Preferential transcription of rabbit Aldh1a1 in the cornea: implication of hypoxia-related pathways.

Here we examine the molecular basis for the known preferential expression of rabbit aldehyde dehydrogenase class 1 (ALDH1A1) in the cornea. The rabbit Aldh1a1 promoter-firefly luciferase reporter transgene (-3519 to +43) was expressed preferentially in corneal cells in transfection tests and in transgenic mice, with an expression pattern resembling that of rabbit Aldh1a1. The 5' flanking region of the rabbit Aldh1a1 gene resembled that in the human gene (60.2%) more closely than that in the mouse (46%) or rat (51.5%) genes. We detected three xenobiotic response elements (XREs) and one E-box consensus sequence in the rabbit Aldh1a1 upstream region; these elements are prevalent in other highly expressed corneal genes and can mediate stimulation by dioxin and repression by CoCl(2), which simulates hypoxia. The rabbit Aldh1a1 promoter was stimulated fourfold by dioxin in human hepatoma cells and repressed threefold by CoCl(2) treatment in rabbit corneal stromal and epithelial cells. Cotransfection, mutagenesis, and gel retardation experiments implicated the hypoxia-inducible factor 3alpha/aryl hydrocarbon nuclear translocator heterodimer for Aldh1a1 promoter activation via the XREs and stimulated by retinoic acid protein 13 for promoter repression via the E-box. These experiments suggest that XREs, E-boxes, and PAS domain/basic helix-loop-helix transcription factors (bHLH-PAS) contribute to preferential rabbit Aldh1a1 promoter activity in the cornea, implicating hypoxia-related pathways.

Adaptive evolution of small heat shock protein/alpha B-crystallin promoter activity of the blind subterranean mole rat, Spalax ehrenbergi.

Blind mole rats have degenerated subcutaneous eyes that are visually nonfunctional. In this investigation, we have compared the tissue specificity of the small heat shock protein (shsp)/alphaB-crystallin promoter of the mole rat superspecies, Spalax ehrenbergi, with that of the mouse. Earlier experiments showed that mouse shsp/alphaB-crystallin promoter/enhancer activity is high in the lens and moderate in the heart and skeletal muscle of transgenic mice. Here, we show in transgenic mouse experiments using the firefly luciferase reporter gene that, despite relatively few changes in sequence, the mole rat shsp/alphaB-crystallin promoter/enhancer has selectively lost lens activity after 13.5 days of embryogenesis (E13.5). The ratios of mole rat/mouse promoter activity were 0.01 for lens, 1.7 for heart, and 13.6 for skeletal muscle in 8-wk-old transgenic mice. Our data indicate that the shsp/alphaB-crystallin promoter/enhancer has undergone adaptive changes corresponding to the subterranean evolution of the blind mole rat. We speculate that selective pressures on metabolic economy may have contributed to these tissue-specific modifications of promoter/enhancer function during adaptation to life underground.

J3-crystallin of the jellyfish lens: similarity to saposins.

J3-crystallin, one of the three major eye-lens proteins of the cubomedusan jellyfish (Tripedalia cystophora), shows similarity to vertebrate saposins, which are multifunctional proteins that bridge lysosomal hydrolases to lipids and activate enzyme activity. Sequence alignment of deduced J3-crystallin indicates two saposin-like motifs arranged in tandem, each containing six cysteines characteristic of this protein family. The J3-crystallin cDNA encodes a putative precursor analogous to vertebrate prosaposins. The J3-crystallin gene has seven exons, with exons 2-4 encoding the protein. Exon 3 encodes a circularly permutated saposin motif, called a swaposin, found in plant aspartic proteases. J3-crystallin RNA was found in the cubomedusan lens, statocyst, in bands radiating from the pigmented region of the ocellus, in the tentacle tip by in situ hybridization, and in the embryo and larva by reverse transcription-PCR. Our data suggest a crystallin role for the multifunctional saposin protein family in the jellyfish lens. This finding extends the gene sharing evolutionary strategy for lens crystallins to the cnidarians and indicates that the putative primordial saposin/swaposin J3-crystallin reflects both the chaperone and enzyme connections of the vertebrate crystallins.

Enigma of the abundant water-soluble cytoplasmic proteins of the cornea: the "refracton" hypothesis.

