Insights into the morphological pattern of erythrocytes' aging: Coupling quantitative AFM data to microcalorimetry and Raman spectroscopy.

Erythrocytes (RBCs) constitute a very interesting class of cells both for their physiological function and for a variety of peculiarities. Due to their exceptionally strong relationship with the environment, the morphology and nanoscale characteristics of these cells can reveal their biochemical status and structural integrity. Among the possible subjects of investigations, the RBCs' ageing is of the utmost importance. This is a fundamental phenomenon that, in physiological conditions, triggers the cell turnover and ensures the blood homeostasis. With these premises, in recent years, we have presented an atomic force microscopy-based methodology to characterize the patterns of RBC ageing from the morphological point of view. In the present work, we used an ageing protocol more similar to the physiological conditions and we used differential scanning calorimetry and atomic force microscopy to probe the cross correlation between important structural and functional proteins. We also assessed the role played by fundamental structural and membrane proteins in the development of the most relevant morphological intermediates observed along the ageing. Furthermore, we coupled the morphological ageing patterns to the (bio)chemical alterations detected by Raman spectroscopy. This allowed identifying the chronology of the ageing morphologies and the metabolic pathways most involved in their development. As a whole, the present study provides the base to correlate specific molecular alterations to the development of structural anomalies, and these latter to the functional status of blood cells.

Heat- and light-induced detachment of the light-harvesting antenna complexes of photosystem I in isolated stroma thylakoid membranes.

The multisubunit pigment-protein complex of photosystem I (PSI) consists of a core and peripheral light-harvesting antenna (LHCI). PSI is thought to be a rather rigid system and very little is known about its structural and functional flexibility. Recent data, however, suggest LHCI detachment from the PSI supercomplex upon heat and light treatments. Furthermore, it was suggested that the splitting off of LHCI acts as a safety valve for PSI core upon photoinhibition (Alboresi et al., 2009). In this work we analyzed the heat- and light-induced reorganizations in isolated PSI vesicles (stroma membrane vesicles enriched in PSI). Using differential scanning calorimetry we revealed a stepwise disassembly of PSI supercomplex above 50°C. Circular dichroism, sucrose gradient centrifugation and 77K fluorescence experiments identified the sequence of events of PSI destabilization: 3min heating at 60°C or 40min white light illumination at 25°C resulted in pronounced Lhca1/4 detachment from the PSI supercomplex, which is then followed by the degradation of Lhca2/3. The similarity of the main structural effects due to heat and light treatments supports the notion that thermo-optic mechanism, structural changes induced by ultrafast local thermal transients, which has earlier been shown to be responsible for structural changes in the antenna system of photosystem II, can also regulate the assembly and functioning of PSI antenna.

Toward successful migration to computerized physician order entry for chemotherapy.

BACKGROUND: Computerized physician order entry (cpoe) systems allow for medical order management in a clinical setting. Use of a cpoe has been shown to significantly improve chemotherapy safety by reducing the number of prescribing errors. Usability of these systems has been identified as a critical factor in their successful adoption. However, there is a paucity of literature investigating the usability of cpoe for chemotherapy and describing the experiences of cancer care providers in implementing and using a cpoe system. METHODS: A mixed-methods study, including a national survey and a workshop, was conducted to determine the current status of cpoe adoption in Canadian oncology institutions, to identify and prioritize knowledge gaps in cpoe usability and adoption, and to establish a research agenda to bridge those gaps. Survey respondents were representatives of cancer care providers from each Canadian province. The workshop participants were oncology clinicians, human factors engineers, patient safety researchers, policymakers, and hospital administrators from across Canada, with participation from the United States. RESULTS: A variety of issues related to implementing and using a cpoe for chemotherapy were identified. The major issues concerned the need for better understanding of current practices of chemotherapy ordering, preparation, and administration; a lack of system selection and procurement guidance; a lack of implementation and maintenance guidance; poor cpoe usability and workflow support; and other cpoe system design issues. An additional three research themes for addressing the existing challenges and advancing successful adoption of cpoe for chemotherapy were identified: The need to investigate variances in workflows and practices in chemotherapy ordering and administrationThe need to develop best-practice cpoe procurement and implementation guidance specifically for chemotherapyThe need to measure the effects of cpoe implementation in medical oncology. CONCLUSIONS: Addressing the existing challenges in cpoe usability and adoption for chemotherapy, and accelerating successful migration to cpoe by cancer care providers requires future research focusing on workflow variations, chemotherapy-specific cpoe procurement needs, and implementation guidance needs.

