Biomedical subjects

C W Kao

Publications and source records attributed to C W Kao.

At least 19 recordsLinked to original sources

PubMed2001

TGFbeta2 in corneal morphogenesis during mouse embryonic development.

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.

Acetyltransferases↗

PubMed2001

Characterization of Corn1 mice: Alteration of epithelial and stromal cell gene expression.

PURPOSE: Corn1 is an autosomal recessive mutation characterized by corneal epithelial hyperplasia and stromal neovascularization. The aim of the present study is to examine the expression patterns of specific epithelial and stromal proteins in corn/corn1 mutant mice. METHODS: Immunohistochemistry with antibodies directed against keratins 1, 4, 5, 12, and 14 as well as loricrin, filaggrin, and involucrin were performed in corn1/corn1 and wild type, A.By/SnJ strain, mice at 4 weeks of age. Western blot hybridization was performed to confirm the presence of involucrin in corneas. In situ and northern blot hybridization were used to evaluate the expression of keratin 12, lumican, and keratocan in these mice. RESULTS: In corn1/corn1 mice, focal areas of corneal epithelial hyperplasia alternate with epithelium with normal appearance. Both regions of normal and hyperplastic corneal epithelium were labeled by anti-keratin 12 antibodies through all corneal epithelial layers. The anti-keratin 14 antibody only labeled the basal cell layer in normal epithelial areas, whereas it labeled both basal and suprabasal cell layers in hyperplastic areas. In wild type mice, anti-keratin 12 antibodies labeled all corneal epithelial layers, whereas anti-keratin 14 labeled the basal corneal epithelial cells only. Positive staining by anti-involucrin antibody was demonstrated in the basal corneal epithelial layer of wild type mice and normal areas of corn1/corn1 mice. Similarly, as observed with anti-keratin 14 antibody, the anti-involucrin antibody labeled both basal and suprabasal cell layers of hyperplastic corneal epithelium of corn1/corn1 mice. Antibodies against keratin 1, keratin 4, loricrin, and fillagrin did not label the corneas of wild type mice or corn1/corn1 mice. Northern hybridization indicated that the expressions of keratocan and lumican mRNA levels were up regulated in corn1/corn1 mice, but keratin 12 mRNA remained similar to that of the wild type mice. In situ hybridization revealed that the lumican mRNA was detected in epithelial and stromal cells of corn1/corn1 mice, whereas keratocan mRNA was only detected in stromal cells. CONCLUSIONS: Hyperproliferative epithelial cells of corn1/corn1 mice have increased levels of expression of keratin 14 and involucrin, but do not exhibit the phenotypical characteristics of cornification. These observations indicate that factors associated with the phenotypes of corn1/corn1 mice do not alter the cornea-type epithelial differentiation of keratin 12 expression, but cause aberrant expression of lumican by corneal epithelial cells.

Animals↗

PubMed2000

Role of lumican in the corneal epithelium during wound healing.

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e. g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum(-/-) mice was significantly delayed compared with Lum(+/-) mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.

Amino Acid Sequence↗

PubMed1998

The cloning of mouse keratocan cDNA and genomic DNA and the characterization of its expression during eye development.

Keratan sulfate proteoglycans (KSPGs) play a pivotal role in the development and maintenance of corneal transparency. Keratocan, lumican, and mimecan (osteoglycin) are the major KSPGs in vertebrate corneas. To provide a better understanding of the structure/function relationship of keratocan, we have cloned both the mouse keratocan gene and its cDNA. We have also examined its expression during embryonic development. The mouse keratocan gene spans approximately 6.5 kilobases of the mouse genome and contains three exons and two introns. Northern blotting and in situ hybridization were employed to examine keratocan gene expression during mouse development. Unlike lumican gene, which is expressed by many tissues other than cornea, keratocan mRNA is more selectively expressed in the corneal tissue of the adult mouse. During embryonic development, keratocan mRNA was first detected in periocular mesenchymal cells migrating toward developing corneas on embryonic day 13.5 (E13.5). Its expression was gradually restricted to corneal stromal cells on E14. 5 approximately E18.5. Interestingly, keratocan mRNA can be detected in scleral cells of E15.5 embryos, but not in E18.5 embryos. In adult eyes, keratocan mRNA can be detected in corneal keratocytes, but not in scleral cells.