It is accepted that the taxon-specific, multifunctional crystallins (small heat-shock proteins and enzymes) serve structural roles contributing to the transparent and refractive properties of the lens. The transparent cornea also accumulates unexpectedly high proportions of taxon-specific, multifunctional proteins particularly, but not only, in the epithelium. For example, aldehyde dehydrogenase 3 (ALDH3) is the main water-soluble protein in corneal epithelial cells of most mammals (but ALDH1 predominates in the rabbit), whereas gelsolin predominates in the zebrafish corneal epithelium. Moreover, some invertebrates (e.g., squid and scallop) accumulate proteins in their corneas that are similar to their lens crystallins. Pax-6, among other transcription factors, is implicated in development and tissue-specific gene expression of the lens and cornea. Environmental factors appear to influence gene expression in the cornea, but not the lens. Although no direct proof exists, the diverse, abundant corneal proteins may have evolved a crystallinlike role, in addition to their enzymatic or cytoskeletal functions, by a gene sharing mechanism similar to the lens crystallins. Consequently, it is proposed that the cornea and lens be considered as a single refractive unit, called here the "refracton," to emphasize their similarities and common function.

Dual use of the transcriptional repressor (CtBP2)/ribbon synapse (RIBEYE) gene: how prevalent are multifunctional genes?

Vertebrates have ribbon synapses in the retina and in other sensory structures that are specialized for rapid, tonic release of synaptic vesicles (1). The lamellar sheets of the ribbon situated at right angles to the plasma membrane are lined with synaptic vesicles that undergo exocytosis under the influence of Ca(2+). Synaptic ribbons act as a conveyer belt to accelerate the release of this ready supply of synaptic vesicles at the presynaptic membranes. Although the protein composition of the terminals of ribbon synapses is generally similar to that of ordinary synapses in nervous tissue, much less is known about the composition of the ribbons themselves. RIM, a universal component of presynaptic active zones that interacts with rab3 on the synaptic vesicle, has been localized to the ribbons (2). In addition, the kinesin motor protein, KIF3A, is associated with the ribbons and other organelles in presynaptic nerve terminals (3). Recently, an approximately 120 kDa protein called RIBEYE has been identified in purified ribbons of bovine retina. The RIBEYE cDNA was cloned and its gene identified in the database.

The mouse beta B1-crystallin promoter: strict regulation of lens fiber cell specificity.

Previous studies have shown that the chicken beta B1-crystallin promoter (-434/+30) contains all of the signals necessary to specifically direct high level expression of heterologous genes to the lens fiber cells of mice. In the present study, the mouse beta B1-crystallin gene was cloned, and its regulation was investigated to further elucidate the mechanisms controlling lens fiber cell-specific gene expression. Phylogenetic footprinting analysis of the 5' flanking sequence from the mouse, rat, human and chicken beta B1-crystallin genes identified several known and putative functional cis elements including the PL2 element which is required for lens-specific expression of the chicken beta B1 promoter. Surprisingly, however, all six mouse beta B1-crystallin/CAT constructs tested (-1493/+44, -1493/+30, -870/+30, -250/+30, -135/+30 and -98/+30) were inactive in three different mammalian lens-derived cell lines while only the -870/+30 and -98/+30 constructs were active in chicken primary patched lens epithelial cells. In contrast, the chicken beta B1-crystallin promoter (-434/+30) was transcriptionally active in all lens-derived cells tested. Transgenic mice harboring a mouse beta B1-crystallin -1493/+44 CAT construct did express the transgene specifically in lens fiber cells, however, at lower levels than that previously reported for a chicken -434/+30 CAT construct. These data suggest that, as in other crystallin genes, the regulatory signals controlling lens fiber cell-specific expression are conserved between chicken and mouse. However, the inability of the mouse beta B1-crystallin promoter to function in mammalian lens-derived cultured cells implies that this gene has acquired additional cis-regulatory elements to ensure lens fiber cell specificity.

Transketolase gene expression in the cornea is influenced by environmental factors and developmentally controlled events.

