Effects of polysialic acid on sensory innervation of the cornea.

Sensory trigeminal growth cones innervate the cornea in a coordinated fashion during embryonic development. Polysialic acid (polySia) is known for its important roles during nerve development and regeneration. The purpose of this work is to determine whether polySia, present in developing eyefronts and on the surface of sensory nerves, may provide guidance cues to nerves during corneal innervation. Expression and localization of polySia in embryonic day (E)5-14 chick eyefronts and E9 trigeminal ganglia were identified using Western blotting and immunostaining. Effects of polySia removal on trigeminal nerve growth behavior were determined in vivo, using exogenous endoneuraminidase (endoN) treatments to remove polySia substrates during chick cornea development, and in vitro, using neuronal explant cultures. PolySia substrates, made by the physical adsorption of colominic acid to a surface coated with poly-d-lysine (PDL), were used as a model to investigate functions of the polySia expressed in axonal environments. PolySia was localized within developing eyefronts and on trigeminal sensory nerves. Distributions of PolySia in corneas and pericorneal regions are developmentally regulated. PolySia removal caused defasciculation of the limbal nerve trunk in vivo from E7 to E10. Removal of polySia on trigeminal neurites inhibited neurite outgrowth and caused axon defasciculation, but did not affect Neural Cell Adhesion Molecule (NCAM) expression or Schwann cell migration in vitro. PolySia substrates in vitro inhibited outgrowth of trigeminal neurites and promoted their fasciculation. In conclusion, polySia is localized on corneal nerves and in their targeting environment during early developing stages of chick embryos. PolySias promote fasciculation of trigeminal axons in vivo and in vitro, whereas, in contrast, their removal promotes defasciculation.

Resistance of corneal RFUVA­cross-linked collagens and small leucine-rich proteoglycans to degradation by matrix metalloproteinases.

PURPOSE: Extracellular matrix metalloproteinases (MMPs) are thought to play a crucial role in corneal degradation associated with the pathological progression of keratoconus. Currently, corneal cross-linking by riboflavin and ultraviolet A (RFUVA) has received significant attention for treatment of keratoconus. However, the extent to which MMPs digest cross-linked collagen and small leucine-rich proteoglycans (SLRPs) remains unknown. In this study, the resistance of RFUVA-cross-linked collagens and SLRPs to MMPs has been investigated. METHODS: To investigate the ability of MMPs to digest cross-linked collagen and SLRPs, a model reaction system using purified collagen type I, type IV, and nonglycosylated, commercially available recombinant SLRPs, keratocan, lumican, mimecan, decorin, and biglycan in solution in vitro has been compared using reactions inside an intact bovine cornea, ex vivo. RESULTS: Our data demonstrate that corneal cross-linked collagen type I and type IV are resistant to cleavage by MMP-1, MMP-2, MMP-9, and MMP-13, whereas non-cross-linked collagen I, IV, and natively glycosylated SLRPs are susceptible to degradation by MMPs. In addition, both cross-linked SLRPs themselves and cross-linked polymers of SLRPs and collagen appear able to resist degradation. These results suggest that the interactions between SLRPs and collagen caused by RFUVA protect both SLRPs and collagen fibrils from cleavage by MMPs. CONCLUSIONS: A novel approach for understanding the biochemical mechanism whereby RFUVA cross-linking stops keratoconus progression has been achieved.

Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation.

PURPOSE: Sensory trigeminal nerve growth cones innervate the cornea in a highly coordinated fashion. The purpose of this study was to determine if extracellular matrix glycosaminoglycans (ECM-GAGs), including keratan sulfate (KS), dermatan sulfate (DS), and chondroitin sulfate A (CSA) and C (CSC), polymerized in developing eyefronts, may provide guidance cues to nerves during cornea innervation. METHODS: Immunostaining using antineuron-specific-ß-tubulin and monoclonal antibodies for KS, DS, and CSA/C was performed on eyefronts from embryonic day (E) 9 to E14 and staining visualized by confocal microscopy. Effects of purified GAGs on trigeminal nerve growth cone behavior were tested using in vitro neuronal explant cultures. RESULTS: At E9 to E10, nerves exiting the pericorneal nerve ring grew as tight fascicles, advancing straight toward the corneal stroma. In contrast, upon entering the stroma, nerves bifurcated repeatedly as they extended anteriorly toward the epithelium. KS was localized in the path of trigeminal nerves, whereas DS and CSA/C-rich areas were avoided by growth cones. When E10 trigeminal neurons were cultured on different substrates comprised of purified GAG molecules, their neurite growth cone behavior varied depending on GAG type, concentration, and mode of presentation (immobilized versus soluble). High concentrations of immobilized KS, DS, and CSA/C inhibited neurite growth to varying degrees. Neurites traversing lower, permissive concentrations of immobilized DS and CSA/C displayed increased fasciculation and decreased branching, whereas KS caused decreased fasciculation and increased branching. Enzymatic digestion of sulfated GAGs canceled their effects on trigeminal neurons. CONCLUSIONS: Data herein suggest that GAGs may direct the movement of trigeminal nerve growth cones innervating the cornea.

Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--molecular mechanisms.

PURPOSE: Tissue glue containing fibrinogen (FIB) and riboflavin (RF), upon exposure to long wavelength ultraviolet light (UVA, 365 nM) has been proposed potentially to solve long-standing problems presented by corneal wound and epithelial ingrowth side-effects from laser-assisted in situ keratomileuis (LASIK). Data presented in a previous study demonstrated an ability of FIB + RF + UVA to adhere two stromal surfaces; however, to our knowledge no molecular mechanisms have been proposed to account for interactions occurring between corneal extracellular matrix (ECM) and tissue glue molecules. Here, we document several covalent and noncovalent interactions between these classes of macromolecules. METHODS: SDS-PAGE and Western blot techniques were used to identify covalent interactions between tissue glue molecules and corneal ECM molecules in either the presence or absence of RF and UVA, in vitro and ex vivo. Surface plasmon resonance (SPR) was used to characterize noncovalent interactions, and obtain k(a), k(d), and K(D) binding affinity values. RESULTS: SDS-PAGE and Western blot analyses indicated that covalent interactions occurred between neighboring FIB molecules, as well as between FIB and collagen type I (Coll-I) proteins (in vitro and ex vivo). These interactions occurred only in the presence of RF and UVA. SPR data demonstrated the ability of FIB to bind noncovalently to corneal stroma molecules, Coll-I, decorin, dermatan sulfate, and corneal basement membrane molecules, laminin and heparan sulfate--only in the presence of Zn(2+). CONCLUSIONS: Covalent and (zinc-mediated) noncovalent mechanisms involving FIB and stromal ECM molecules contribute to the adhesion created by FIB + RF + UVA.

Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--conditions for tissue adhesion.

PURPOSE: Laser-assisted in situ keratomileus (LASIK) creates a permanent flap that remains non-attached to the underlying laser-modified stroma. This lack of permanent adhesion is a liability. To immobilize a corneal flap, a protocol using fibrinogen (FIB), riboflavin (RF), and ultraviolet (UVA) light (FIB+RF+UVA) was devised to re-adhere the flap to the stroma. METHODS: A model flap was created using rabbit (Oryctolagus cuniculus) and shark (Squalus acanthias) corneas. Solutions containing FIB and RF were applied between corneal strips as glue. Experimental corneas were irradiated with long wavelength (365 nm) UVA. To quantify adhesive strength between corneal strips, the glue-tissue interface was subjected to a constant force while a digital force gauge recorded peak tension. RESULTS: In the presence of FIB, substantive non-covalent interactions occurred between rabbit corneal strips. Adhesiveness was augmented if RF and UVA also were applied, suggesting formation of covalent bonds. Additionally, exposing both sides of rabbit corneas to UVA generated more adhesion than exposure from one side, suggesting that RF in the FIB solution catalyzes formation of covalent bonds at only the interface between stromal molecules and FIB closest to the UVA. In contrast, in the presence of FIB, shark corneal strips interacted non-covalently more substantively than those of rabbits, and adhesion was not augmented by applying RF+UVA, from either or both sides. Residual RF could be rinsed away within 1 hour. CONCLUSIONS: Glue solution containing FIB and RF, together with UVA treatment, may aid immobilization of a corneal flap, potentially reducing risk of flap dislodgement.

Effect of the synthetic NC-1059 peptide on diffusion of riboflavin across an intact corneal epithelium.

