Valerie A. Smith

Purification and partial characterization of a gibberellin 2β-hydroxylase fromPhaseolus vulgaris

A gibberellin 2β-hydroxylase has been purified from mature seeds ofPhaseolus vulgaris. The enzyme is of molecular weight 36,000 and has the characteristics of a dioxygenase; the cofactors areα-ketoglu-tarate, Fe2+ and ascorbate, and activity is stimulated by catalase. The Vmax of the enzyme is 6.86 nmole h−1 mg−1, and the Km values for [1,2-3H2]GA1 andα-ketoglutarate are 0.085 μM and 21 μM, respectively. The purified enzyme preparation catalyzes hydroxylation of GA1, GA4, GA9, and GA20 but exhibits a marked preference for the 3-hydroxylated gibberellins as substrate.

Light Transmission in the Human Cornea as a Function of Position across the Ocular Surface: Theoretical and Experimental Aspects

This article investigates the theoretical basis for differences in visible light transmission through the human cornea as a function of distance from the center. Experimentally, transmission decreases approximately linearly up to 3 mm from the central axis, then quadratically beyond this. It is known that collagen fibril number density and collagen fibril radii change from the central region to the corneal periphery. We modeled, using the direct-summation-of-scattered-fields method, the effects these ultrastructural changes would be expected to have on light transmission, accounting for the increase in corneal thickness from center to edge. Fibril positions for the modeling were obtained from electron micrographs of human cornea. Theoretically, transmission remains fairly constant across the central cornea; then, as the fibril diameter increases, the predicted scattering increases. Interfibrillar spacing changes alter the refractive index ratio between matrix and fibril; this was modeled in our theoretical deductions. Fibril number density had a minimal effect on light propagation. Our theoretical deductions were in broad agreement with our experimental data. It is concluded that the reduced transparency in the peripheral stroma is primarily caused by changes in fibril radius and an increase in refractive index ratio between the fibril and the interfibrillar substance.

Does orally administered doxycycline reach the tear film

Background/aims: Orally administered doxycycline, a broad-spectrum antibiotic, is an established treatment for ocular surface diseases, particularly rosacea, meibomian gland dysfunction and recurrent epithelial cell erosion. In recent times, its efficacy in treating these diseases has been ascribed to an ability to inhibit matrix metalloproteinase (MMP) activity and both MMP and interleukin-1 (IL-1) synthesis. Since these functions are concentration-dependent, the aim of this project was to determine whether sufficient doxycycline reached the tear film to fulfil these roles in vivo. Methods: Doxycycline was extracted with 1-butanol from tear and blood plasma samples obtained from patients with ocular surface disease and healthy individuals and quantified spectrophotometrically. The MMPs present in the patients tear films before and during doxycycline treatment were analysed zymographically. Results: The quantity of doxycycline detected in the blood plasma samples of patients undergoing treatment ranged from 1.83 to 13.18 μM. Although doxycycline was not detected in their tear samples, the treatment caused the disappearance of the MMPs symptomatic of disease progression. Conclusion: The inability to detect doxycycline in the tear film of patients undergoing treatment indicates that doxycycline does not directly inhibit MMP activity or the synthesis/secretion of these proteases and IL-1 from corneal epithelial cells.

Enzymes from seeds of Phaseolus vulgaris L.: Hydroxylation of gibberellins A20 and A1 and 2,3-dehydrogenation of gibberellin A20

A time-course study is described relating the enzyme activities for GA20 metabolism with seed development in Phaseolus vulgaris L. Enzyme activity for the 3β-hydroxylation of GA20 to GA1, and for the 2,3-desaturation of GA20 to GA5, was confined to the cotyledons and showed maximal specific activity at 21 d after anthesis. These enzyme activities co-occurred, together with a much lower level of activity for the 2β-hydroxylation of GA20 to GA29. The observed rates of GA1, GA5 and GA29 formation from GA20 were constant under a range of incubation conditions. Enzyme activity for the conversion of GA1 to GA8 was detected only in embryos of seed from 40 d after anthesis. By deuterium-labelling and analysis of the products by gas chromatography-selected ion monitoring it was shown that 2β-hydroxylation of GA1 to GA8 and 3β-hydroxylation of GA20 to GA1 occur with retention of configuration and that the conversion of GA20 to GA5 occurs with loss of the 2β- and 3β-hydrogens. These results establish that GA1 is not formed from GA20 via GA5.

Adenovirus mediated gene delivery of tissue inhibitor of metalloproteinases-3 induces death in retinal pigment epithelial cells

Background: Sorsbys fundus dystrophy (SFD) and age related macular degeneration (ARMD) are retinal diseases associated with a high level of accumulation of mutant and wild type TIMP-3, respectively, in Bruchs membrane. The pathogenic role of TIMP-3 in these diseases is uncertain, but causative mutations have been identified in the TIMP-3 gene of patients with SFD. Recent reports that TIMP-3 causes apoptosis in certain cell types and not in others prompted the authors to investigate whether TIMP-3 causes apoptosis in cultured retinal pigment epithelium (RPE) cells. Methods: RPE and MCF-7 cells (as a positive control) were initially infected with replication deficient adenovirus, to overexpress β-galactosidase (RAdLacZ) or TIMP-3 (RAdTIMP-3). TIMP-3 was detected by western blotting and ELISA. Cell viability was defined by cell counts. ISEL was used to investigate the mechanism of cell death. Results: Cultured RPE cells produced small quantities of endogenous TIMP-3 and remained viable. However, overexpression of TIMP-3 caused a dose related death of RPE cells. The mechanism of cell death was apoptosis. Conclusion: The previously unreported finding of TIMP-3 induced apoptosis of RPE cells may account for some of the early features seen in SFD and ARMD.