Amino Acid Sequence↗

PubMed1998

Healing of corneal epithelial defects in plasminogen- and fibrinogen-deficient mice.

PURPOSE: The local deposition of fibrinogen and other plasma products from tears within corneal wounds and the expression of plasminogen activator by corneal epithelial cells suggest that the coagulation and fibrinolytic systems play an important role in corneal wound healing. The authors used mouse lines deficient in plasminogen (Plg), fibrinogen (Fib), or both to elucidate the roles of these key fibrinolytic and coagulation factors in the healing of corneal epithelial defects. METHODS: Mice were anesthetized, and corneal epithelial defects (3 mm) were created with a blade. The authors conducted histologic examination and immunohistochemical analysis on the healing of injured corneas. RESULTS: The corneal epithelial defects of wild-type mice with transparent corneas healed quickly in 7 days, whereas the healing of plasminogen-deficient mice was impaired and complicated by severe and persistent inflammatory responses, the formation of retrocorneal fibrin deposits, corneal cloudiness caused by scar-tissue formation, and often stromal neovascularization. To determine whether these defects in corneal wound repair were specifically related to an impediment in fibrinolysis, corneal wound healing was compared in mice with a combined deficiency in plasminogen and fibrinogen. The loss of fibrinogen in mice lacking plasminogen resulted in the restoration of normal healing with transparent corneas in 7 days, similar to that of wild-type mice. CONCLUSIONS: These results provide direct evidence that hemostatic factors play a crucial role in corneal wound repair despite the lack of local hemorrhage. Furthermore, they demonstrate that the essential role of plasmin in corneal would healing is fibrinolysis. It prevents the adverse inflammatory responses caused by prolonged fibrin and fibrinogen deposition in injured corneas.

Afibrinogenemia↗

PubMed1998

Characterization of Bsk mice: I. The Bsk mutation does not involve a recombination of cornea-specific keratin 12 and skin-specific hair keratin genes.

PURPOSE: Bsk (bare skin) is an autosomal dominant mutation linked to the Krt 1 (type 1 keratin) locus of mouse chromosome 11. The adult Bsk mouse manifests hair loss and corneal opacity. To identify and characterize the keratin genes involved in this mutation, we examined the hypothesis proposing that the Bsk mutation might involve a recombination event between cornea-specific (K12) and hair-specific (mHa 1, 2, 3 and 4) type I keratin genes. METHODS: The Bsk phenotype was examined by histochemical analysis, using light and electron microscopy. RFLP was used for their genotyping, and possible keratin gene expression was examined by immunohistochemical staining, Western analysis, RT-PCR and Northern hybridization. RESULTS: Northern hybridization, RT-PCR and Western blot analysis revealed that mHa 1, 2, 3 and 4 keratins are expressed in the skin, but not in cornea, whereas the expression of K12 is limited to the corneas of the Bsk mice. These data ruled out the hypothesis that Bsk phenotype results from a recombination event between K12 and mHa 1, 2, 3 and 4. Ultrastructural and biochemical analyses also indicated that Bsk does not involve negative dominant mutations of keratin 12, mHa 1, 2, 3 and 4, epidermal-specific keratin 10, or basal cell-specific keratin 14. Expression of an acidic 50 kD keratin, recognized by monoclonal antibody AK 2, was up-regulated in the injured corneas of normal mice as well as Bsk corneas. CONCLUSION: The gene linked to the Bsk mutation remains unknown. The pathological changes in the skin and corneas may be secondary to the loss of protecting hairs and lashes by an unknown mechanism.

