Grafting of an autologous tissue-engineered human corneal epithelium to a patient with limbal stem cell deficiency (LSCD).

PURPOSE: In this study, we evaluated the feasibility of recovering the corneal surface integrity in a patient suffering from unilateral LSCD through the transplantation of cultured autologous corneal epithelial cells. METHODS: Human corneal epithelial cells (HCECs) were isolated from a limbal biopsy of the contralateral eye of a patient with unilateral LSCD and cultured in monolayer in the presence of an irradiated human fibroblasts feeder layer (iHFL). To produce a cultured autologous corneal epithelium (CACE), HCECs were seeded on a fibrin substrate and maintained in culture until confluence. The in vitro obtained CACE was then used to treat the affected eye of the patient. Two years later, a successful penetrating keratoplasty was performed. RESULTS: Efficient restoration of the corneal epithelium was achieved following transplantation of CACE indicating probable re-colonization of the cornea by stem cells. Corneal transparency was restored after removing the scarred stroma by performing a penetrating keratoplasty. CONCLUSION: CACE produced in vitro was shown to restore a normal corneal surface capable of sustaining a viable and clear penetrating keratoplasty and reestablished a near normal vision in a unilateral LSCD patient.

The presence of a feeder layer improves human corneal endothelial cell proliferation by altering the expression of the transcription factors Sp1 and NFI.

Based on the use of tissue-cultured human corneal endothelial cells (HCECs), cell therapy is a very promising avenue in the treatment of corneal endothelial pathologies such as Fuchs' dystrophy, and post-surgical corneal edema. However, once in culture, HCECs rapidly lose their phenotypic and physiological characteristics, and are therefore unsuitable for the reconstruction of a functional endothelial monolayer. Expression of NFI, a transcription factor that can either function as an activator or a repressor of gene transcription, has never been examined in endothelial cells. The present study therefore aimed to determine the impact of a non-proliferating, lethally irradiated i3T3 feeder layer on the maintenance of HCEC's morphological characteristics, and both the expression and stability of Sp1 (a strong transcriptional activator) and NFI in such cells. The typical morphology of endothelial cells was best maintained when 8 × 103/cm2 HCECs were co-cultured in the presence of 2 × 104 cells/cm2 i3T3. HCECs were found to express both Sp1 and NFI in vitro. Also, the presence of i3T3 led to higher levels of Sp1 and NFI in HCECs, with a concomitant increase in their DNA binding levels (assessed by electrophoretic mobility shift assays (EMSA)). Specifically, i3T3 increased the expression of the NFIA, NFIB and NFIC isoforms, without a noticeable increase in their mRNAs (as revealed by gene profiling on microarray). Gene profiling analysis also identified a few feeder layer-dependent, differentially regulated genes whose protein products may contribute to improving the properties of HCECs in culture. Therefore, co-culturing HCECs with an i3T3 feeder layer clearly improves their morphological characteristics by maintaining stable levels of Sp1 and NFI in cell culture.

The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing.

Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing.

Combined increases in mitochondrial cooperation and oxygen photoreduction compensate for deficiency in cyclic electron flow in Chlamydomonas reinhardtii.

During oxygenic photosynthesis, metabolic reactions of CO2 fixation require more ATP than is supplied by the linear electron flow operating from photosystem II to photosystem I (PSI). Different mechanisms, such as cyclic electron flow (CEF) around PSI, have been proposed to participate in reequilibrating the ATP/NADPH balance. To determine the contribution of CEF to microalgal biomass productivity, here, we studied photosynthesis and growth performances of a knockout Chlamydomonas reinhardtii mutant (pgrl1) deficient in PROTON GRADIENT REGULATION LIKE1 (PGRL1)-mediated CEF. Steady state biomass productivity of the pgrl1 mutant, measured in photobioreactors operated as turbidostats, was similar to its wild-type progenitor under a wide range of illumination and CO2 concentrations. Several changes were observed in pgrl1, including higher sensitivity of photosynthesis to mitochondrial inhibitors, increased light-dependent O2 uptake, and increased amounts of flavodiiron (FLV) proteins. We conclude that a combination of mitochondrial cooperation and oxygen photoreduction downstream of PSI (Mehler reactions) supplies extra ATP for photosynthesis in the pgrl1 mutant, resulting in normal biomass productivity under steady state conditions. The lower biomass productivity observed in the pgrl1 mutant in fluctuating light is attributed to an inability of compensation mechanisms to respond to a rapid increase in ATP demand.

Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells.

PURPOSE: To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. METHODS: Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or α- or ß-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly. RESULTS: Three or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (α and ß) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin. CONCLUSIONS: This collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell-cell contacts and the existence of polarized morphology of these layers over corneal equivalents.

A tissue-engineered corneal wound healing model for the characterization of reepithelialization.

Progress in tissue engineering has led to the discovery of technologies allowing reconstruction of autologous tissues from the patient's own cells and the development of new in vitro models to study cellular and molecular mechanisms implicated in wound healing. The outer surface of the eye, the cornea, is involved in the sense of sight, thus an adequate reepithelialization process after wounding is essential in order to maintain corneal function. In this chapter, protocols to generate a new in vitro three-dimensional human corneal wound healing model suitable for studying the different components that play important roles in corneal reepithelialization are described in details. The methods include extraction and culture of human corneal epithelial cells (HCECs), human corneal fibroblasts, a complete description of the cornea reconstructed by tissue-engineering as well as the corneal wound healing model.

Expression of the α5 integrin gene in corneal epithelial cells cultured on tissue-engineered human extracellular matrices.

The integrin α5ß1 plays a major role in corneal wound healing by promoting epithelial cell adhesion and migration over the fibronectin matrix secreted as a cellular response to corneal damage. Expression of α5 is induced when rabbit corneal epithelial cells (RCECs) are grown in the presence of fibronectin. Here, we examined whether α5 expression is similarly altered when RCECs or human corneal epithelial cells (HCECs) are grown on a reconstructed stromal matrix used as an underlying biomaterial. Mass spectrometry and immunofluorescence analyses revealed that the biomaterial matrix produced by culturing human corneal fibroblasts with ascorbic acid (ECM/35d) contains several types of collagens, fibronectin, tenascin and proteoglycans. Results from transfection of CAT/α5-promoter plasmids, Western blot and EMSA analyses indicated that ECM/35d significantly increase expression of α5 in HCECs as a result of alteration in the expression and DNA binding of the transcription factors NFI, Sp1, AP-1 and PAX6. The biological significance of this biomaterial substitute on the expression of the α5 gene may therefore contribute to better understand the function played by the α5ß1 integrin during corneal wound healing.

Heavy metal accumulation by recombinant mammalian metallothionein within Escherichia coli protects against elevated metal exposure.

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins known to provide protection against toxic heavy metals such as cadmium. In an attempt to increase the ability of bacterial cells to accumulate heavy metals, sheep MTII was produced in fusion with the maltose binding protein (MBP) and localized to the cytoplasmic or periplasmic compartments of Escherichia coli. For all metals tested, higher levels of bioaccumulation were measured with strains over-expressing MBP-MT in comparison with control strains. A marked bioaccumulation of Cd, As, Hg and Zn was observed in the strain over-expressing MBP-MT in the cytoplasm, whereas Cu was accumulated to higher levels when MBP-MT was over-expressed in the periplasm. Metal export systems may also play a role in this bioaccumulation. To illustrate this, we over-expressed MBP-MT in the cytoplasm of two mutant strains of E. coli affected in metal export. The first, deficient in the transporter ZntA described to export numerous divalent metal ions, showed increasing quantities of Zn, Cd, Hg and Pb being bioaccumulated. The second, strain LF20012, deficient in As export, showed that As was bioaccumulated in the form of arsenite. Furthermore, high quantities of accumulated metals, chelated by MBP-MT in the cytoplasm, conferred greater metal resistance levels to the cells in the presence of added toxic metals, such as Cd or Hg, while other metals showed toxic effects when the export systems were deficient. The strain over-expressing MBP-MT in the cytoplasm, in combination, with disruption of metal export systems, could be used to develop strategies for bioremediation.

Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves.