Sorsby's fundus dystrophy mutant tissue inhibitors of metalloproteinase-3 induce apoptosis of retinal pigment epithelial and MCF-7 cells.

C‐terminal domain tissue inhibitor of metalloproteinases‐3 (TIMP‐3) mutations cause the rare hereditary blindness Sorsbys fundus dystrophy (SFD), which involves loss of retinal pigment epithelial (RPE) cells. Since wild‐type TIMP‐3 causes apoptosis, we investigated whether SFD TIMP‐3 might kill RPE and other cells. Plasmid‐mediated overexpresion of Ser‐156, Gly‐167, Tyr‐168 and Ser‐181 SFD mutant TIMP‐3 decreased RPE viability to 22±8, 20±6, 32±5, 30±12% (SFD mutants all P<0.01 versus wild‐type 50±8%) and similarly increased propidium iodide staining and in situ end labelling. Adenovirus‐mediated overexpression of the Gly‐167 mutant also caused RPE apoptosis dose‐dependently. Apoptosis of RPE cells might therefore contribute to the pathology of SFD.

Matrix Metalloproteinase 2 Activation in Cultured Corneas

Purpose: Corneas that are maintained in tissue culture medium shed their epithelial cells and repopulation following graft surgery is an essential facet of the healing process. Failure to do so may be a result of structural damage to the epithelial basement membrane of a donor cornea. The purpose of the present investigation was to ascertain whether MMP-2, the matrix metalloproteinase produced by corneal keratocytes, may be activated during storage and hence cleave the type IV collagen component of the epithelial cell basement membrane. Methods: Fresh and transplant rejected corneas that had been stored in culture medium for varying time periods and of known donor age were collected. The soluble protein fractions of these corneas were obtained. Their MMP-2 proteins were visualised by zymography on SDS gelatin polyacrylamide gels and assayed for activity against nitrophenyl acetate and denatured [3H]type I collagen. Results: The stromal tissue of fresh, normal corneas produced inactive MMP-2 of Mr 66,000. Although the cultured corneas did not up-regulate MMP-2 production, they contained additional MMP-2 activities of Mr 62,000 and Mr 43,000. The appearance of these additional MMP-2 activities correlated with corneal culture time but not donor age. The ability to cleave denatured [3H]type I collagen correlated with the appearance of the Mr 43,000 activity but not the Mr 62,000 activity. Conclusion: Activated MMP-2 is produced in cultured corneas. For this reason the corneas donated for all graft procedures should not be held in culture medium for periods exceeding 4 weeks.

Localisation of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy

The use of picrosirius red to localise connective tissue in thin tissue sections viewed by bright-field microscopy is well documented. Its use on thin tissue sections imaged by fluorescence confocal microscopy has also been reported. Here we describe modifications to published procedures that allow picrosirius red staining of thick 60-microm sections and their subsequent analysis by confocal microscopy. The use of phosphomolybdic acid pre-treatment was found to be essential for confocal analysis; in addition to preventing non-specific staining, it also quenched tissue autofluorescence. By incubating sections free-floating, pre-treating them with phosphomolybdic acid for 30 min and imaging them using an argon ion laser we were able to use confocal microscopy to image the entire depth of 60-microm human optic nerve and nerve head sections stained with picrosirius red. The application of this modified picrosirius red and confocal microscopy technique should be useful for analysing the three-dimensional structure of the optic nerve and other tissues with a similarly complex arrangement of connective tissue.

Immunochromatographic purification of gibberellins from vegetative tissues of Cucumis sativus L: Separation and identification of 13-hydroxy and 13-deoxy gibberellins

Immunological methods are described for the separation and purification of 13-hydroxy and 13-deoxy-gibberellins of Cucumis sativus. Qualitative and quantitative data show that 13-deoxygibberellins predominate over 13-hydroxygibberellins in stems and leaves of this species.

Evaluation of an Animal Product-Free Variant of MegaCell™ MEM as a Storage Medium for Corneas Destined for Transplantation

Purpose: The traditional medium for storing corneas in European Eye Banks is Gibco’s MEM (Eagle’s) with Earle’s salts and Hepes containing 2% fetal calf serum, glutamine and antimycotics. Although serum-free MegaCell™ MEM has been reported to be more suitable for this purpose, it contains components of animal origin that potentially pose health risks to corneal recipients. The possibility of removing or replacing these components has therefore been investigated. Methods: A MegaCell basal medium (DME) and a formulation of this (MegaCell DCS), which contains no components of animal origin, have been prepared. The viability of the endothelial, epithelial and stromal cells of corneas held in these media has been assessed, their stress levels monitored and water content determined. Results: The endothelial cell count and morphology of corneas held in MegaCell DME and DCS for 30 days remained little changed. Their epithelial and stromal cells also retained their ability to proliferate in culture. Neither DME nor DCS prevented corneal swelling but the lack of endogenous MMP-2 activation indicated that the corneas were not subject to metabolic stress. Conclusion: MegaCell DCS is an animal product-free medium formulated for corneal storage. The quality of corneas held in this medium is similar to those held in MegaCell MEM.