PURPOSE: To investigate the effect of the peptide NC-1059 on riboflavin (RF) diffusion across an intact corneal epithelium into the stroma. METHODS: NC-1059 peptide was synthesized by solid-phase synthesis with 9-fluorenylmethoxycarbonyl chemistry, characterized by reversed-phase HPLC, and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. The diffusion of RF across embryonic day 18 chick corneal epithelium ex vivo was monitored using confocal microscopy. The depth distributions of RF in the corneal stroma were calculated using a group of linear equations based on the relationship between RF fluorescence intensity and concentration. RESULTS: Data presented in this study demonstrate that the NC-1059 peptide can transiently open the intact epithelial barrier to allow the permeation of RF into the stroma. The effect of NC-1059 peptide on RF diffusion across the corneal epithelium was concentration and time dependent. The amount of RF reaching a 50-µm depth of chick corneal stoma increased dramatically after exposure to NC-1059 for 10 minutes, reaching a plateau by 30 minutes. The concentrations of RF in the presence of NC-1059 at corneal stromal depths of 50, 100, and 150 µm were significantly higher than in the absence of the peptide, and almost as high as in corneas in which the epithelium first had been physically removed. In addition, a cell viability assay indicated that the NC-1059 peptide did not kill corneal epithelial cells. CONCLUSIONS: NC-1059 peptide significantly enhances the diffusion of RF across intact corneal epithelium into the stroma.

Development and mineralization of embryonic avian scleral ossicles.

PURPOSE: To investigate the development and mineralization of avian scleral ossicles using fluorescence microscopy in combination with field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS). METHODS: The anterior halves of whole eyeballs from chickens on embryonic (E) days E10 to E21 and Japanese quail on embryonic days E8 to E17 were fixed in 100% methanol for 1 min, stained with Giemsa solution for 5 min, destained with distilled water for 30 min, and then viewed by epifluorescence. Propidium iodide (PI) was used to detect the nuclei of osteocytes in scleral ossicles. FESEM and EDS were then used to show areas of mineralization and to identify differences in the elemental composition of different regions of the ossicles. RESULTS: Using Giemsa as a fluorescence stain, it was possible to observe the detailed morphology and development of both chicken and quail scleral ossicles. In chickens, bone microporosities first became visible at E15. Each microporosity contained a single nucleus, likely that of an osteocyte. The amount of carbon in ossicles steadily decreased during embryogenesis and post-hatching, while the concentration of oxygen showed a distinct increase over this time period. Calcium and phosphate levels in the ossicles increased gradually during embryonic and post-hatching stages. CONCLUSIONS: A novel approach to study the development and mineralization of avian scleral ossicles during embryogenesis is presented. This methodology was validated by studying two different species, both important models for avian developmental research.

Expression and localization of neural cell adhesion molecule and polysialic acid during chick corneal development.

PURPOSE: To assay for expression and localization of neural cell adhesion molecule (NCAM) and polysialic acid (polySia) in the chick cornea during embryonic and postnatal development. METHODS: Real time quantitative PCR and Western blot analyses were used to determine NCAM expression and polysiaylation in embryonic, hatchling, and adult chick corneas. Immunofluorescence staining for NCAM and polySia was conducted on cryosections of embryonic and adult corneas, whole embryonic corneas, and trigeminal neurons. RESULTS: NCAM and ST8SiaII mRNA transcripts peaked by embryonic day (E)9, remained steady between E10 and E14 and slowly decreased thereafter during embryonic development. Both gene transcripts showed > 190-fold decline in the adult chick cornea compared with E9. In contrast, ST8SiaIV expression gradually decreased 26.5-fold from E6 to E19, increased thereafter, and rose to the early embryonic level in the adult cornea. Western blot analysis revealed NCAM was polysialylated and its expression developmentally changed. Other polysiaylated proteins aside from NCAM were also detected by Western blot analysis. Five NCAM isoforms including NCAM-120, NCAM-180 and three soluble NCAM isoforms with low molecular weights (87-96 kDa) were present in chick corneas, with NCAM-120 being the predominate isoform. NCAM was localized to the epithelium, stroma, and stromal extracellular matrix (ECM) of the embryonic cornea. In stroma, NCAM expression shifted from anterior to posterior stroma during embryonic development and eventually became undetectable in 20-week-old adult cornea. Additionally, both NCAM and polySia were detected on embryonic corneal and pericorneal nerves. CONCLUSIONS: NCAM and polySia are expressed and developmentally regulated in chick corneas. Both membrane-associated and soluble NCAM isoforms are expressed in chick corneas. The distributions of NCAM and polySia in cornea and on corneal nerves suggest their potential functions in corneal innervation.