BACKGROUND: When cultivated under stress conditions, many microalgae species accumulate both starch and oil (triacylglycerols). The model green microalga Chlamydomonas reinhardtii has recently emerged as a model to test genetic engineering or cultivation strategies aiming at increasing lipid yields for biodiesel production. Blocking starch synthesis has been suggested as a way to boost oil accumulation. Here, we characterize the triacylglycerol (TAG) accumulation process in Chlamydomonas and quantify TAGs in various wild-type and starchless strains. RESULTS: In response to nitrogen deficiency, Chlamydomonas reinhardtii produced TAGs enriched in palmitic, oleic and linoleic acids that accumulated in oil-bodies. Oil synthesis was maximal between 2 and 3 days following nitrogen depletion and reached a plateau around day 5. In the first 48 hours of oil deposition, a ~80% reduction in the major plastidial membrane lipids occurred. Upon nitrogen re-supply, mobilization of TAGs started after starch degradation but was completed within 24 hours. Comparison of oil content in five common laboratory strains (CC124, CC125, cw15, CC1690 and 11-32A) revealed a high variability, from 2 µg TAG per million cell in CC124 to 11 µg in 11-32A. Quantification of TAGs on a cell basis in three mutants affected in starch synthesis (cw15sta1-2, cw15sta6 and cw15sta7-1) showed that blocking starch synthesis did not result in TAG over-accumulation compared to their direct progenitor, the arginine auxotroph strain 330. Moreover, no significant correlation was found between cellular oil and starch levels among the twenty wild-type, mutants and complemented strains tested. By contrast, cellular oil content was found to increase steeply with salt concentration in the growth medium. At 100 mM NaCl, oil level similar to nitrogen depletion conditions could be reached in CC124 strain. CONCLUSION: A reference basis for future genetic studies of oil metabolism in Chlamydomonas is provided. Results highlight the importance of using direct progenitors as control strains when assessing the effect of mutations on oil content. They also suggest the existence in Chlamydomonas of complex interplays between oil synthesis, genetic background and stress conditions. Optimization of such interactions is an alternative to targeted metabolic engineering strategies in the search for high oil yields.

Corneal endothelial toxicity of air and SF6.

PURPOSE: The authors conducted in vivo assessment of corneal endothelial toxicity of air and SF6 in the feline model. This research was motivated by the increased use of air in anterior segment surgery in human subjects. METHODS: This was a prospective masked study. The eyes of 16 healthy adult cats were randomly assigned for the injection of 0.7 mL air into the anterior chamber of one eye and SF6 in the contralateral eye. Daily examination included slit lamp photographs, pachymetry, and tonometry. Specular microscopy was performed before, 7 days after, and 10 days after injection. The animals were euthanatized, and the corneas were processed for alizarin red-trypan blue staining and for light and electron microscopy. RESULTS: SF6 remained in the anterior chamber significantly longer than air. Both groups showed postinjection inflammation, which on average was maximal at day 2 and more severe with SF6. No difference in IOP was observed between the two groups. Specular microscopy showed significant endothelial cell loss in the SF6 group (mean postinjection cell loss, 132 ± 50 cells/mm(2)) but not in the group injected with air. Alizarin red staining revealed significant regional differences in cell density only in the SF6 group and more pronounced endothelial cell loss in the superior area. CONCLUSIONS: These results indicate that both air and SF6 injected into the anterior chamber of the eye can induce intraocular reaction in the feline model and that SF6 is more toxic than air in terms of endothelial cell loss and anterior chamber inflammation.

Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.

PURPOSE: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. METHODS: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. RESULTS: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1. CONCLUSIONS: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.

Tissue engineering of skin and cornea: Development of new models for in vitro studies.

Human beings are greatly preoccupied with the unavoidable nature of aging. While the biological processes of senescence and aging are the subjects of intense investigations, the molecular mechanisms linking aging with disease and death are yet to be elucidated. Tissue engineering offers new models to study the various processes associated with aging. Using keratin 19 as a stem cell marker, our studies have revealed that stem cells are preserved in human skin reconstructed by tissue engineering and that the number of epithelial stem cells varies according to the donor's age. As with skin, human corneas can also be engineered in vitro. Among the epithelial cells used for reconstructing skin and corneas, significant age-dependent variations in the expression of the transcription factor Sp1 were observed. Culturing skin epithelial cells with a feeder layer extended their life span in culture, likely by preventing Sp1 degradation in epithelial cells, therefore demonstrating the pivotal role played by this transcription factor in cell proliferation. Finally, using the human tissue-engineered skin as a model, we linked Hsp27 activation with skin differentiation.

Surface topography induces 3D self-orientation of cells and extracellular matrix resulting in improved tissue function.

