for Levo-Benzedrine (RAT) transmitting toward the outside of cell along the functional molecular channel within D₃R is 91.4 kJ mol^?1, while it is 117.7 kJ mol^?1 for RAT to permeate into the POPC bilayer membrane along the protective molecular channel within D₃R. The values of free energy suggest that SAT relatively prefers likely to pass through the functional molecular channel within D₃R for increasing the release of dopamine molecules resulting in a variety of functional effects for SAT. The obtained results show that the pharmacology and addiction mechanisms of SAT as a drug are closely related to the molecular dynamics and mechanism for SAT transmitting along molecular channels within D₃R.     

Field-acclimated Gossypium hirsutum cultivars exhibit genotypic and seasonal differences in photosystem II thermostability

Previous investigations have demonstrated that photosystem II (PSII) thermostability acclimates to prior exposure to heat and drought, but contrasting results have been reported for cotton (Gossypium hirsutum). We hypothesized that PSII thermotolerance in G. hirsutum would acclimate to environmental conditions during the growing season and that there would be differences in PSII thermotolerance between commercially-available U.S. cultivars. To this end, three cotton cultivars were grown under dryland conditions in Tifton Georgia, and two under irrigated conditions in Marianna Arkansas. At Tifton, measurements included PSII thermotolerance (T₁₅, the temperature causing a 15% decline in maximum quantum yield), leaf temperatures, air temperatures, midday (1200 to 1400 h) leaf water potentials (Ψ_MD), leaf-air vapor pressure deficit (VPD), actual quantum yield (Φ_PSII) and electron transport rate through PSII (ETR) on three sample dates. At Marianna, T₁₅ was measured on two sample dates. Optimal air and leaf temperatures were observed on all sample dates in Tifton, but PSII thermotolerance increased with water deficit conditions (Ψ_MD = −3.1 MPa), and ETR was either unaffected or increased under water-stress. Additionally, T₁₅ for PHY 499 was ∼5 °C higher than for the other cultivars examined (DP 0912 and DP 1050). The Marianna site experienced more extreme high temperature conditions (20–30 days T_max ≥ 35 °C), and showed an increase in T₁₅ with higher average T_max. When average T₁₅ values for each location and sample date were plotted versus average daily T_max, strong, positive relationships (r² from .954 to .714) were observed between T_max and T₁₅. For all locations T₁₅ was substantially higher than actual field temperature conditions. We conclude that PSII thermostability in G. hirsutum acclimates to pre-existing environmental conditions; PSII is extremely tolerant to high temperature and water-deficit stress; and differences in PSII thermotolerance exist between commercially-available cultivars.     

High resolution 13C NMR spectra on oriented lipid bilayers: from quantifying the various sources of line broadening to performing 2D experiments with 0.2-0.3 ppm resolution in the carbon dimension

¹³C NMR spectra routinely performed on oriented lipid bilayers display linewidth of 1–2 ppm, although T₂ measurements indicate that 0.1–0.2 ppm could be obtained. We have prepared a DMPC – ¹³C₄-cholesterol (7/3) sample, and oriented the lipid bilayers between glass plates so that the bilayer normal makes an angle of 90° (or of the magic angle) with B₀. We have measured T₂s, CSAs, and linewidths for the choline ¹³C--methyl, the cholesterol-C₄ carbons and the lipid head group phosphorus, at both angles and 313 K. The magnetic field distribution within the sample was calculated using the surface current formalism. The line shapes were simulated as a function of B₀ field inhomogeneities and sample mosaic spread. Both effects contribute to the experimental linewidth. Using three signals of different CSA, we have quantified both contributions and measured the mosaic spread accurately. Direct shimming on a sample signal is essential to obtain sharp resonances and ¹³C labelled choline methyl resonance of DMPC is a good candidate for this task. After optimisation of the important parameters (shimming on the choline resonance, mosaic spread of ±0.30° ), ¹³C linewidth of 0.2–0.3 ppm have been obtained. This newly achieved resolution on bilayers oriented at 90°, has allowed to perform two 2D experiments, with a good sensitivity: 2D PELF (correlation of carbon chemical shifts and C-H dipolar couplings) and 2D D-resolved experiment (correlation of carbon chemical shifts and C-C dipolar couplings). A C-C dipolar coupling of 35 ± 2 Hz between the choline methyl carbons was determined.     

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E_m) of one-electron oxidation for all Car (E_m(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (Car_D1) in van der Waals contact with the chlorin ring of Chl_Z(D1). Each Car_D1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between Car_D1 and plastoquinone/Q_A may imply a direct charge recombination of Car⁺Q_A⁻. The calculated E_m(Car_D1) and E_m(Chl_Z(D1)) are, respectively, 83 and 126 mV higher than E_m(Car_D2) and E_m(Chl_Z(D2)), which could explain why Car_D2⁺ and Chl_Z(D2)⁺ are observed rather than the corresponding Car_D1⁺ and Chl_Z(D1)⁺.     

Ca binding to sarcoplasmic reticulum ATPase phosphorylated by Pi reveals four thapsigargin-sensitive Ca sites in the presence of ADP

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase was phosphorylated by P_i at pH 8.0 in the presence of dimethyl sulfoxide (Me₂SO). Under these conditions, it was possible to measure transient ⁴⁵Ca²⁺ binding to the phosphoenzyme. Binding reached 1.2 Ca²⁺ per phosphoenzyme (E-PCa_x) within 10 min in 30% Me₂SO, 20 mM MgCl₂ and 0.1 mM P_i and the phosphoenzyme only decreased by 23% during this period. This Ca²⁺ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca²⁺-ATPase. At 40% Me₂SO, simultaneous addition of Ca²⁺ and ADP increased Ca²⁺ binding up to almost four Ca²⁺ per phosphoenzyme (ADPE-PCa_y), revealing a species bearing simultaneously four Ca²⁺ sites. Both E-PCa_x and ADPE-PCa_y were further identified as distinct species by (2′,3′-O-2(2,4,6-trinitrophenyl)adenosine 5′-triphosphate) fluorescence, which revealed long-range modifications in the Ca²⁺-transport sites induced by ADP binding to E-P. In addition, E-PCa_x was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me₂SO was diluted. These findings indicate that more than two functional Ca²⁺-sites exist on the functional Ca²⁺-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca²⁺-ATPase allowing simultaneous binding of Ca²⁺ at lumenal and cytosolic sites. The stoichiometries for Ca²⁺ binding found here could either be interpreted as binding of four Ca²⁺ on a Ca²⁺-ATPase monomer considered as the functional unit or as binding of two Ca²⁺ per monomer of a functional dimer.     

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18^T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985^T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043^T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18^T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, and C_16:1ω7c/C_16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18^T (= CCM 8464 = CECT 8192^T = IBRC-M 10767^T = LMG 27461^T).