Nerve repulsion by the lens and cornea during cornea innervation is dependent on Robo-Slit signaling and diminishes with neuron age.

The cornea, the most densely innervated tissue on the surface of the body, becomes innervated in a series of highly coordinated developmental events. During cornea development, chick trigeminal nerve growth cones reach the cornea margin at embryonic day (E)5, where they are initially repelled for days from E5 to E8, instead encircling the corneal periphery in a nerve ring prior to entering on E9. The molecular events coordinating growth cone guidance during cornea development are poorly understood. Here we evaluated a potential role for the Robo-Slit nerve guidance family. We found that Slits 1, 2 and 3 expression in the cornea and lens persisted during all stages of cornea innervation examined. Robo1 expression was developmentally regulated in trigeminal cell bodies, expressed robustly during nerve ring formation (E5-8), then later declining concurrent with projection of growth cones into the cornea. In this study we provide in vivo and in vitro evidence that Robo-Slit signaling guides trigeminal nerves during cornea innervation. Transient, localized inhibition of Robo-Slit signaling, by means of beads loaded with inhibitory Robo-Fc protein implanted into the developing eyefield in vivo, led to disorganized nerve ring formation and premature cornea innervation. Additionally, when trigeminal explants (source of neurons) were oriented adjacent to lens vesicles or corneas (source of repellant molecules) in organotypic tissue culture both lens and cornea tissues strongly repelled E7 trigeminal neurites, except in the presence of inhibitory Robo-Fc protein. In contrast, E10 trigeminal neurites were not as strongly repelled by cornea, and presence of Robo-Slit inhibitory protein had no effect. In full, these findings suggest that nerve repulsion from the lens and cornea during nerve ring formation is mediated by Robo-Slit signaling. Later, a shift in nerve guidance behavior occurs, in part due to molecular changes in trigeminal neurons, including Robo1 downregulation, thus allowing nerves to find the Slit-expressing cornea permissive for growth cones.

The role of nonenzymatic glycation and carbonyls in collagen cross-linking for the treatment of keratoconus.

PURPOSE: Corneal cross-linking (CXL) is a treatment for keratoconus that eliminates the need for keratoplasty in most patients. However, its molecular mechanisms remain under study. Advanced glycation end products (AGEs) have been suggested by many studies as the causative strengthening agent during CXL, though no studies to date have directly tested this hypothesis. METHODS: Corneas of young rabbits and sharks were pretreated with pyridoxal hydrochloride and copper ions before CXL. Two known inhibitors of AGE formation, aminoguanidine and rifampicin, were applied during CXL in the treatment solution. Tensile strength tests were conducted after these experiments to detect diminished or accentuated corneal stiffening after CXL. SDS-PAGE was performed on type I collagen cross-linked in the absence and presence of AGE inhibitors. RESULTS: Pretreatment with pyridoxal hydrochloride resulted in significantly higher corneal stiffening after CXL. AGE inhibitors significantly diminished cross-linking as detected by both tensile strength measurements using whole corneas and gel electrophoresis of in vitro cross-linking of type I collagen in solution, in the presence and absence of the inhibitors. Rifampicin inhibited CXL more significantly than aminoguanidine in gel electrophoresis and tensile strength tests, confirming recent findings on its efficacy as an AGE inhibitor. CONCLUSIONS: Data presented here suggest that CXL is carbonyl dependent and involves the formation of AGE cross-links. Six possible cross-linking mechanisms are discussed.

Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking.

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and ß chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

Proteomic analysis of potential keratan sulfate, chondroitin sulfate A, and hyaluronic acid molecular interactions.

