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.

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.

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.

Molecular dynamics simulations of human glutathione transferase P1-1: conformational fluctuations of the apo-structure.

We have investigated by molecular dynamics simulations the conformational fluctuations of the monomer of human apo-glutathione transferase P1-1. After attainment of steady-state dynamics, the structural fluctuations involve mainly the protein segments that participate also in the holo-apo transition discussed in the accompanying article (Stella et al., 1999:37:1-9.). The most mobile region is the C-terminal segment of helix 2. In contrast, helices 1, 6, 7, and 8 constitute a relatively rigid protein core. An "essential dynamics" analysis of the simulation shows that the largest fluctuations involve specific regions of glutathione transferases. In such regions, atomic motions are correlated. Motions of helix 2 are accounted for by the second most prominent principal component, which reveals a fluctuation between two distinct conformations. The residues that constitute the H-site undergo a breathing motion, possibly relevant during the binding of hydrophobic cosubstrates. Based on our simulation, several experimental findings can be rationalized, including the viscosity-dependent reactivity of Cys 47 and Cys 101 as well as the selective proteolysis of the peptide bond between Lys 44 and Ala 45. We have also modeled the structural changes that lead to the formation of an intrachain disulfide bridge between cysteines 47 and 101 and to the inactivation of the enzyme. The resulting structure maintains essentially the native fold except for helix 2, which closes the G-site. Proteins 1999;37:10-19.

Molecular dynamics simulations of human glutathione transferase P1-1: analysis of the induced-fit mechanism by GSH binding.

We report here a 1-ns molecular dynamics simulation on the ligand-free monomer of human glutathione transferase P1-1 in bulk water. The average conformation obtained from the last 500 ps of simulation is taken as a model for the apo-structure of this protein and compared to the available crystallographic data. Remarkable changes in the tertiary structure take place during the simulation and are ascribed to the removal of the ligand. They support an induced fit mechanism occurring upon glutathione binding, whose major features can be described in detail. A portion of helix 2 (residues 42-50), which participates in the formation of the active site, undergoes the most prominent conformational changes. Other protein segments, such as the C-terminal loop and helix 4, also show relevant structural rearrangements. All these transitions cause a significant shielding from the solvent of the hydrophobic binding site of the co-substrate, whose exposed surface goes from 4.6 nm(2) in the holo-structure to about 3.1 nm(2) in the apo-conformation. The results of this simulation are consistent with numerous experimental observations previously obtained on GST P1-1 and provide new insights for their explanation at the molecular level. Proteins 1999;37:1-9.

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).

Erythrocytic proteases: preferential degradation of alpha hemoglobin chains.

Proteolytic activity against native hemoglobin polypeptide chains is demonstrated, under strictly physiological conditions, in human reticulocytes of both normal subjects and individuals suffering from a variety of pathologic conditions involving erythrocytes, including beta-thalassemia. Two thirds of the activity are found in the cytoplasm and the remainder of it is associated with the reticulocyte membrane. That this proteolytic activity is due to contamination by WBC is excluded. The activity preferentially degrades the alpha-hemoglobin chains. An increase in this substrate within the erythroid cells, as observed in beta-thalassemia, does not enhance proteolysis. Protease inhibitors produce a variable decrease in proteolysis. None inhibit completely, thus showing that several enzymes, with different specificities, are involved.