The organization of cells and extracellular matrix (ECM) in native tissues plays a crucial role in their functionality. However, in tissue engineering, cells and ECM are randomly distributed within a scaffold. Thus, the production of engineered-tissue with complex 3D organization remains a challenge. In the present study, we used contact guidance to control the interactions between the material topography, the cells and the ECM for three different tissues, namely vascular media, corneal stroma and dermal tissue. Using a specific surface topography on an elastomeric material, we observed the orientation of a first cell layer along the patterns in the material. Orientation of the first cell layer translates into a physical cue that induces the second cell layer to follow a physiologically consistent orientation mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in an oriented self-assembled tissue sheet. These self-assembled tissue sheets were then used to create 3 different structured engineered-tissue: cornea, vascular media and dermis. We showed that functionality of such structured engineered-tissue was increased when compared to the same non-structured tissue. Dermal tissues were used as a negative control in response to surface topography since native dermal fibroblasts are not preferentially oriented in vivo. Non-structured surfaces were also used to produce randomly oriented tissue sheets to evaluate the impact of tissue orientation on functional output. This novel approach for the production of more complex 3D tissues would be useful for clinical purposes and for in vitro physiological tissue model to better understand long standing questions in biology.

Impact of cell source on human cornea reconstructed by tissue engineering.

PURPOSE: To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro. METHODS: Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined. RESULTS: The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis. CONCLUSIONS: The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis.

Tissue engineering of feline corneal endothelium using a devitalized human cornea as carrier.

The difficulties in obtaining good quality tissue for the replacement of corneas of patients suffering from endothelial dysfunctions have prompted us to evaluate the feasibility of producing a tissue-engineered (TE) corneal endothelium using devitalized human stromal carriers. Thus, corneal substitutes were produced by seeding cultured feline corneal endothelial cells on top of previously frozen human corneal stromas. After two weeks of culture to allow attachment and spreading of the seeded cells, the TE corneal endothelium was stained with alizarin red for endothelial cell count and fixed for histology, immunofluorescence labeling, scanning and transmission electron microscopy. Histology and Hoechst staining showed that there were no remaining cells in the devitalized stroma. After seeding, histology and transmission electron microscopy showed that the TE corneal endothelium formed a monolayer of tightly packed cells that were well adhered to Descemet's membrane. Scanning electron microscopy corroborated that the cells covered the entire posterior corneal surface and had an endothelial morphology. Alizarin staining showed that mean cell counts were 2272 +/- 344 cells/mm(2), indicating that the cell density was appropriate for grafting. The TE feline corneal endothelium also expressed the function-related proteins Na(+)/HCO(3)(-), ZO-1, and Na(+)/K(+)-ATPase alpha1, and could easily be marked with a fluorescent tracker. This study demonstrates the feasibility of reconstructing a highly cellular and healthy corneal endothelium on devitalized human corneal stromas.

Regeneration of skin and cornea by tissue engineering.

Progress in tissue engineering has led to the development of technologies allowing the reconstruction of autologous tissues from the patient's own cells. Thus, tissue-engineered epithelial substitutes produced from cultured skin epithelial cells undergo long-term regeneration after grafting, indicating that functional stem cells were preserved during culture and following grafting. However, these cultured epithelial sheets reconstruct only the upper layer of the skin and lack the mechanical properties associated to the connective tissue of the dermis. We have designed a reconstructed skin entirely made from human cutaneous cells comprising both the dermis and the epidermis, as well as a well-organized basement membrane by a method named the self-assembly approach. In this chapter, protocols to generate reconstructed skin and corneal epithelium suitable for grafting are described in details. The methods include extraction and culture of human skin keratinocytes, human skin fibroblasts as well as rabbit and human corneal epithelial cells, and a complete description of the skin reconstructed by the self-assembly approach and of corneal epithelium reconstructed over a fibrin gel.

Characterization of wound reepithelialization using a new human tissue-engineered corneal wound healing model.

PURPOSE: The reepithelialization of the corneal surface is an important process for restoring the imaging properties of this tissue. The purpose of the present study was to characterize and validate a new human in vitro three-dimensional corneal wound healing model by studying the expression of basement membrane components and integrin subunits that play important roles during epithelial cell migration and to verify whether the presence of exogenous factors could accelerate the reepithelialization. METHODS: Tissue-engineered human cornea was wounded with a 6-mm biopsy punch, and the reepithelialization from the surrounding margins was studied. Biopsy samples of the reepithelialized surface were harvested 3 days after wounding and were processed for histologic, electron microscopic, and immunofluorescence analyses. The effects of fibrin and epithelial growth factor (EGF) on wound reepithelialization were also studied. RESULTS: Results demonstrated that this in vitro model allowed the migration of human corneal epithelial cells on a natural extracellular matrix. During reepithelialization, epithelial cell migration followed a consistent wavelike pattern similar to that reported for human corneal wound healing in vivo. This model showed a histologic appearance similar to that of native tissue as well as expression and modulation of basement membrane components and the integrin subunits known to be main actors during the wound healing process. It also allowed quantification of the reepithelialization rate, which was significantly accelerated in the presence of fibrin or EGF. The results indicated that alpha v beta6 integrin expression was increased in the migrating epithelial cells compared with the surrounding corneal tissue. CONCLUSIONS: The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing.