PURPOSE: Corneal stroma extracellular matrix (ECM) glycosaminoglycans (GAGs) include keratan sulfate (KS), chondroitin sulfate A (CSA), and hyaluronic acid (HA). Embryonic corneal keratocytes and sensory nerve fibers grow and differentiate according to chemical cues they receive from the ECM. This study asked which of the proteins that may regulate keratocytes or corneal nerve growth cone immigration interact with corneal GAGs. METHODS: Biotinylated KS (bKS), CSA (bCSA), and HA (bHA) were prepared and used in microarray protocols to assess their interactions with 8268 proteins and a custom microarray of 85 extracellular epitopes of nerve growth-related proteins. Surface plasmon resonance (SPR) was performed with bKS and SLIT2, and their ka, kd, and KD were determined. RESULTS: Highly sulfated KS interacted with 217 microarray proteins, including 75 kinases, several membrane or secreted proteins, many cytoskeletal proteins, and many nerve function proteins. CSA interacted with 24 proteins, including 10 kinases and 2 cell surface proteins. HA interacted with 6 proteins, including several ECM-related structural proteins. Of 85 ECM nerve-related epitopes, KS bound 40 proteins, including SLIT, 2 ROBOs, 9 EPHs, 8 Ephrins (EFNs), 8 semaphorins (SEMAs), and 2 nerve growth factor receptors. CSA bound nine proteins, including ROBO2, 2 EPHs, 1 EFN, two SEMAs, and netrin 4. HA bound no ECM nerve-related epitopes. SPR confirmed that KS binds SLIT2 strongly. The KS core protein mimecan/osteoglycin bound 15 proteins. CONCLUSIONS: Corneal stromal GAGs bind, and thus could alter the availability or conformation of, many proteins that may influence keratocyte and nerve growth cone behavior in the cornea.

Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA).

PURPOSE: Treatment of de-epithelialized human corneas with riboflavin (RF) + long-wavelength ultraviolet light (UVA; RFUVA) increases corneal stroma tensile strength significantly. RFUVA treatment retards the progression of keratoconus, perhaps by cross-linking of collagen molecules, but exact molecular mechanisms remain unknown. Research described here tested possible chemical mechanisms of cross-linking. METHODS: Corneas of rabbits and spiny dogfish sharks were de-epithelialized mechanically, subjected to various chemical pretreatments, exposed to RFUVA, and then subjected to destructive tensile stress measurements. Tensile strength was quantified with a digital force gauge to measure degree of tissue cross-linking. RESULTS: For both rabbit and shark corneas, RFUVA treatment causes significant cross-linking by mechanism(s) that can be blocked by the presence of sodium azide. Conversely, such cross-linking is greatly enhanced in the presence of deuterium oxide (D(2)O), even when RF is present at only one tenth the currently used clinical concentrations. Blocking carbonyl groups preexisting in the stroma with 2,4-dinitrophenylhydrazide or hydroxylamine blocks essentially all corneal cross-linking. In contrast, blocking free amine groups preexisting in the stroma with acetic anhydride or ethyl acetimidate does not affect RFUVA corneal cross-linking. When both carbonyl groups are blocked and singlet oxygen is quenched, no RFUVA cross-linking occurs, indicating the absence of other cross-linking mechanisms. CONCLUSIONS: RFUVA catalyzes cross-linking reactions that require production of singlet oxygen ((1)O(2)), whose half-life is extended by D(2)O. Carbonyl-based cross-linking reactions dominate in the corneal stroma, but other possible reaction schemes are proposed. The use of D(2)O as solution media for RF would enable concentration decreases or significant strength enhancement in treated corneas.

Embryonic corneal Schwann cells express some Schwann cell marker mRNAs, but no mature Schwann cell marker proteins.