PURPOSE: Transketolase (TKT) has been proposed to be a corneal crystallin, and its gene and protein are abundantly expressed in the corneal epithelium of several mammals. A marked up-regulation of TKT gene expression coincides with the time of eyelid opening in the mouse. Here, we examined whether exposure to incident light contributes to the up-regulation of TKT gene expression during cornea maturation. METHODS: Mice were raised in either standard light/dark cycling conditions or total darkness. In some cases, subcutaneous injections of epidermal growth factor (EGF) were given beginning on the day of birth to induce early eyelid opening. RNA was prepared from the corneas of mothers and pups and subjected to Northern blot analyses. In addition, the relative levels of TKT mRNA and/or enzyme activity were examined in the corneas of human, bovine, rat, chicken, and zebrafish. RESULTS: TKT mRNA levels were 2.1-fold higher in the corneas of 25-day-old mouse pups ( 12 days after eyelid opening) that had been born and raised in light/dark conditions compared to pups born and raised in total darkness. By contrast, the level of TKT mRNA in the mature corneas of adult mice maintained in the dark for 2-8 weeks did not vary greatly from those of mice maintained in light/dark conditions. Interestingly, TKT mRNA levels in the corneas of dark-raised mice, although reduced, did exhibit the increase characteristically observed before and after eyelid opening. In addition, TKT mRNA levels were elevated fivefold in the corneas of 28-day-old mice raised in darkness and injected with EGF compared to uninjected mice also deprived of light. The EGF-injected mice opened their eyes 3 days early, and their corneal epithelium did not grossly differ from that of control mice. TKT mRNA and/or enzyme activity was found to be much higher in the corneas than in other tissues of humans, bovines, and rats but was extremely low in the corneas of chicken and zebrafish. CONCLUSION: Our studies suggest that both exposure to incident light and events surrounding the process of eyelid opening play a role in the up-regulation of TKT gene expression observed during corneal maturation in mice. Light appears to play a less important role in the mature cornea in maintaining high levels of TKT gene expression. The low levels of TKT in the cornea of chicken and zebrafish support the notion that TKT acts as a taxon-specific enzyme-crystallin in mammals. The involvement of environmental signals for this putative, mammalian cornea crystallin contrasts with the purely developmental signals involved in the up-regulation of the crystallin genes of the lens.

Expression of betaB(2)-crystallin mRNA and protein in retina, brain, and testis.

PURPOSE: To evaluate the expression of betaB(2)-crystallin mRNA and protein in rat, bovine, and human nonlens and nonocular tissues. METHODS: betaB(2)-crystallin mRNA levels were detected by RT-PCR. betaB(2)-crystallin protein was purified from rat and bovine tissues by FPLC chromatography. FPLC fractions were analyzed by immunoblotting. The identity of betaB(2)-crystallin protein, isolated from the retina, was confirmed by protein microsequencing. RESULTS: betaB(2)-crystallin transcript was detected in rat brain, rat testis, and human retina by RT-PCR. betaB(2)-crystallin transcript was not found in rat lung, heart, ovary, spleen, thymus, kidney, and liver or in human brain and testis. betaB(2)-crystallin protein was partially purified from and its identity confirmed in rat brain, rat testis, and bovine retina. The bovine retinal protein was further confirmed to be authentic betaB(2)-crystallin by protein microsequencing. CONCLUSIONS: These results establish that betaB(2)-crystallin mRNA and protein are expressed in tissues outside of the lens and outside of the eye including retina, brain, and testis. Extralenticular and extraocular expression of betaB(2)-crystallin, coupled with its participation in phosphorylation pathways, suggests that it has nonrefractive functions in these tissues.

Omega -crystallin of the scallop lens. A dimeric aldehyde dehydrogenase class 1/2 enzyme-crystallin.

While many of the diverse crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Omega-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Omega-crystallin (officially designated ALDH1A9) is 55-56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/eta-crystallin of elephant shrews. Like other enzyme-crystallins, scallop Omega-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Omega-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Omega-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Omega-crystallins, which are tetrameric proteins, scallop Omega-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.

Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of (alpha)5(beta)1 integrin expression.