Animals↗

PubMed1998

A CAT reporter construct containing 277bp GNAT2 promoter and 214bp IRBP enhancer is specifically expressed by cone photoreceptor cells in transgenic mice.

PURPOSE: The alpha-subunit of human cone transducin plays an important role in interacting with visual pigment and activating the cGMP-dependent phosphodiesterase (cGMP-PDE). The human GNAT2 gene (cone transducin alpha-subunit) has been cloned and characterized by Fong et al. In this report, we describe the use of transgenic mice to characterize the tissue specificity of the GNAT2 promoter. METHODS: A chimeric reporter gene construct which consists of a 277 bp 5'-flanking fragment of the GNAT2 gene at 5' end of the chloramphenicol acetyltransferase (CAT) gene and a 214 bp enhancer region from the human interphotoreceptor retinoid-binding protein (IRBP) gene at the 3' end of the CAT gene was used to generate transgenic mice. Transgenic mice were identified by Southern blot hybridization and polymerase chain reaction (PCR) analysis using tail DNA from experimental animals. Immunostaining was used to study the developmental expression of CAT and the endogenous GNAT2 gene. RESULTS: Analysis of four transgenic mouse lines revealed that three lines had low CAT activity in the retina. The CAT gene, along with the endogenous GNAT2 gene, was expressed at high levels in cone photoreceptor cells in the fourth transgenic mouse line as determined by CAT enzyme assays and immunostaining. CONCLUSION: The results show that the 277 bp 5'-flanking sequence from the human GNAT2 gene coupled with the 214 bp IRBP enhancer can direct a tissue-specific expression pattern of CAT reporter gene in mouse retina, which parallels the expression pattern of endogenous GNAT2.

Animals↗

PubMed1998

Identification of the cornea-specific keratin 12 promoter by in vivo particle-mediated gene transfer.

PURPOSE: Keratin 12 (K12) is a cornea epithelial cell-specific intermediate filament component. To provide a better understanding of its expression, it is necessary to identify and characterize the promoter of Krt1.12 gene. METHODS: The 2.5-kb DNA 5' to Krt1.12 gene was sequenced. Krt1.12 promoter-beta-gal DNA constructs were prepared and used in vivo to transfect rabbit corneas, conjunctivas, and skin by particle-mediated gene transfer (Gene Gun). In vitro, the DNA constructs were transfected into cultured T-antigen-transformed rabbit corneal epithelial (RCE-T) cells and human fibrosarcoma HT-1080 fibroblasts with lipofectamine. The promoter activity was assessed by measuring beta-gal (beta-galactosidase) activity using histochemical staining with 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside and enzyme assay with o-nitrophenyl beta-D-galactopyranoside. RESULTS: There are four Pax-6 pair box binding elements found between -910 and -2000 bp 5'-flanking the transcription initiation site of the Krt1.12 gene. None of promoter constricts can be expressed by HT-1080 cells. Cotransfection of Pax-6 cDNA with K12 promoter-beta-gal constructs containing Pax-6 elements results in a fourfold increase of beta-gal activities in RCE-T cells but not HT-1080 fibroblasts. The data of in vivo transfection in the rabbit by Gene Gun indicate that reporter gene constructs containing 0.6-kb and longer DNA fragments 5'-flanking Krt1.12 gene are effectively expressed in corneal, but not conjunctival or epidermal epithelial cells. CONCLUSIONS: The particle-mediated gene transfer is a suitable technique for in vivo delivery of transgenes to corneal epithelial cells. The 2.5-kb DNA fragment 5'-flanking Krt1.12 contains corneal epithelial cell-specific regulatory cis-DNA elements. Pax-6 is a positive transcription factor essential for keratin 12 expression.

Animals↗

PubMed1997

Characterization and expression of the mouse lumican gene.

Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3' noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5'-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency.

Amino Acid Sequence↗

PubMed1997

Stromal fibroblasts are associated with collagen IV in scar tissues of alkali-burned and lacerated corneas.