PURPOSE: Embryonic chick nerves encircle the cornea in pericorneal tissue until embryonic day (E)9, then penetrate the anterior corneal stroma, invade the epithelium, and branch over the corneal surface through E20. Adult corneal nerves, cut during transplantation or LASIK, never fully regenerate. Schwann cells (SCs) protect nerve fibers and augment nerve repair. This study evaluates SC differentiation in embryonic chick corneas. METHODS: Fertile chicken eggs were incubated from E0 at 38 degrees C, 45% humidity. Dissected permeabilized corneas plus pericorneal tissue were immunostained for SC marker proteins. Other corneas were paraffin embedded, sectioned, and processed by in situ hybridization for corneal-, nerve-related, and SC marker gene expression. E9 to E20 corneas, dissected from pericorneal tissue, were assessed by real-time PCR (QPCR) for mRNA expression. RESULTS: QPCR revealed unchanging low to moderate SLIT2/ROBO and NTN/UNC5 family, BACE1, and CADM3/CADM4 expressions, but high NEO1 expression. EGR2 and POU3F1 expressions never surpassed PAX3 expression. ITGNA6/ITGNB4 expressions increased 20-fold; ITGNB1 expression was high. SC marker S100 and MBP expressions increased; MAG, GFAP, and SCMP expressions were very low. Antibodies against the MPZ, MAG, S100, and SCMP proteins immunostained along pericorneal nerves, but not along corneal nerves. In the cornea, SLIT2 and SOX10 mRNAs were expressed in anterior stroma and epithelium, whereas PAX3, S100, MBP, and MPZL1 mRNAs were expressed only in corneal epithelium. CONCLUSIONS: Embryonic chick corneas contain SCs, as defined by SOX10 and PAX3 transcription, which remain immature, at least in part because of stromal transcriptional and epithelial translational regulation of some SC marker gene expression.

Integrated genomic approaches implicate osteoglycin (Ogn) in the regulation of left ventricular mass.

Left ventricular mass (LVM) and cardiac gene expression are complex traits regulated by factors both intrinsic and extrinsic to the heart. To dissect the major determinants of LVM, we combined expression quantitative trait locus1 and quantitative trait transcript (QTT) analyses of the cardiac transcriptome in the rat. Using these methods and in vitro functional assays, we identified osteoglycin (Ogn) as a major candidate regulator of rat LVM, with increased Ogn protein expression associated with elevated LVM. We also applied genome-wide QTT analysis to the human heart and observed that, out of 22,000 transcripts, OGN transcript abundance had the highest correlation with LVM. We further confirmed a role for Ogn in the in vivo regulation of LVM in Ogn knockout mice. Taken together, these data implicate Ogn as a key regulator of LVM in rats, mice and humans, and suggest that Ogn modifies the hypertrophic response to extrinsic factors such as hypertension and aortic stenosis.

Expression studies of osteoglycin/mimecan (OGN) in the cochlea and auditory phenotype of Ogn-deficient mice.

Genes involved in the hearing process have been identified through both positional cloning efforts following genetic linkage studies of families with heritable deafness and by candidate gene approaches based on known functional properties or inner ear expression. The latter method of gene discovery may employ a tissue- or organ-specific approach. Through characterization of a human fetal cochlear cDNA library, we have identified transcripts that are preferentially and/or highly expressed in the cochlea. High expression in the cochlea may be suggestive of a fundamental role for a transcript in the auditory system. Herein we report the identification and characterization of a transcript from the cochlear cDNA library with abundant cochlear expression and unknown function that was subsequently determined to represent osteoglycin (OGN). Ogn-deficient mice, when analyzed by auditory brainstem response and distortion product otoacoustic emissions, have normal hearing thresholds.

On-target derivatization of keratan sulfate oligosaccharides with pyrenebutyric acid hydrazide for MALDI-TOF/TOF-MS.

In the present work, a rapid and novel method of on-target plate derivatization of keratan sulfate (KS) oligosaccharides for subsequent analysis by matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is described. MALDI-(time-of-flight)-TOF spectra of labeled KS oligosaccharides revealed that significantly improved ionization can be accomplished through derivatization with pyrenebutyric acid hydrazide (PBH), and the most abundant peak in each spectrum corresponds to the singly charged molecular ion [M - H]- or [M + (n - 1)Na - nH]-, where n = the number of sulfates (n = 1, 2, 3...). The high-energy collision-induced dissociation (heCID) spectra of labeled KS oligosaccharides displayed fragments of compounds similar to those observed with laser-induced dissociation (LID) analysis, suggesting that both heCID and LID fragmentations can be used to analyze KS oligosaccharides. Moreover, fragmentation analysis of all labeled KS oligosaccharides was performed by MALDI-TOF/TOF-MS. With LID mode, sodium adducts showed fragmentation of glycosidic linkages with mainly Y/B/C ions, as well as various cross-ring cleavages providing exact information for the positions of sulfate groups along the KS oligosaccharide chains. This one-step on-target derivatization method makes MALDI-TOF/TOF-MS identification of KS fast, simple and highly throughput for trace amounts of biological samples.

