Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Morphological description of limbal epithelium: searching for stem cells crypts in the dog, cat, pig, cow, sheep and horse.

The cornea provides protection and transparency to the eye, allowing an optimal sharpness view. In some pathological conditions the cornea is able to regenerate thanks to the presence of a stem cells reservoir present at the level of the transition area between cornea and sclera (limbus). Corneal cell therapies in Veterinary Medicine are really limited due to the lacking of knowledge about the anatomy of the limbal area, the putative presence of stem cells and their identification in domestic species. The aim of this study was to provide an overview of the main distinctive structural features of the sclero-corneal junction and conjunctival-corneal junction areas in some species of veterinary importance, using optic microscope observations of histological sections. The resulting data were compared with cornea from humans adapting protocols already used to identify stem cells by means of a specific cellular marker. We tested the expression of ΔNp63α isoform in the cornea basal cells, trying to correlate the distribution profile with areas of highly proliferative turnover. The results obtained from this study represent a first step towards the identification of a corneal stem cells reservoir in different animals.

Protein dynamics. An overview on flash-photolysis over broad temperature ranges.

Ligand binding kinetics to heme-proteins between 40 and 300 K point to a regulatory role of protein dynamics. A protein-specific susceptibility of the heme-iron reactivity to dynamic fluctuations emerges from the distribution of reaction enthalpies derived from flash-photolysis measurements below ca. 180 K; we quantify it in terms of 'intramolecular viscosity', postulating that narrow low-temperature enthalpy distributions correspond to low internal viscosity and vice versa. The thermal evolution of ligand binding kinetics suggests, with other results, an interplay between high-frequency transitions of the amino acid side chains and low-frequency collective motions as a possible regulatory mechanism of protein dynamics.

Kinetic properties of cobalt--iron hybrid hemoglobins.

The replacement of O2 with CO was studied on cobalt-iron hemoglobin hybrids. Both proto- and mesocobalt hemes were used for the reconstitution. In the oxy quaternary conformation no difference is observed between alpha- and beta-subunits when only proto hemes are present in the hybrid (k4 = 30 s-1, k'4/l'4 = 2.5). If Co-meso heme is present on the beta-chains the binding properties of the partner subunit are modified (k'4/l'4 = 4).

Electron paramagnetic resonance properties of liver fluke (Dicrocoelium dendriticum) nitrosyl hemoglobin.

The electron paramagnetic resonance properties of the nitric oxide derivative of liver fluke (Dicrocoelium dendriticum) hemoglobin (DD-Hb) have been investigated in the pH range from 4.8 to 7.8. In the neutral and alkaline regions the spectra have a rhombic shape, with gx = 2.09, gy = 1.99 and gz = 2.009, and a triplet hyperfine structure of 2.2 mT, due to the nitrogen of the bound NO molecule, in the center resonance. No superhyperfine lines in the gz region, related to the interaction of the iron with the proximal histidine, are detected, suggesting a large distance between the metal and the N epsilon of the imidazole. By lowering the pH the EPR spectrum undergoes a reversible change showing a 3-line pattern in the high-field region. Such a spectrum is fully formed at pH 4.8 and is interpreted in terms of a dissociation of the proximal histidine from the heme iron.

Development of a hemicornea from human primary cell cultures for pharmacotoxicology testing.

We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen-glycosaminoglycan-chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63DeltaNalpha, and the expression of keratin 3 and 14-3-3sigma in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell-matrix interactions on epithelial cell morphogenesis.

Deformability of the hemoglobin molecule as the basis of its functional behavior.

It has been proposed that the process of ligand binding to hemoproteins is governed by sequential potential barriers as depicted in the following scheme [Austin et al., Biochemistry 14, pp. 5355-5373, 1975; Ansari et al., Proc. natn. Acad. Sci. USA. pp. 5000-5004, 1985]: (formula; see text) At sufficiently high temperatures (approximately 300 K), the binding process is exponential in time and its velocity proportional to the concentration of the ligand in the solvent. At low temperatures (typically less than 210 K), the combination velocity is not correlated with the amount of free ligand in the solvent and the time course is nonexponential (approximately power law). This implies that (a) proteins are flexible structures and fluctuate between many substates, each having a different ligand binding velocity, and (b) that the rate of interconversion between the various conformational substates is high compared to that of ligand binding at and above room temperature and extremely slow below approximately 200 K. We have investigated the temperature dependence of CO binding after a photolysing pulse to a variety of myoglobins and hemoglobin subunits. The results show that for all myoglobins investigated, the most probable activation energy for the inner process (B----A) is in the order of 10 kJ/mol, while for the hemoglobin subunits it varies between 2.5 and 5 kJ/mol. The distribution of barrier heights is very broad in myoglobins, somewhat narrower in the non-alpha-chains and narrowest in the alpha-hemoglobin subunits. This finding implies that different degrees of flexibility exist between storage and transport proteins and that hemoglobin cooperativity may be related to the presence of subunits with different flexibility within the tetramer.

Dynamic properties of monomeric insect erythrocruorin III from Chironomus thummi-thummi: relationships between structural flexibility and functional complexity.

