Agnes E. Ostafin

Combined deletion of mouse dematin-headpiece and β-adducin exerts a novel effect on the spectrin-actin junctions leading to erythrocyte fragility and hemolytic anemia

Dematin and adducin are actin-binding proteins of the erythrocyte “junctional complex.” Individually, they exert modest effects on erythrocyte shape and membrane stability, and their homologues are expressed widely in non-erythroid cells. Here we report generation and characterization of double knock-out mice lacking β-adducin and the headpiece domain of dematin. The combined mutations result in altered erythrocyte morphology, increased membrane instability, and severe hemolysis. Peripheral blood analysis shows evidence of severe hemolytic anemia with reduced number of erythrocytes/hematocrit/hemoglobin and an ∼12-fold increase in the number of circulating reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes correlates with increased osmotic fragility and reduced in vivo life span. Despite the apparently normal protein composition of the mutant erythrocyte membrane, the retention of the spectrin-actin complex in the membrane under low ionic strength conditions is significantly reduced by the double mutation. Atomic force microscopy reveals an increase in grain size and a decrease in filament number of the mutant membrane cytoskeleton, although the volume parameter is similar to wild type erythrocytes. Aggregated, disassembled, and irregular features are visualized in the mutant membrane, consistent with the presence of large protein aggregates. Importantly, purified dematin binds to the stripped inside-out vesicles in a saturable manner, and dematin-membrane binding is abolished upon pretreatment of membrane vesicles with trypsin. Together, these results reveal an essential role of dematin and adducin in the maintenance of erythrocyte shape and membrane stability, and they suggest that the dematin-membrane interaction could link the junctional complex to the plasma membrane in erythroid cells.

Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes. The changes are quantitatively described using the pixel height histogram and window size grain analysis.

Fluorescence of Cascade Blue™ inside nano-sized porous shells of silicate

Abstract Cascade Blue™ (CB) dye at a concentration as high as 0.227 M was encapsulated within nano-sized porous silicate shells, and its relative fluorescence yield determined over the pH range of 1.8–12.3, using 380 nm as the excitation wavelength. The results were compared with those obtained in aqueous solution using similar pH and total dye concentration. Near neutral pH, the relative fluorescence yields of CB inside the shells exhibited little fluorescence quenching, even though a high concentration of the dye was trapped inside the particles, while the peak wavelength of fluorescence was shifted from 420 nm in solution to 430 nm in shells. Both in shells and solution, the relative fluorescence intensity decreased as the solution pH was raised from 2 to 4, and in shells it nearly disappeared at about pH 3–4. As the pH was further increased, the red shift of fluorescence peak in the shell-trapped dyes was evident at pH 5 and its fluorescence intensity regained equal to that in acid. In the neutral pH range, the fluorescence intensity of CB in the shells was similar to that of the equivalent total concentration of the CB in solution. In solution, a similar red shift of the fluorescence maximum of CB to 430 nm was observed only above pH 9. These observations suggest that the fluorescence intensities of dyes trapped inside nano-sized porous silicate shells can be equal to or higher than that observed in solution under comparable conditions, leading to several hundreds times more fluorescent intensity when it is measured per single shell rather than per unit fluorophore.

Antibody-conjugated soybean oil-filled calcium phosphate nanoshells for targetted delivery of hydrophobic molecules

Hollow calcium phosphate nanoparticles capable of encapsulating poorly water-soluble molecules were produced by self-assembly. Previously reported were solid calcium phosphate nanoparticles and water-filled calcium phosphate nanocapsules suited for encapsulating mostly hydrophilic, but not hydrophobic compounds. Here, calcium phosphate was deposited around 100 nm diameter, 1,2-dioleoyl-sn-glycero-3-phosphate stabilized soybean oil nanoemulsions using either calcium chloride or NaOH titrations to achieve shell thickness between 20–70 nm. The surface was functionalized with carboxylic acid via the addition of carboxyethylphosphonic acid to attach Molecular Probes AB-594C antibody using sulpho-n-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride with an efficiency of ∼70%, while retaining near complete antibody function. Hydrophobic pyrene was encapsulated with an efficiency of 95%, at concentrations much higher than its water solubility limit, and exhibited spectral features characteristic of a hydrophobic environment. These materials can be used in the targeted delivery of many useful, yet poorly water-soluble pharmaceutical and nutraceutical compounds.

