Graded potential of neural crest to form cornea, sensory neurons and cartilage along the rostrocaudal axis

Neural crest cells arising from different rostrocaudal axial levels form different sets of derivatives as diverse as ganglia, cartilage and cornea. These variations may be due to intrinsic properties of the cell populations, different environmental factors encountered during migration or some combination thereof. We test the relative roles of intrinsic versus extrinsic factors by challenging the developmental potential of cardiac and trunk neural crest cells via transplantation into an ectopic midbrain environment. We then assess long-term survival and differentiation into diverse derivatives, including cornea, trigeminal ganglion and branchial arch cartilage. Despite their ability to migrate to the periocular region, neither cardiac nor trunk neural crest contribute appropriately to the cornea, with cardiac crest cells often forming ectopic masses on the corneal surface. Similarly, the potential of trunk and cardiac neural crest to form somatosensory neurons in the trigeminal ganglion was significantly reduced compared with control midbrain grafts. Cardiac neural crest exhibited a reduced capacity to form cartilage, contributing only nominally to Meckle's cartilage, whereas trunk neural crest formed no cartilage after transplantation, even when grafted directly into the first branchial arch. These results suggest that neural crest cells along the rostrocaudal axis display a graded loss in developmental potential to form somatosensory neurons and cartilage even after transplantation to a permissive environment. Hox gene expression was transiently maintained in the cardiac neural tube and neural crest at 12 hours post-transplantation to the midbrain, but was subsequently downregulated. This suggests that long-term differences in Hox gene expression cannot account for rostrocaudal differences in developmental potential of neural crest populations in this case

Effects of Silver and Other Metals on the Cytoskeleton

Directly or indirectly, trace concentrations of silver ion (Ag(+)) stabilize microtubules (Conrad, A.H., et al. Cell Motil. & Cytoskel. 27:117-132), as does taxol (Conrad, A.H., et al. J. Exp. Zool. 262:154-165), an effect with major consequences for cellular shape changes and development. Polymerization of microtubules is gravity-sensitive (Tabony and Job, Proc. Natl. Acad. Sci. USA 89:6948-6952), so trace amounts of Ag(+) may alter cellular ability to respond to gravity. If Ag electrolysis is used to purify water on NASA space vehicles, plants and animals/astronauts will be exposed continuously to Ag(+), a regimen with unknown cellular and developmental consequences. Fertilized eggs of the marine mudsnail, Ilyanassa obsoleta, are the cells in which the effects of A(+) on microtubules were discovered. They distribute visible cytoplasmic contents according to gravity and contain cytoplasmic morphogenetic determinants for heart development. The objectives are to determine if the effects of Ag(+), AU(3+), (of biosensor relevance), or Gd(3+) (inhibitor of some stretch-activated ion channels) on the cytoskeleton (in the presence and absence of mechanical loading) will affect cellular responses to gravity

Effects of Microgravity on Quail Eye Development

During embryonic development, the most exposed tissue of the eye, the cornea, becomes differentially bulged outward because of constant intraocular pressure (IOP). The component cells of the cornea secrete a unique, paracrystalline extracellular matrix (the stroma) composed of orthogonal plies of collagen fibrils and proteoglycans. The cornea remains avascular, becomes transparent, and becomes more densely innervated than any other region on the surface of the body. Corneas from chicken embryos that flew on STS-47 contain many more cellular processes in the outermost region of the stroma (Bowman's Layer) than any corresponding region of control corneas. These processes appear to be cross-sections of cytoplasmic extensions of cells and are found in that region of Bowman's Layer immediately beneath the basal lamina of the corneal epithelium. Here, we propose to compare corneas of quail that flew in space on Mir-1 with those of ground controls to determine if the same unusual cellular processes are seen as in the space-flown chicken corneas. In the central regions of such space-flown corneas, the processes appear to be either portions of basal epithelial cells whose pseudopodial extensions have migrated down through their own basal lamina into the stroma, or corneal nerves that have innervated the corneal stroma in an unusual manner. Eyeballs of embryos fixed on Mir-1, control embryos fixed at KSC and clinostated embryos fixed at KSU, will provide corneas for this study. Electron microscopy will be used to assess the distribution of the cellular processes in Bowman's Layer in the central region of each cornea. Attempts also will be made to determine the relative glycosaminoglycan distributions in the corneal stromas by indirect immunofluorscence and to record whole-mount staining patterns of the corneal nerves

Detection and Quantification of 3,5,3′-Triiodothyronine and 3,3′,5′-Triiodothyronine by Electrospray Ionization Tandem Mass Spectrometry

A novel and rapid method for identifying and quantifying 3,5,3′-triiodothyronine (T3) and 3,3′,5′-triiodothyronine (rT3; reverse T3) has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate T3 and rT3. Quantification of the T3 and rT3 isomers under the negative ionization mode is also achieved without prior separation by HPLC

Resistance of corneal RFUVA-cross-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

Does prenatal methylmercury exposure from fish consumption affect blood pressure in childhood?

INTRODUCTION: Prenatal exposure to organic methylmercury (MeHg) from seafood consumption has been reported to increase children's blood pressure (BP). A report from the Faroe Islands noted significantly increased diastolic and systolic BP in 7-year-old children as prenatal MeHg exposure increased. The Faroese diet includes sea mammals that contain MeHg, cadmium, and other pollutants. We examined this relationship in the Seychelles Islands to determine if it was present in a society exposed primarily to MeHg from consuming ocean fish. METHODS: We obtained BP at ages 12 and 15 years on children with known prenatal MeHg exposure enrolled in the Seychelles Child Development Study (SCDS). We examined the association between prenatal MeHg exposure and BP using longitudinal models and linear regression adjusted for relevant covariates. RESULTS: Blood pressure at both ages was associated with BMI, height and maternal hypertension during pregnancy as expected. No association between prenatal MeHg exposure and BP was present in girls at either age or in either sex at age 12 years. At age 15 years diastolic BP in boys increased with increasing prenatal MeHg exposure, while systolic BP was unaffected. SUMMARY: It is unclear whether the association between prenatal MeHg exposure and diastolic BP seen in 15-year-old boys is of biological significance or if it is a chance finding. However, the finding is intriguing and deserves further study

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

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 ka, kd, and KD 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 Zn2+. Conclusions. Covalent and (zinc-mediated) noncovalent mechanisms involving FIB and stromal ECM molecules contribute to the adhesion created by FIB + RF + UVA