Brassinosteroids regulate the thylakoid membrane architecture and the photosystem II function.

Brassinosteroids (BRs) are plant steroid hormones known to positively affect photosynthesis. In this work we investigated the architecture and function of photosynthetic membranes in mature Arabidopsis rosettes of BR gain-of-function (overexpressing the BR receptor BR INSENSITIVE 1 (BRI1), BRI1OE) and loss-of-function (bri1-116 with inactive BRI1 receptor, and constitutive photomorphogenesis and dwarfism (cpd) deficient in BR biosynthesis) mutants. Data from atomic force microscopy, circular dichroism, fluorescence spectroscopy and polarographic determination of oxygen yields revealed major structural (enlarged thylakoids, smaller photosystem II supercomplexes) and functional (strongly inhibited oxygen evolution, reduced photosystem II quantum yield) changes in all the mutants with altered BR response compared to the wild type plants. The recorded thermal dependences showed severe thermal instability of the oxygen yields in the BR mutant plants. Our results suggest that an optimal BR level is required for the normal thylakoid structure and function.

Interfacial water structure controls protein conformation.

A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacteriorhodopsin and myoglobin. The experimental results support the ability of the new formalism to account for the diverse manifestations of salt effects on protein conformation, dynamics, and stability, and to resolve the puzzle of chaotropes stabilizing certain proteins (and other anomalies). It is also shown that the relation between interfacial tension and protein structural stability is straightforwardly linked to protein conformational fluctuations, providing a keystone for the microscopic interpretation of Hofmeister effects. Implications of the results concerning the use of Hofmeister effects in the experimental study of protein function are discussed.

Protein-coated beta-ferric hydrous oxide particles. An electrokinetic and electrooptic study.

Using microelectrophoresis and electric light scattering techniques, we investigated the adsorption characteristics, surface coverage and surface electric parameters of superstructures from two isoforms of plastocyanin, PCa and PCb, in an oxidized state adsorbed on beta-ferric hydrous oxide particles. The surface electric charge and electric dipole moments of the composite particles and the thickness of the protein adsorption layer are determined in a wide pH range, at different ionic strengths and concentration ratios of PC to beta-FeOOH. The adsorption of the two proteins was found to shift the particles' isoelectric point and to alter the total electric charge and the electric dipole moments of the oxide particles to different extent. A "reversal" in the direction of the permanent dipole moment is observed at lower pH for PCb- than for PCa-coated oxide particles. Strict correlation is found between the changes in the electrokinetic charge of the composite particles and the variation in their "permanent" dipole moments. Data suggest that the adsorption of the proteins is driven by electrostatic and/or hydrophobic interactions with the oxide surfaces dependent on pH. The adsorption behaviour is consistent with the involvement of the "eastern" and "northern" patches of the plastocyanin molecules in their adsorption on the oxide surfaces that are differently charged depending on pH.

Excitation of the L intermediate of bacteriorhodopsin: electric responses to test x-ray structures.

The L intermediate of bacteriorhodopsin was excited, and its electrical response was measured. Two positive components were found in it with respect to the direction of proton pumping: an unresolved fast component, and a slower one (tau=7 micros) of small amplitude. The fast component was assigned to a charge motion corresponding to reisomerization of the retinal moiety, whereas the slow one was attributed to charge rearrangements reestablishing the ground state. Because three x-ray crystallographic structures have recently been reported for the L intermediate, it seemed important to calculate the intramolecular dipole moment changes associated to bR-->L for all three structures, so as to compare them with similar quantities determined from the electrical signals. The results are discussed in terms of amino acid side chains possibly contributing to the observed effect. We propose to use electrical signals as a verification tool for intermediate structures of the photocycle, and thus for molecular models of proton pumping.