Transcriptional regulation of the human alpha6 integrin gene by the transcription factor NFI during corneal wound healing.

PURPOSE: Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human alpha6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human alpha6 gene, and its regulatory influence was characterized in corneal epithelial cells. METHODS: Plasmids bearing the alpha6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the alpha6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. RESULTS: All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of alpha6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of alpha6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. CONCLUSIONS: Repression of alpha6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane.

Metal binding and antioxidant properties of chimeric tri- and tetra-domained metallothioneins.

An unusual tri-domained (alpha-beta-beta) natural oyster metallothionein (MT) is known, and non-oxidative MT dimers occur in vivo in mollusk species and in mammals. To assess the respective role of the MT domains, two chimeric MTs were constructed: a tetra-domained oyster MT corresponding to the alpha-beta-alpha-beta structure, in order to mimic the natural non-oxidative dimeric form, and a tri-domained alpha-beta-alpha oyster MT. Metal binding and putative antioxidant properties of these two chimeric MTs were investigated using expression of the related genes in the bacteria Escherichia coli. In a wild-type strain these MTs could efficiently bind Cd. In a superoxide dismutase (sodA sodB) null mutant, the tri-domained MT was found to exacerbate Cd toxicity whereas the tetra-domained MT efficiently protected bacteria from Cd. The paradoxical toxicity displayed by the tri-domained MT upon Cd contamination was linked to the generation of superoxide radicals generated by a mechanism which most probably involves a copper-redox cycling reaction, since a Cd-contaminated sodA sodB strain expressing this MT produced 4 times more O2(-) than the control bacteria, and MT toxicity disappeared in the presence of bathocuproine disulfonic acid, a copper chelator. In contrast, the tetra-domained form did not. Interestingly, in bacteria producing superoxide dismutase but hypersensitive to oxidative stress due to either mutations in thioredoxin and glutathione reductase pathways (WM104 mutant) or to a lack of gamma-glutamylcysteine synthetase (gshA mutant), both chimeric MTs were protecting against Cd toxicity. However, an unexpected lack of antioxidant function was observed for both chimeric MTs, which were found to enhance the toxicity of hydrogen peroxide in WM104, or that of menadione in QC1726. Altogether, our results suggest that superoxide dismutase activity counteracts the potential prooxidative effect of the tri-domained MT mediated by Cu ions and that the tetra-domained form is a very efficient protector against metal toxicity in vivo.

Heavy metal tolerance in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an aerobic, non-fermentative Gram-negative bacterium widespread in the environment. S. maltophilia Sm777 exhibits innate resistance to multiple antimicrobial agents. Furthermore, this bacterium tolerates high levels (0.1 to 50 mM) of various toxic metals, such as Cd, Pb, Co, Zn, Hg, Ag, selenite, tellurite and uranyl. S. maltophilia Sm777 was able to grow in the presence of 50 mM selenite and 25 mM tellurite and to reduce them to elemental selenium (Se(0)) and tellurium (Te(0)) respectively. Transmission electron microscopy and energy dispersive X-ray analysis showed cytoplasmic nanometer-sized electron-dense Se(0) granules and Te(0) crystals. Moreover, this bacterium can withstand up to 2 mM CdCl(2) and accumulate this metal up to 4% of its biomass. The analysis of soluble thiols in response to ten different metals showed eightfold increase of the intracellular pool of cysteine only in response to cadmium. Measurements by Cd K-edge EXAFS spectroscopy indicated the formation of Cd-S clusters in strain Sm777. Cysteine is likely to be involved in Cd tolerance and in CdS-clusters formation. Our data suggest that besides high tolerance to antibiotics by efflux mechanisms, S. maltophilia Sm777 has developed at least two different mechanisms to overcome metal toxicity, reduction of oxyanions to non-toxic elemental ions and detoxification of Cd into CdS.