The PAX6 gene, a key regulator of eye development, produces two major proteins that differ in paired domain structure: PAX6 and PAX6(5a). It is known that an increase in the PAX6(5a) to PAX6 ratio leads to multiple ocular defects in humans. Here, transgenic mice were created that overexpress human PAX6(5a) in the lens. These mice develop cataracts with abnormalities in fiber cell shape as well as fiber cell/lens capsule and fiber cell/fiber cell interactions. While the structure of the actin cytoskeleton appeared relatively normal, the cataractous lens expresses increased amounts of paxillin and p120(ctn) as well as large aggregates of (alpha)5(beta)1 integrin in the dysgenic fiber cells. The elevated amounts of these proteins in the transgenic lens correlated well with elevated levels of their respective mRNAs. To investigate the role of Pax-6(5a) in the upregulation of these genes, a series of gel shift experiments using truncated proteins and consensus oligonucleotides demonstrated the complexity of Pax-6 and Pax-6(5a) binding to DNA, aiding our identification of potential binding sites in the human (&agr;)5- and (beta)1-integrin promoters. Consequent gel shift analysis demonstrated that these putative regulatory sequences bind Pax-6 and/or Pax-6(5a) in lens nuclear extracts, suggesting that the human (alpha)5 and (beta)1 integrin promoters contain PAX6/PAX6(5a) binding sites and maybe directly regulated by this transcription factor in the transgenic lens. We conclude that these transgenic mice are good models to study a type of human cataract and for identifying batteries of genes that are directly or indirectly regulated by both forms of Pax-6.

Review: A case for corneal crystallins.

It is established that the diverse, multifunctional crystallins are responsible for the optical properties of the cellular, transparent lens of the complex eyes of vertebrates and invertebrates. Lens crystallins often differ among species and may be enzymes or stress proteins. I present here the idea that abundant water-soluble enzymes and other proteins may also be used for cellular transparency in the epithelial cells and, possibly, stromal keratocytes of the cornea. Aldehyde dehydrogenases and transketolase are among the putative "corneal crystallins" in mammals, and gelsolin may be a corneal crystallin in the zebrafish. In invertebrates, the glutathione S-transferase-related S-crystallins of the lens appear to be used also as corneal crystallins in the squid, and an aldehyde dehydrogenase-related protein is the crystallin in the lens and, possibly, cornea of the scallop. The use of abundant, taxon-specific water-soluble proteins as crystallins for cellular transparency in the cornea would provide a new conceptual link between this tissue and the lens.

Evidence for gelsolin as a corneal crystallin in zebrafish.

We have shown that gelsolin is one of the most prevalent water-soluble proteins in the transparent cornea of zebrafish. There are also significant amounts of actin. In contrast to actin, gelsolin is barely detectable in other eye tissues (iris, lens, and remaining eye) of the zebrafish. Gelsolin cDNA hybridized intensely in Northern blots to RNA from the cornea but not from the lens, brain, or headless body. The deduced zebrafish gelsolin is approximately 60% identical to mammalian cytosolic gelsolin and has the characteristic six segmental repeats as well as the binding sites for actin, calcium, and phosphatidylinositides. In situ hybridization tests showed that gelsolin mRNA is concentrated in the zebrafish corneal epithelium. The zebrafish corneal epithelium stains very weakly with rhodamine-phalloidin, indicating little F-actin in the cytoplasm. In contrast, the mouse corneal epithelium contains relatively little gelsolin and stains intensely with rhodamine-phalloidin, as does the zebrafish extraocular muscle. We propose, by analogy with the diverse crystallins of the eye lens and with the putative enzyme-crystallins (aldehyde dehydrogenase class 3 and other enzymes) of the mammalian cornea, that gelsolin and actin-gelsolin complexes act as water-soluble crystallins in the zebrafish cornea and contribute to its optical properties.

Enhancer-independent promoter activity of the mouse alphaB-crystallin/small heat shock protein gene in the lens and cornea of transgenic mice.