Uptake of calcium phosphate nanoshells by osteoblasts and their effect on growth and differentiation

The influence of calcium phosphate nanoshell materials on the uptake, viability, and mineralization of human fetal osteoblast cultures was evaluated. Proliferation rates and alkaline phosphatase activity of the cultures were unaffected by the addition of nanoshells to the growth media, but mineralization levels were enhanced by nearly 40%, in contrast to media prepared without nanoshells, or with other calcium phosphate nanomaterials. Nanoshells were internalized by macropinocytosis, and migrated toward the cell nucleus at a rate of 0.34 microm hr(-1). Dye-loaded nanoshells maintained high light emission intensity for over five days while inside the cells, where they could be used as intracellular markers for in vitro microscopic imaging. From these results, it appears that the CaP nanoshells could be developed into a safe sensor and delivery vehicle for osteoblast cell culture studies, whereas the carrier itself has intrinsic bioactivity and may itself upregulate the formation of new bone.

Cryogenic charge transport in oxidized purple bacterial light-harvesting 1 complexes

We report on the analysis of the inter-bacteriochlorophyll a (BChla) charge-transport process that occurs in oxidized purple bacterial light-harvesting 1 (LH1) complexes. Experimentally, charge migration within oxidized LH1 is monitored by following the temperature-dependent changes of the BChla(•)(+) electron paramagnetic resonance (EPR) line-shape characteristics. At 6 K, a Gaussian-shaped spectrum with a 1.3-mT width is detected. These characteristics indicate that at extremely low temperatures charge transport is substantially slowed so that the unpaired electron is localized on one or two BChlas. At higher temperatures, the spectra exhibit non-Gaussian line shapes and decreased line widths. These characteristics are engendered by charge migration. We have analyzed the temperature dependence of the transport process through EPR spectral simulations. The simulations incorporated a nonadiabatic model for electron transfer. The temperature dependence could be adequately described on the basis of an electron-transfer model that accounts for the effects of slow medium relaxation, whereas a satisfactory description could not be obtained on the basis of conventional multimode models for transport. The results of our analysis are consistent with the notion that the protein functions as the primary solvent for the redox centers and are in accord with the view that the protein behaves as a frozen glass, even at room temperature, with respect to the low-frequency vibrational motions coupled to electron transfer.

Quinone exchange at the A1 site in Photosystem I in spinach and cyanobacteria

Abstract The electron spin polarization (ESP) EPR signal arising from the P-700+ A1− radical pair in Photosystem I (PS I) consisting of the oxidized PS I primary donor, P-700 , and the reduced vitamin K1 (K1) acceptor, A1, is studied as a function of isotopic labelling of the native A1 acceptor. No pre-extraction of the native A1 acceptor is utilized, instead, exchange of the native acceptor with protonated or perdeuterated K1 is accomplished by incubation of the reaction centers in solution (incubation/exchange). The incubation/exchange process is studied in PS I of spinach and in two cyanobacteria, the thermophilic Synechococcus lividus (S. lividus) and the non-thermophilic Synechococcus leopoliensis (S. leopoliensis). A complete data set, HPHA1, HPDA1, DPHA1, DPDA1 (where P refers to the primary chlorophyll donor, A1, the quinone acceptor, and H and D denote protonated and deuterated, respectively), is obtained for S. lividus. The correlated radical pair polarization model (CRPP) is used to reproduce the experimental lineshapes for the four different isotopic combinations of the P-700+A1− radical pair. The experimental ESP EPR spectra are well predicted using the geometric and energetic parameters which have been deduced for perdeuterated PS I reaction centers in whole cells of S. lividus (Kothe, G. et al. (1994) J. Phys. Chem. 98, 2706–2712). The results suggest that the isotopically labelled K1 binds in the A1 site with the same orientation as the native acceptor in unexchanged PS I. No evidence for electron transfer to K1 in configurations other than the A1 binding site is observed. Incubation/exchange is studied as a function of incubation temperature. For spinach and non-thermophilic S. leopoliensis the effect of incubation/exchange is most efficient for 1/2 h incubations at temperatures around and above 30–35°C, but requires somewhat higher incubation temperatures, above 40–45°C, for thermophilic S. lividus. Similar changes in the ESP spectrum are also demonstrated for PS I reaction centers in thylakoid membranes of S. lividus.