PURPOSE: Corneal wound healing frequently leads to the formation of opaque scar tissue. We examined whether stromal fibroblastic cells of injured corneas express collagen IV and contributes to the formation of a basal lamina-like structure. METHODS: Rabbits were anesthetized, and central corneal alkali burn (8 mm in diameter; 1 M NaOH, 1 min) or laceration (8 mm long) were produced. The injured corneas, which had healed for 1, 7, 21 and 45 days, were subjected to histological and immunohistochemical studies with goat anti-collagen IV antibodies, using light and electron microscopy, and in situ hybridization with an antisense digoxigenin-labeled riboprobe of collagen alpha 1(IV) mRNA. For comparison, twenty-day-old fetal corneas were subjected to immunohistochemical study and transmission electron microscopy (TEM). RESULTS: TEM examinations revealed that the stromal collagenous matrix was organized in orthogonal lamellae during corneal development, whereas that of alkali-burned cornea, which had healed for 3 weeks, was disorganized. The stroma of twenty-day-old fetal cornea was not labeled by the anti-collagen IV antibodies. In contrast, one week after injury, specific collagen IV immunostaining was detected in the injured stroma. As the healing proceeded (21-45 days), the antibodies reacted with fibroblastic cells and the extracellular matrix of scar tissues located in the anterior portion of alkali-burned corneas, as well as the posterior portion of lacerated corneas. The middle portion of the stromal tissues was weakly labeled by the anti-collagen IV antibodies with the exception of the blood vessel wall. Immuno-electron microscopic study showed that collagen IV and fibronectin were closely associated with the fibroblastic cells. In situ hybridization demonstrated that epithelial and endothelial cells and fibroblastic cells in the wounded corneal stroma and retro-corneal membrane expressed alpha 1(IV) mRNA, whereas in normal corneas the expression of alpha 1(IV) mRNA was limited to epithelial and endothelial cells. CONCLUSIONS: The enhanced expression of collagen IV by the fibroblastic cells in the stroma of injured corneas is consistent with the notion that they may contribute to the formation of basal lamina-like structures in injured corneas.

Animals↗

PubMed1996

Conjunctival epithelial cells can resurface denuded cornea, but do not transdifferentiate to express cornea-specific keratin 12 following removal of limbal epithelium in mouse.

Limbal stem cell deficiency contributes to recurrent corneal epithelial defects. We examined whether the conjunctival epithelium can transdifferentiate to corneal epithelium following surgically induced limbal stem cell deficiency. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital. Partial or total epithelial removal was produced with a no. 69 Beaver blade under a dissecting microscope. The wounds were allowed to heal for 0-28 days, and the mice were examined every other day to evaluate re-epithelialization. Corneas were then subjected to histological, immunohistochemical studies and Western blot analysis with epitope-specific anti-keratin 12 antibodies. Partial epithelial defects re-epithelialized within 2 days and were normal in appearance and expressed cornea-specific keratin 12. In eyes with limbal deficiency, re-epithelialization progressed more slowly and was characterized by opacification; epithelial closure usually occurred by the 7th day. This epithelium differed from normal corneal epithelium in basic morphology, cell shape, and the presence of goblet cells at 2 weeks after injury. The epithelium at the center of injured corneas with total defect at 4 weeks had cornealike morphology and was devoid of goblet cells. These epithelial cells derived from conjunctiva did not express the cornea-specific keratin 12 as determined by immunohistochemistry, Western blot analysis and in situ hybridization. As evidenced by differences in morphology and the expression of cornea-specific keratin 12, conjunctival transdifferentiation does not occur in conjunctical overgrowth after the removal of limbal epithelium.

Amino Acid Sequence↗

PubMed1996

Appearance of immune cells and expression of MHC II DQ molecule by fibroblasts in alkali-burned corneas.