Actinic light-energy dependence of proton release from bacteriorhodopsin.

Measuring the light-density (fluence) dependence of proton release from flash excited bacteriorhodopsin with two independent methods we found that the lifetime of proton release increases and the proton pumping activity, defined as a number of protons per number of photocycle, decreases with increasing fluence. An interpretation of these results, based on bending of purple membrane and electrical interaction among the proton release groups of bacteriorhodopsin trimer, is presented.

Irreversible temperature-induced changes in purple membranes studied by electrooptics.

The thermal stability of purple membranes is studied by electric light scattering. Information on the polarization mechanisms is obtained. Incomplete recovery of the initial electric state (i.e., of permanent dipole moment, p( perpendicular), and electric polarizability, gamma(fast, ||)) after the membranes are heated at temperatures above 60 degrees C is revealed. Additional slow polarizability components, gamma(slow, perpendicular) and gamma(slow, ||), relaxing at different characteristic frequencies than the fast longitudinal polarizability gamma(fast, ||) appear in the temperature range where the order-disorder transition takes place. The slow polarizability gamma(slow, perpendicular) is probably connected with counterion displacement in the electrical double layer perpendicular to the disk plane. The results are important for understanding the polarization mechanisms and the origin of slow orienting moments.

Thermal stability and DPPC/Ca2+ interactions of pulmonary surfactant SP-A from bulk-phase and monolayer IR spectroscopy.

Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein, is implicated in multiple biological functions including surfactant homeostasis, biophysical activity, and host defense. SP-A forms ternary complexes with lipids and Ca2+ which are important for protein function. The current study uses infrared (IR) transmission spectroscopy to investigate the bulk-phase interaction between SP-A, 1,2-dipalmitoylphosphatidylcholine (DPPC), and Ca2+ ions along with IR reflection-absorption spectroscopy (IRRAS) to examine protein secondary structure and lipid orientational order in monolayer films in situ at the air/water interface. The amide I contour of SP-A reveals two features at 1653 and 1636 cm(-1) arising from the collagen-like domain and a broad feature at 1645 cm(-1) suggested to arise from the carbohydrate recognition domain (CRD). SP-A secondary structure is unchanged in lipid monolayers. Thermal denaturation of SP-A in the presence of either DPPC or Ca2+ ion reveals a sequence of events involving the initial melting of the collagen-like region, followed by formation of intermolecular extended forms. Interestingly, these spectral changes were inhibited in the ternary system, showing that the combined presence of both DPPC and Ca2+ confers a remarkable thermal stability upon SP-A. The ternary interaction was revealed by the enhanced intensity of the asymmetric carboxylate stretching vibration. The IRRAS measurements indicated that incorporation of SP-A into preformed DPPC monolayers at a surface pressure of 10 mN/m induced a decrease in the average acyl chain tilt angle from 35 degrees to 28 degrees. In contrast, little change in chain tilt was observed at surface pressures of 25 or 40 mN/m. These results are consistent with and extend the fluorescence microscopy studies of Keough and co-workers [Ruano, M. L. F., et al. (1998) Biophys. J. 74, 1101-1109] in which SP-A was suggested to accumulate at the liquid-expanded/liquid-condensed boundary. Overall these experiments reveal the remarkable stability of SP-A in diverse, biologically relevant environments.

Buffer effects on electric signals of light-excited bacteriorhodopsin mutants.