Characterization of expressed pigmented core light harvesting complex (LH 1) in a reaction center deficient mutant of Blastochloris viridis

The utility of photosynthetically defective mutants in the purple photosynthetic bacterium Blastochloris viridis (formerly Rhodopseudomonas viridis)was demonstrated with construction of a reaction-center deficient mutant, LH 1-H. This LH 1-H mutant has a photosynthetic apparatus in which most of the puf operon genes were deleted, resulting in an organism containing only the genes for the light harvesting polypeptides and the H subunit of the reaction center. This B. viridisstrain containing a truncation of the puf operon was characterized by gel electrophoresis, lipid-to-protein ratio analysis, optical spectroscopy, electron paramagnetic resonance and transmission electron microscopy. Optical and electron paramagnetic resonance spectroscopies revealed no photoactivity in this LH 1-H mutant consistent with the absence of intact reaction centers. Electron paramagnetic resonance evidence for assembled LH 1 complexes suggested that the interactions between light harvesting polypeptide complexes in membranes were largely unchanged despite the absence of their companion reaction center cores. The observed increase in the lipid-to-protein ratio was consistent with modified interactions between LH 1s, a view supported by transmission electron microscopy analysis of membrane fragments. The results show that B. viridis can serve as a practical system for investigating structure-function relationships in membranes and photosynthesis through the construction of photosynthetically defective mutants.

A SAMPLE CELL AND LOW-TEMPERATURE ACCESSORY DESIGN FOR FLUORESCENCE-MODE X-RAY ABSORPTION SPECTROSCOPY OF AIR-SENSITIVE AND CORROSIVE COMPOUNDS

A sample cell and low-temperature cooling unit design suitable for x-ray absorption spectroscopy (XAS) of oxygen and/or water sensitive compounds is presented. The cell is a simplified and optimized modification of a previously suggested design. The low-temperature accessory is specifically designed for good thermal isolation and equilibration at liquid nitrogen temperatures, eliminating the need for a more expensive commercial cryostat. The entire construction can be directly attached to an ion chamber and either a standard Lytle detector or energy-discriminating germanium-element solid-state detector via helium gas-purged adaptor flanges. The apparatus was tested at liquid nitrogen temperatures and gave good temperature stability within ±0.1 K. The XAS signal from an air-sensitive potassium/pyrene complex at 100 K taken at the X-6B beam line at the National Synchrotron Light Source facility at Brookhaven National Laboratory is presented.

Temperature dependent UV-Vis spectral changes in hydrogen- and deuterium-bonded photosynthetic reaction centers of Rhodobacter sphaeroides

The UV-Vis absorption spectra of detergent-isolated hydrogen-and deuterium-bonded reaction centers (RCs) from Rhodobacter sphaeroides PUC 705Ba were examined as a function of temperature between 20 and 55 °C. The enthalpy and entropy of denaturation for the specimens was determined, revealing that their process of thermal denaturation is significantly different. Deuterium-bonded RCs are most stable at 37 °C, rather than at room temperature, and undergo a “cold denaturation” as the temperature is lowered to room temperature. At room temperature the addition of 1,3,5-heptanetriol brought the deuterium-bonded RC back to its more stable configuration. Hence the hydrogen bonding interactions in the RC do influence its conformation and this is reflected in the microenvironment of its associated pigments.