We have investigated the kinetics of geminate carbon monoxide binding to the monomeric component III of Chironomus thummi-thummi erythrocruorin, a protein that undergoes pH-induced conformational changes linked to a pronounced Bohr effect. Measurements were performed from cryogenic temperatures to room temperature in 75% glycerol and either 0.1 M potassium phosphate (pH 7) or 0.1 potassium borate (pH 9) after nanosecond laser photolysis. The distributions of the low temperature activation enthalpy g(H) for geminate ligand binding derived from the kinetic traces are quite narrow and are influenced by temperature both below and above approximately 170 K, the glass transition temperature. The thermal evolution of the CO binding kinetics between approximately 50 K and approximately 170 K indicates the presence of some degree of structural relaxation, even in this temperature range. Above approximately 220 K the width of the g(H) progressively decreases, and at 280 K geminate CO binding becomes exponential in time. Based on a comparison with analogous investigations of the homodimeric hemoglobin from Scapharca inaequivalvis, we propose a link between dynamic properties and functional complexity.

Penetration and interaction with haemoglobin of corynebacteria-like microorganisms into erythrocytes in vitro.

Following 24 h incubation of normal blood in the presence of the microorganism, the evolution of cell wall deficient forms within the erythrocytes and a process of oxidation of the haemoglobin may be observed.

Kinetics of oxygen and carbon monoxide binding to liver fluke (Dicrocoelium dendriticum) hemoglobin. An extreme case?

The kinetics of oxygen and carbon monoxide binding to the monomeric liver fluke (Dicrocoelium dendriticum) hemoglobin have been studied. The ligand association rates are approximately 1 X 10(8) and approximately 3 X 10(8) M-1 s-1, respectively, for CO and O2 and show no pH dependence. On the contrary the ligand dissociation rates decrease by lowering the pH below 7, the pK of the transition being around 5.5. These findings, together with spectroscopic properties of the protein, are discussed in relation to the fact that, in this hemoglobin, the distal histidine is replaced by a glycine.

Encapsulation of hemoglobin in phospholipid liposomes: characterization and stability.

Hemoglobin is encapsulated in liposomes of different lipid composition. The resulting dispersion consists primarily of multilamellar liposomes (hemosomes) of a wide particle size distribution (diameter ranging mainly between 0.1 and 1 micron). The encapsulation efficiency is significantly larger with liposomes containing negatively charged lipids as compared to liposomes made of phosphatidylcholine. The integrity of the phospholipid bilayer is maintained in the presence of hemoglobin. The reaction rate of CO binding to encapsulated hemoglobin is reduced compared to that of free hemoglobin, but it is still greater than that observed in red blood cells. Hemoglobin encapsulated in liposomes made from negatively charged phospholipids is less stable than hemoglobin entrapped in isoelectric phosphatidylcholine. The instability of hemoglobin is due to the protein interacting with the negatively charged lipid bilayer. This interaction leads in turn to hemoglobin denaturation, possibly involving the dissociation of the heme group from the heme-globin complex. The nature of the negatively charged phospholipid is important in promoting the interaction with hemoglobin, the effect being in the order phosphatidic acid greater than phosphatidylinositol congruent to phosphatidylglycerol greater than phosphatidylserine. The presence of equimolar amounts of cholesterol in the phospholipid bilayer has a stabilizing effect on hemoglobin. This effect is pronounced with saturated phospholipids, but it is also observed, though to a lesser extent, with unsaturated ones, indicating that the bilayer fluidity has a modulating effect. The presence of cholesterol possibly interferes with secondary interactions following the binding of hemoglobin to the negatively charged lipid bilayer.

Isocyanide binding kinetics to monomeric hemoproteins. A study on the ligand partition between solvent and heme pocket.

The kinetics of methyl-, ethyl-, iso-propyl-, and ter-butyl-isocyanide binding to Aplysia limacina myoglobin (distal His----Lys) and the isolated beta chains from hemoglobin Zurich (distal His----Arg) have been investigated by flash photolysis at various temperatures above 0 degrees C. Sperm whale (Physter catodon) myoglobin and the isolated beta chains from normal adult hemoglobin have been used as references. In most reaction systems investigated the apparent extent of photolysis increases with temperature. For sperm whale myoglobin and the normal beta chains the increase is of the same magnitude and not correlated to the type of ligand used. On the contrary, for the two proteins lacking the distal histidine, the phenomenon is dependent on the size of the alkyl side chain of the ligand. The results, analyzed on the basis of the multibarrier model (Austin, R.H., K.W. Beeson, L. Eisenstein, H. Frauenfelder, and I.C. Gunsalus, 1975, Biochemistry, 16:5355-5373), suggest that the partition of the ligand molecules between the solvent and the heme pocket, occurring during the photolysis process, is primarily determined by interactions between the ligand and residues in the heme cavity rather than by diffusion through the protein matrix.

Isoelectric focusing of cytochrome P450: isolation of six phenobarbital-inducible rat liver microsomal isoenzymes.

A procedure for the isolation of native proteins from membranes by isoelectric focusing is described. It was used to resolve into six components the major fraction of cytochrome P450, obtained from liver microsomes of phenobarbital-treated rats, after chromatography on DE-52 cellulose. When eluted from the gel, these proteins are in a native form as shown by (a) the light absorption spectra of the Soret region of their reduced carbonyl derivatives, all characterized by maxima around 450 nm, and (b) their enzymatic activities toward three different substrates. Characterization by a monoclonal antibody and partial sequence analysis of tryptic peptides reveal that three of the IEF-purified proteins have P450IIB1 character, whereas the other three are related to P450IIB2.