The effects of glycyl-glycine and bis-trispropane buffers on the light-excited electric signals due to proton motion in the molecule were studied for the bacteriorhodopsin (bR) mutants D38R, D96N, E204Q, R227Q, D85N, D85T, R82Q/D85N, and D85N/D96N in purple membranes and for delipidated purple membrane containing the wild-type bR. The results show additional charge motion caused by the buffers in all cases. Arrhenius parameters calculated from the temperature dependence of the difference signals (with buffer minus without buffer) are similar to the parameters found for the wild-type bR in the case of these buffers: the values of the activation enthalpies are mostly in the range 25-50 kJ/mol; all the activation entropies are negative. The results are evaluated with the cluster hypothesis outlined previously.

Electric signals of light excited bacteriorhodopsin mutant D96N.

The study of mutant D96N played an important role in understanding proton translocation by light driven bacteriorhodopsin. Our measurement of photoelectric current for single and double flash illumination revealed new details of the photocycle of this mutant. With double flash excitation we found an intermediate absorbing near the wavelength of the ground state of bacteriorhodopsin (bR) but pumping in the opposite direction. This intermediate has the same lifetime as the species described by Zimányi et al. [Proc. Natl. Acad. Sci. USA 96 (1999) 4414-4419] and was assigned to early recovery of a fraction of the ground state after excitation. Because the electric response does not reconcile with that of the ground state, we tentatively assign it to the L intermediate or to an intermediate similar in absorption to bR (bR').

Contribution of LHC II complex to the electric properties of thylakoid membranes: an electric light scattering study of Chl b-less barley mutant.

Electric light scattering measurements demonstrate a strong decline in the permanent electric dipole moment and electric polarizability of both thylakoid membranes and photosystem II-enriched particles of the Chlorina f2 mutant which has severely reduced levels of light-harvesting chlorophyll a/b-binding proteins compared to the wild type barley chloroplasts. The shift in the electric polarizability relaxation to higher frequencies in thylakoids and photosystem II particles from Chlorina f2 reflects higher mobility of the interfacial charges of the mutant than that of the wild type membranes. The experimental data strongly suggest that the major light-harvesting complex of photosystem II directly contribute to the electric properties of thylakoid membranes.

Differential effects of surfactant protein A on regional organization of phospholipid monolayers containing surfactant protein B or C.

Epifluorescence microscopy combined with a surface balance was used to study monolayers of dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (PG) (8:2, mol/mol) plus 17 wt % SP-B or SP-C spread on subphases containing SP-A in the presence or absence of 5 mM Ca(2+). Independently of the presence of Ca(2+) in the subphase, SP-A at a bulk concentration of 0.68 microg/ml adsorbed into the spread monolayers and caused an increase in the molecular areas in the films. Films of DPPC/PG formed on SP-A solutions showed a pressure-dependent coexistence of liquid-condensed (LC) and liquid-expanded (LE) phases. Apart from these surface phases, a probe-excluding phase, likely enriched in SP-A, was seen in the films between 7 mN/m < or = pi < or = 20 mN/m. In monolayers of SP-B/(DPPC/PG) spread on SP-A, regardless of the presence of calcium ions, large clusters of a probe-excluding phase, different from probe-excluding lipid LC phase, appeared and segregated from the LE phase at near-zero surface pressures and coexisted with the conventional LE and LC phases up to approximately 35 mN/m. Varying the levels of either SP-A or SP-B in films of SP-B/SP-A/(DPPC/PG) revealed that the formation of the probe-excluding clusters distinctive for the quaternary films was influenced by the two proteins. Concanavalin A in the subphase could not replace SP-A in its ability to modulate the textures of films of SP-B/(DPPC/PG). In films of SP-C/SP-A/(DPPC/PG), in the absence of calcium, regions consisting of a probe-excluding phase, likely enriched in SP-A, were detected at surface pressures between 2 mN/m and 20 mN/m in addition to the lipid LE and LC phases. Ca(2+) in the subphase appeared to disperse this phase into tiny probe-excluding particles, likely comprising Ca(2+)-aggregated SP-A. Despite their strikingly different morphologies, the films of DPPC/PG that contained combinations of SP-B/SP-A or SP-C/SP-A displayed similar distributions of LC and LE phases with LC regions occupying a maximum of 20% of the total monolayer area. Combining SP-A and SP-B reorganized the morphology of monolayers composed of DPPC and PG in a Ca(2+)-independent manner that led to the formation of a separate potentially protein-rich phase in the films.