Corneal alkali burns are characterized by persistent inflammatory response and recurrent epithelial erosions. We examine whether immune cell types, i.e., T-cells and B-cells, play a role in this devastating process. Rabbit alkali-burned corneas that healed for 1-49 days were subjected to immunostaining with monoclonal antibodies (mAb) L11/135 (anti-T-cells), and 2C4 (anti-MHC II DQ). Serum was collected weekly and subjected to Western blot immunostaining to detect antibodies against denatured corneal proteins. Our observations demonstrated that all injured corneas reepithelialized within 3 days but then developed recurrent erosions. Immunohistochemical studies revealed that PMN, monocytes, and B-cells labeled by 2C4 mAb and T-cells labeled by L11/135 mAb appeared in the periphery to the cornea at 1 day after alkali burn. Many of these myeloid and lymphoid cells invaded the central stroma after 2 weeks of injuries when the alkali-burned corneas were heavily vascularized. In addition, some fibroblastic cells also expressed the MHC II DQ molecules in the alkali-burned corneas that had healed for > 2 weeks. Plasma cells appeared in granulation tissue of injured corneas that had healed for > 3 weeks. Western blot analysis demonstrated a production of heterogeneous antibodies in a majority of the rabbits (11 of 14) to various denatured corneal proteins (between 80 kDa and 25 kDa) at 5 weeks of alkali burn. Inflammatory cell types, i.e., PMN, macrophages could be found underneath the detached epithelium. These observations are consistent with the notion that the myeloid and lymphoid cells may participate in and complicate the healing of corneal alkali burns.

Alkalies↗

PubMed1996

Keratin 12-deficient mice have fragile corneal epithelia.

PURPOSE: Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. METHODS: One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. RESULTS: The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice. CONCLUSIONS: The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.

Animals↗

PubMed1994

Characterization and chromosomal localization of the cornea-specific murine keratin gene Krt1.12.

Keratins are a group of water-insoluble proteins constituting paired acidic and basic keratin molecules that form 10-nm intermediate filaments in epithelial cells. Expression of the K3/K12 keratin pair characterizes the cornea-type differentiation. However, the mechanism that regulates this cornea-specific K12 expression remains unknown. To provide a better understanding of the cornea-specific expression, we have cloned the K12 cDNA (Liu, C.-Y., Zhu, G., Westerhausen-Larson, A., Converse, R., Kao, C. W.-C., Sun, T.-T., and Kao, W. W.-Y. (1993) Curr. Eye Res. 12, 963-974). In present studies, the murine K12 keratin gene (Krt1.12) was isolated and characterized. The murine Krt1.12 gene spans 6,567 base pairs of genomic DNA, and the mRNA encoding K12 keratin is distributed into eight exons. Chromosome mapping reveals that murine Krt1.12 is located within the Krt1 complex of mouse chromosome 11. In addition to the production of authentic K12 mRNA, the Krt1.12 gene gives rise to several alternate poly(A)+ RNAs by the use of alternative splicing in intron 2, an alternative promoter in intron 1, and/or both. Sequence analysis indicates that the transcripts derived from alternative splicing and/or the alternative promoter do not have a long open reading frame for keratin or keratin-like molecules. It is not known whether these alternate K12 poly(A)+ RNAs have any biological functions, e.g. regulation of K12 gene expression.

Amino Acid Sequence↗

PubMed1993

Effects of polymorphonuclear neutrophils on protein synthesis by alkali-injured rabbit corneas. A preliminary study.

