Molecular and cellular mechanisms of ultraviolet corneal crosslinking

In the presented mainly experimental work, the regularities and mechanisms of ultrastructural transformation and changes in corneal metabolism under ultraviolet exposure are established. High-quality saturation of the stroma with riboflavin is necessary to perform safe and effective UV crosslinking of the cornea, which can be achieved mainly due to a sufficient area of de-epithelization (at least 8-9 mm). It was found that UV crosslinking of the cornea with the use of developed riboflavin solutions (Dextralink, Ribolink and Khitolink) as photosensitizers increases its strength properties associated with changes in the structural state of collagen fibrils. It is proved that the riboflavin-UV effect on the optical shell of the eyeball is caused by a short-term local increase in free radical processes, a decrease in the overall antioxidant status, and the absence of necrotic and significant inflammatory reactions. It is shown that the lack of riboflavin in the stroma forms a cascade of pathomorphological events of an irreversible nature, leading to the loss of the native structure of the cornea. On the basis of the conducted studies, the necessity of a differentiated approach to the use of riboflavin-containing solutions during ultraviolet crosslinking of the cornea is justified. Key words: ultraviolet corneal crosslinking, molecular-cellular mechanisms, photosensitizers, riboflavin.

Changes in Ultrastructure and Pigment Content During Development and Senescence of Fruits of Normal and rin and nor Mutant Tomatoes

Fruits of the normal Rutgers tomato cultivar and of the rin and nor mutants were examined at the following times: Rutgers at 17, 21, 32, 45, 47, 50 and 55 days after anthesis; rin and nor at 17, 21, 32, 50, 65, 78 and 95 days after anthesis. Ripening in Rutgers fruits began at about 47 days, and fruits were fully ripe by 55 days after anthesis. Full size was reached in both Rutgers and in the mutant fruits about 50 days after anthesis. Rin fruits subsequently turned yellow, and nor fruits developed some pink colour, but the fruits of both mutants remained sound and comparatively firm for up to 95 days after anthesis. No unique differences in ultrastructure were noted between Rutgers and mutant fruits except that myelin figures were observed in 21- and 32-day nor fruits and spiral membrane tubules were found in the epidermis of 95-day rin fruits. In Rutgers fruits, the major change was the transformation of chloroplasts to chromoplasts during ripening; this transformation was completed within 5 days. A similar, but much slower and less complete, transformation was noted in the mutants. Some grana and chlorophyll were present even in 95-day fruits, although rin and nor fruits began to lose chlorophyll and to accumulate coloured carotenoids from the time the fruits reached full size. The slow changes in ultrastructural transformation of plastids, paralleled by the changes in colour, suggest either suppression of nuclear action or lack of capacity to produce cellular components essential for normal ripening. The latter suggestion was favoured by current information on the physiology of the mutants.

ENERGY-LINKED ULTRASTRUCTURAL TRANSFORMATIONS IN ISOLATED LIVER MITOCHONDRIA AND MITOPLASTS

An investigation was carried out in which microsamples of isolated rat liver mitochondria and freshly prepared mitoplasts in defined energy states were freeze-cleaved. Parallel microsamples were fixed with osmium tetroxide and with glutaraldehyde followed by osmium tetroxide as previously used in this laboratory for the preservation of energy-linked mitochondrial configurations. The details of the orthodox configuration of energized mitochondria and the condensed configuration of de-energized mitochondria, as revealed previously by chemical fixation, are confirmed in this report for nonfixed, freeze-cleaved mitochondria. The precise agreement in preservation of configuration obtained by the physical fixation of rapid freezing and by chemical fixation establishes unequivocally that mitochondria undergo energy-linked ultrastructural transformation between the condensed and the orthodox configurations which are thus natural structural states related to the metabolic activity of the mitochondrion. Configurations observed by freeze-cleaving and by chemical fixation reveal that mitoplasts also undergo a specific and dramatic ultrastructural transformation with the induction of oxidative phosphorylation. The transformation appears to be isovolumetric and therefore is thought to be mediated through energized conformational activity in the surface electron-transport membrane of the mitoplast. Passively swollen, spherical, osmotically active mitoplasts could not be fixed rapidly enough by chemical fixatives as normally used without altering the spherical form. In this special case preservation of configurational form required rapid freezing or chemical fixatives of low osmolar concentration.

OXIDATIVE PHOSPHORYLATION AND ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA IN THE INTACT ASCITES TUMOR CELL

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90° light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox → condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90° light-scattering increase, which also occurs at this time, showed a t ½ of only 25 sec which agreed temporally with a slower orthodox → maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox → condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.