The alphaB-crystallin/small heat shock protein gene is expressed very highly in the mouse eye lens and to a lesser extent in many other nonocular tissues, including the heart, skeletal muscle and brain. Previously we showed in transgenic mice that lens-specific alphaB-crystallin promoter activity is directed by a proximal promoter fragment (-164/+44) and that non-lens promoter activity depends on an upstream enhancer (-427/-259) composed of at least 5 cis-control elements. Here we have used truncated alphaB-crystallin promoter-CAT transgenes to test by biphasic CAT assays and/or histochemistry for specific expression in the cornea and lens. Deletion either of 87 bp (-427/-340) from the 5' end of the alphaB-crystallin enhancer or of the whole enhancer (-427/-258) abolished alphaB-crystallin promoter activity in all tissues except the lens and corneal epithelium when examined by the biphasic CAT assay in 4-5-week-old transgenic mice. These truncations also lowered promoter strength in the lens. The -426/+44-CAT, -339/+44-CAT and -164/+44-CAT (previously thought to be lens-specific in transgenic mice) transgenes were all expressed in the 4-6-week-old corneal epithelium when examined histochemically. Immunohistochemical staining confirmed the presence of endogenous alphaB-crystallin in the mature corneal epithelial cells. CAT gene expression driven by the alphaB-crystallin promoter with or without the enhancer was evident in the embryonic and 4-6-week-old lens. By contrast, activity of the alphaB-crystallin promoter/enhancer-CAT transgene was not detectable in the corneal epithelium before birth. Taken together, these results indicate that the intact enhancer of the alphaB-crystallin/small heat shock protein gene is required for promoter activity in all tissues tested except the lens and cornea.

Truncated forms of Pax-6 disrupt lens morphology in transgenic mice.

PURPOSE: Extensive literature shows that Pax-6 is critical for lens development and that Paxb mutations can result in aniridia in humans. In addition, it has been reported that truncated Pax-6 molecules can act as dominant-negative repressors of wild-type Pax-6 activity in cultured cells. This study was designed to determine whether Pax-6 molecules without either the activation domain (AD) or the homeodomain (HD) and the AD can function as dominant-negative repressors in vivo and alter the phenotype of the lens. METHODS: Transgenic mice were created harboring the alphaA-crystallin promoter linked to a cDNA encoding either a truncated Pax-6 without the C terminus (paired domain [PD] + homeodomain) or Pax-6 consisting of only the PD. The phenotype of the resultant animals was investigated by light and electron microscopy as well as atomic absorption spectroscopy. RESULTS: Two lines of PD + HD mice and three lines of PD mice were generated, all of which exhibit posterior nuclear and/or cortical cataracts of variable severity. The lenses from mice transgenic for either Pax-6 truncation are smaller and more hydrated than normal. Morphologically, the mice expressing the PD + HD of Pax-6 have swollen lens fibers with attenuated ball-and-socket junctions. In contrast, the lenses from mice overexpressing the PD of Pax-6 have posterior nuclear cataracts composed of cell debris, whereas the remaining fiber cells appear generally normal. CONCLUSIONS: The presence of truncated Pax-6 protein in the lens is sufficient to induce cataract in a wild-type genetic background. The simplest explanation for this phenomenon is a dominant-negative effect; however, a number of other possible mechanisms are presented.

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.

Regulation of alphaA-crystallin gene expression. Lens specificity achieved through the differential placement of similar transcriptional control elements in mouse and chicken.

The lens-preferred mouse alphaA-crystallin gene contains a conserved stretch (proximal element 2, +24/+43) in its 5'-noncoding region that we have previously shown binds nuclear proteins of lens and non-lens cells. The 5'-half of this sequence (PE2A, +25/+32) has consensus binding sites for AP-1 and other transcription factors. We show here by deletion experiments that PE2A is important for activity of the mouse alphaA-crystallin promoter and mediates phorbol ester and c-Jun responsiveness of this promoter in transfected lens cells. In vitro protein binding studies suggest that AP-1 complexes are capable of binding to PE2A. Our findings suggest that PE2A plays a role in mouse alphaA-crystallin gene expression through AP-1-mediated regulatory mechanisms. We propose that the mouse and chicken alphaA-crystallin genes are expressed with lens specificity using a similar assortment of transcription factors but with a different physical arrangement of their respective cis-elements within the promoter region. A fundamental role for AP-1 in lens-preferred expression of crystallin genes is consistent with the idea that a redox-sensitive mechanism is a selective force for recruiting lens crystallins.

Pax-6 interactions with TATA-box-binding protein and retinoblastoma protein.