Thyroxine increases the rate but does not alter the pattern of innervation during embryonic chick corneal development.

PURPOSE: Embryonic chick corneal nerves reach limbal mesenchyme by embryonic day (E)5, encircle the cornea in several days, then defasciculate into the stroma simultaneously from all sides, while extracellular keratan sulfate proteoglycan (KSPG) accumulates from posterior to anterior stroma. Precocious thyroxine (T4)-induced increases in corneal thinning/transparency are blocked by 2-thiouracil (2-TU) inhibition of T3 synthesis. The hypothesis for this study was that precocious T4 exposure increases corneal innervation similarly. METHODS: E8 embryos received T4, 2-TU, T4+2-TU, or buffer; corneas were harvested on E12. Corneal nerves were stained with neuronal beta-tubulin-specific TuJ1 antibody or chick nerve-specific CN antibody. Corneal thickness was determined from cryostat sections, and mRNA expression was measured by real-time PCR. RESULTS: Nerves avoided the cornea until E9, then entered the anterior stroma, extended toward and reached the cornea center by E14, and never invaded posterior stroma. E7 to E18 corneal expressions of nerve growth factor and neurotrophin-3 genes were unchanged; receptor gene expressions rose. E7 to E12 semaphorin 3A and 3F and ephrin A2 and A5 expressions did not change significantly; semaphorin and ephrin/eph expressions increased from E9 to E18. E8 T4 administration increased nerve extension by E11, but did not alter circumferential penetration, anterior-only penetration, or neurotrophin expressions. 2-TU prevented T4-induced precocious corneal thinning, but augmented T4 nerve stimulation. CONCLUSIONS: No changes in corneal neurotrophin or nerve pathfinding gene expressions accompany corneal transition to nerve growth cone permissiveness. T4 increases corneal nerve penetration rates by a non-T3-dependent mechanism. Results are consistent with possible roles for corneal KSPGs in regulating corneal nerve growth.

Keratan sulfate and chondroitin/dermatan sulfate in maximally recovered hypocellular stromal interface scars of postmortem human LASIK corneas.

PURPOSE: To analyze the amounts and distributions of nonsulfated and sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in the interface wound of human postmortem LASIK corneas in comparison with normal control corneas. METHODS: Corneal stromal tissue samples from central and paracentral hypocellular primitive stromal interface scars of human LASIK corneas and from similar regions of normal control corneas were collected by laser capture microdissection (LCM) and subsequently were digested with specific glycosidase enzymes. Digests were directly analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: Concentrations of both monosulfated GlcNAc(6S)-beta-1,3-Gal (MSD2) and disulfated Gal (6S)-beta-1,4-GlcNAc(6S) (DSD) KS disaccharides from the LASIK interface scars were significantly lower than in normal control corneal stromas. No significant difference was found for the concentration of nonsulfated (NSD) KS disaccharides in LASIK interface scars compared with normal controls. The concentration of DeltaUA-beta-1,3-GalNAc(6S) (Deltadi-6S) CS/DS disaccharides from the LASIK interface scar was significantly higher than normal corneal stroma, whereas concentrations of DeltaUA-beta-1,3-GalNAc(4S) (Deltadi-4S) and nonsulfated Deltadi-0S CS/DS disaccharides demonstrated no significant differences from normal corneas. CONCLUSIONS: The profiles of KS and CS/DS disaccharides in LASIK interface scars are significantly different from those in normal cornea stromal tissue, as revealed by LCM and ESI-MS/MS.

Differentiation of monoiodothyronines using electrospray ionization tandem mass spectrometry.

In this work two monoiodothyronines, 3-T1 and 3'-T1, have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS. MS2 spectra in either negative or positive ion mode can be used to differentiate 3-T1 and 3'-T1. Based on the relative abundance of fragment ions in MS2 spectra in the negative ion mode, quantification of the 3-T1 and 3'-T1 isomers in mixtures is achieved without prior separation. Solid-phase extraction in combination with ESI-MS/MS provides a practicable procedure that can be used to determine the molar ratio of 3-T1 and 3'-T1 in human serum with an error less than 3%. The detection limits for 3-T1 and 3'-T1 were 0.5 and 0.7 pg/microL, respectively.