Bacteriorhodopsin thermal stability: influence of bound cations and lipids on the intrinsic protein fluorescence.

Temperature-induced changes in protein intrinsic fluorescence of native, delipidated and deionized purple membranes are investigated. It is found that the removal of cations most strongly affects the protein and its thermal stability. The denaturation of dei-BR completes at 70 degrees C, while delipidated and native BR still maintain their native structure at this temperature. Both the quantum yield and the fluorescence maximum suggest correlation between the Trp-retinal coupling and protein structural stability. The low red shift of the fluorescence maximum caused by increasing of temperature indicates limited unfolding of bacteriorhodopsin upon denaturation.

Adsorption of pulmonary surfactant protein SP-A to monolayers of phospholipids containing hydrophobic surfactant protein SP-B or SP-C: potential differential role for tertiary interaction of lipids, hydrophobic proteins, and SP-A.

Surface balance techniques were used to study the interactions of surfactant protein SP-A with monolayers of surfactant components preformed at the air-water interface. SP-A adsorption into the monolayers was followed by monitoring the increase in the surface pressure Deltapi after injection of SP-A beneath the films. Monolayers of dipalmitoylphosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (8:2, mol/mol) spread at initial surface pressure pi(i) = 5 mN/m did not promote the adsorption of SP-A at a subphase concentration of 0.68 microg/mL as compared to its adsorption to the monolayer-free surface. Surfactant proteins, SP-B or SP-C, when present in the films of DPPC:PG spread at pi(i) = 5 mN/m, enhanced the incorporation of SP-A in the monolayers to a similar extent; the Deltapi values being dependent on the levels of SP-B or SP-C, 3-17 wt %, in the lipid films. Calcium in the subphase did not affect the intrinsic surface activity of SP-A but reduced the Deltapi values produced by the adsorption of the protein to all the preformed films independently of their compositions and charges. The divalent ions likely modified the interaction of SP-A with the monolayers through their effects on the conformation, self-association, and charge state of SP-A. Values of Deltapi produced by adsorption of SP-A to the films of DPPC:PG with or without SP-B or SP-C were a function of the initial surface pressure of the films, pi(i). In the range of pressures 5

Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability.

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.

Electric properties of thylakoid membranes from pea mutants with modified carotenoid and chlorophyll-protein complex composition.

Surface electric properties of thylakoid membranes from wild type and two mutant forms, Coeruleovireus 2/16 and Costata 2/133, of pea are investigated by electric light scattering and microelectrophoretic measurements. Characterization of the chlorophyll-protein complexes in thylakoid membranes reveals that the relative ratio of oligomeric (LHC II(1)) to monomeric (LHC II(3)) forms of the light-harvesting Chl a/b complex of Photosystem II is lower (3.34) in 2/133 mutant and higher (6.62) in 2/16 mutant than in wild type (4.57). This is accompanied by elevated amounts and a considerable reduction of all carotenoids in 2/16 and 2/133 mutant, respectively, as compared to the wild type. The concomitant variations of the permanent dipole moment (transversal charge asymmetry), electric polarizability and electrokinetic charge of the thylakoid membranes from both the mutants are discussed in terms of the differences in the supramolecular (oligomeric) organization of the light-harvesting complexes II within the photosynthetic apparatus.

Photoelectric response of the N intermediate of bacteriorhodopsin and its mutant T46V.

Double flash experiments were performed in order to gain information about the characteristics of the N intermediates of the photocycle of bacteriorhodopsin. The N intermediates of wild-type bacteriorhodopsin and mutant T46V were excited at different delay times after the first laser flash which induced the photocycle and the electric responses were registered. These electric signals revealed that charge motions occurred in both cases, though charge translocation, i.e. H(+) pumping, could not be observed. The delay time dependence of the electric signals is characterized by two distinct processes corresponding to two substates of the N intermediates.

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.