PURPOSE: To identify proteins that physically interact with Pax-6, a paired domain- and homeodomain (HD)-containing transcription factor that is a key regulator of eye development. METHODS: Protein-protein interactions involving Pax-6, TATA-box-binding protein (TPB), and retinoblastoma protein were studied using affinity chromatography with Pax-6 as ligand, glutathione-S-transferase (GST) pull-down assays, and immunoprecipitations. RESULTS: The authors have shown that Pax-6 is a sequence-specific activator of many crystallin genes, all containing a TATA box, in the lens. Others have shown that lens fiber cell differentiation, characterized by temporally and spatially regulated crystallin gene expression, depends on retinoblastoma protein. In the present study it was shown that Pax-6 interacted with the TBP, the DNA-binding subunit of general transcription complex TFIID. GST pull-down assays indicated that this interaction was mediated by the Pax-6 HD, with a substantial role for its N-terminal arm and first two alpha-helices. The experiments also indicated a binding role for the C-terminal-activation domain of the protein. In addition, the present study showed that the HD of Pax-6 interacted with retinoblastoma protein. Immunoprecipitation experiments confirmed retinoblastoma protein/Pax-6 complexes in lens nuclear extracts. CONCLUSIONS: Blending the present results with those in the literature suggests that Pax-6 and retinoblastoma protein participate in overlapping regulatory pathways controlling epithelial cell division, fiber cell elongation, and crystallin gene expression during lens development.

Development of characterization of a immortal and differentiated murine trabecular meshwork cell line.

PURPOSE: To study mouse trabecular meshwork (TM) and to develop a murine TM cell line. METHODS: Mouse TM in situ was studied by light and electron microscopy (EM). In addition, TM was isolated from the H-2K(b)-tsA58 transgenic mouse strain in which promoter sequences of the major histocompatibility complex H-2Kb class 1 gene are fused to sequences of the SV40 mutant temperature-sensitive (ts) strain tsA58. The promoter is inducible by interferon (IFN)-gamma, and the tsA58 gene product is active at 33 degrees C (permissive conditions), but not at 37 degrees C (nonpermissive conditions). The TM explant was cultured in permissive conditions. Outgrowing cells were passaged through two rounds of single-cell cloning. One clonal cell line (MUTM-NEI/1) was characterized in nonpermissive conditions by EM, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and northern blot hybridization. In addition, MUTM-NEI/1 cells were transfected with plasmid DNA. RESULTS: The mouse eye has a circumferentially oriented outflow vessel and a TM that is subdivided in an outer juxtacanalicular or cribriform part and an inner lamellated or trabecular part. From the TM of the H-2Kb-tsA58 mouse, a clonal cell line (MUTM-NEI/1) was established. In permissive conditions, MUTM-NEI/1 cells remained proliferative through at least 80 generations without change in phenotype. In nonpermissive conditions, proliferation was slower, and MUTM-NEI/1 cells differentiated and synthesized collagen types I, III, IV, and VI; laminin; and fibronectin. MUTM-NEI/1 cells were immunoreactive for vimentin, alphaB-crystallin, and neural cell adhesion molecule (NCAM), but not for desmin or cytokeratin. Less than 10% of MUTM-NEI/1 cells stained for alpha-smooth muscle actin, whereas after 3 days of treatment with transforming growth factor-beta1 almost all cells were positive. MUTM-NEI/1 cells expressed mRNA for NCAM, aquaporin 1, myocilin/trabecular meshwork glucocorticoid-inducible protein, and alphaB-crystallin, which was increased after oxidative stress. MUTM-NEI/1 cells could be successfully transfected with plasmid DNA. CONCLUSIONS: The architecture of the murine outflow system is comparable to that in primates. The MUTM-NEI/1 cell line is a clonal, immortal, and differentiated TM cell line that will be an important tool for study of the expression of TM genes.

O-Crystallin, arginine kinase and ferritin from the octopus lens.

Three proteins have been identified in the eye lens of the octopus, Octopus dofleini. A 22 kDa protein comprising 3-5% of the soluble protein of the lens is 35-43% identical to a family of phosphatidylethanolamine-binding proteins of vertebrates. Other members of this family include the immunodominant antigen of the filarial parasite, Onchocerca volvulus, putative odorant-binding proteins of Drosophila and a protein with unknown function of Caenorhabditis elegans. We have called this protein O-crystallin on the basis of its abundance in the transparent lens. O-Crystallin mRNA was detected only in the lens. Two tryptic peptides of another octopus lens protein, less abundant than O-crystallin, showed 80% identity to arginine kinase of invertebrates, a relative of creatine kinase of vertebrates. Finally, ferritin cDNA was isolated as an abundant cDNA from the octopus lens library. Northern blots showed that ferritin mRNA is not lens-specific.