Invasion of polymorphonuclear neutrophils (PMN) into injured cornea is one of the early events in corneal wound-healing. In the present studies, we examine the mutual effects on protein synthesis by PMN and injured and normal corneas when they are cocultured. PMN were labeled with [35S]methionine in the presence or absence of normal or alkali-injured rabbit corneas for 1-5 h. The acid-insoluble 35S-labeled proteins in medium, cells, and tissues were measured. Our data indicate that alkali-injured rabbit corneas induce higher rates of incorporation of [35S]methionine and secretion of 35S-labeled newly synthesized proteins by PMN. The newly synthesized 35S-labeled proteins were then analyzed by two-dimensional PAGE. The results indicate that alkali-injured and normal rabbit corneas enhance the synthesis and secretion of a 18-kD protein by PMN. In contrast, alkali injury greatly reduced the secretion of a group of proteins having molecular weights of approximately 30 kD by rabbit corneas. The alkali-injured corneas synthesize a new 200-kD protein (AC-200) in tissues and increase the secretion of a 120-kD protein (AC-120) into the culture medium. Furthermore, PMN slightly inhibits the incorporation of [35S]methionine and alter the synthesis of several 35S-labeled proteins by normal and injured corneas. For example, incubation with PMN abolishes the synthesis of the AC-200 protein, but enhances the secretion of the AC-120 protein by the alkali-injured corneas. However, the function and nature of these proteins remain largely unknown. Further studies are needed to elucidate the biological roles of these polypeptides during corneal wound healing.

Animals↗

PubMed1993

Cornea-specific expression of K12 keratin during mouse development.

The full-length cDNA of mouse K12 keratin was characterized by sequencing overlapping cDNA clones isolated from a mouse cornea cDNA library. Using Northern blot hybridization, the radio-labeled cDNA hybridized to a 1.9 kb mRNA from adult cornea, but not from other mouse tissues including snout, esophagus, tongue, and skin. During mouse development, corneas do not express K12 mRNA until 4 days postnatal when the epithelium begins to stratify as judged by Northern blot and in situ hybridization. In situ hybridization with 3H-labeled cDNA probe and immunohistochemical studies with antibodies against a synthetic oligo-peptide deduced from rabbit K12 cDNA demonstrate that this mouse K12 keratin is expressed in all cell layers of adult corneal epithelium, and the suprabasal layers, but not the basal layer of the limbal epithelium. Epidermal growth factor (EGF) has been shown to promote epithelium stratification of cultured chicken and human corneas in vitro. To examine whether EGF can promote K12 expression, EGF was administered to neonatal mice. The results indicate that EGF retards K12 expression by corneal epithelial cells, even though it promotes corneal epithelial stratification during mouse development. Taken together, our results demonstrate that the expression of K12 keratin is cornea-specific, differentiation-dependent, and developmentally regulated.

Amino Acid Sequence↗

PubMed1991

Isolation of wound-specific cDNA clones from a cDNA library prepared with mRNAs of alkali-burned rabbit corneas.

Alkali burn is one of the most severe corneal injuries. In order to gain a better understanding of the healing of alkali-burned corneas, it is necessary to identify and characterize proteins that are specifically synthesized by the injured corneal tissues. In this study, we developed a useful procedure to identify and isolate cDNA clones that encode messenger ribonucleic acids (mRNAs) that are specific and/or abundant in alkali-burned rabbit corneas (ARCs), but absent in normal rabbit corneas (NRCs). At first, a cDNA library was prepared by cloning cDNA of mRNA isolated from ARCs into the lambda ORF-8 vector. A differential plaque hybridization was used to screen 2.5 x 10(4) plaque-forming units (pfu) from an ARC cDNA library using 32P-labeled cDNAs prepared from mRNA of ARCs and NRCs. Thirty-seven cDNA clones of mRNAs specific for ARCs were identified and isolated in their pureform. The cDNA inserts of these lambda ORF-8 phages were subcloned into the pSM216 vector by in vivo recombination. The cDNA inserts then were characterized by restriction enzyme digestion, i.e., BamHI, HindIII, and EcoRI. The size of the cDNA inserts ranged from 210 to 5,000 base pairs. Using Northern blot hybridization of total RNA prepared from polymorphonuclear neutrophils, mononuclear leukocytes, alkali-burned corneas, and normal corneas, the cDNA clones were divided into three groups. Five cDNA clones encoded mRNA of corneal cells in ARCs. Twenty-four cDNA clones derived from mRNA of inflammatory cells were present in alkali-burned corneas, but Northern blot hybridization failed to identify mRNA of discrete sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkalies↗

PubMed1988

Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.

Amino Acid Sequence↗