Growth regulation of the mammalian ocular lens by vitreous humor.

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95 degrees C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor-receptor binding is essential for the growth inhibitory effect.     

Domain structure of cartilage proteoglycans revealed by rotary shadowing of intact and fragmented molecules.

The rotary-shadowing technique for molecular electron microscopy was used to study cartilage proteoglycan structure. The high resolution of the method allowed demonstration of two distinct globular domains as well as a more strand-like portion in the core protein of large aggregating proteoglycans. Studies of proteoglycan aggregates and fragments showed that the globular domains represent the part of the proteoglycans that binds to the hyaluronic acid, i.e. the hyaluronic acid-binding region juxtapositioned to the keratan sulphate-attachment region. The strand-like portion represents the chondroitin sulphate-attachment region. Low-Mr proteoglycans from cartilage could be seen as a globule connected to one or two side-chain filaments of chondroitin sulphate.     

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.     

Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing

The structure of the peripheral domains of neurofilaments (NFs) was revealed by rotary shadowing electron microscopy. NFs were isolated from bovine spinal cords by Sepharose CL-4B gel filtration and examined by low angle rotary shadowing. The peripheral domains appeared as thin, flexible, filamentous structures projecting from the intermediate filament core, with a constant density along their entire length. The average length of the projections was approximately 85 nm and the width about 4 nm. These projections appeared from regularly distributed sites, at 22 nm spacing, which seemed to correspond to the typical repeat of the alpha-helix-rich rod domain of the core filament. The density of the projections was found to be 4.1 (+/- 0.6) per 22 nm. We performed reconstitution experiments using purified NF polypeptides to confirm that the projection was indeed the NF peripheral domain. Individual components of the NF triplet, i.e. NF-L, NF-M and NF-H, were purified by DE-52 and Mono-Q anion exchange chromatographies in the presence of 6 M-urea and were assembled in various combinations into filaments. Reassembled filaments were somewhat more slender than the isolated NFs and exhibited a distinct 22 nm axial periodicity. While prominent projections were not observed in the filaments assembled from NF-L alone, reconstructed filaments containing NF-L plus either NF-M or NF-H revealed many projections. The average length of the projections in the filaments reconstructed from NF-L and NF-H was about 63 nm. The projections of reconstructed filaments from NF-L and NF-M were about 55 nm in length. The difference in the lengths of the projections might reflect the difference in the length of the carboxy-terminal tail domain between NF-M and NF-H. The results are interpreted to show that the carboxy-terminal tail domains of NFs project in a regular pattern from the core filament, which is consistent with a half-staggered organization of the tetrameric subunits.     

Dermatan sulphate proteoglycans from sclera examined by rotary shadowing and electron microscopy.

Two dermatan sulphate-containing proteoglycans from bovine sclera were examined by rotary shadowing and electron microscopy, and the results were compared with previous biochemical findings. Both the large iduronate-poor proteoglycan (PGI) and the small iduronate-rich proteoglycan (PGII) possessed a globular proteinaceous region. Whereas PGI had a branched extension from the globular region, with five to eight side chains attached to it, PGII had only a single tail, which was of glycosaminoglycuronan. PGII aggregated via globular-region interactions, which were much diminished by reduction and alkylation. PGI aggregated via side chains and globular-region interactions. Although a few PGI aggregates were large, and similar to the hyaluronan-cartilage proteoglycan aggregates [Weidemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333], hyaluronan did not cause enhanced aggregation. PGII is very similar in shape to the small cartilage chondroitin sulphate proteoglycan, whereas PGI somewhat resembles the large cartilage chondroitin sulphate proteoglycan, although with many fewer glycosaminoglycan side chains, and probably only one globular region as opposed to two in the cartilage proteoglycan.     

Phosphorylation of small molecular weight polypeptides in the iris-ciliary complex, aqueous humor and vitreous humor

The polypeptides of vitreous humor, aqueous humor and iris-ciliary complex cells of eyes were phosphorylated with [gamma-32P]ATP without exogenous protein kinase. Phosphorylated polypeptides were analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The phosphorylated polypeptides of rabbit vitreous humor showed many high molecular weight prominent bands, but no detectable phosphoproteins were found in the 12 kDa or lower range. Bovine vitreous humor has predominantly acidic polypeptides and some of them are below 20 kDa. Rabbit and bovine iris-ciliary complex and rabbit aqueous humor showed a prominent common 4 kDa phosphopolypeptide which could also be synthesized by cloned populations of cells from the bovine iris and the rabbit iris-ciliary body. It is possible that the 4 kDa phosphopolypeptide of the aqueous humor is synthesized by the iris-ciliary complex cells.     

Increased nonenzymatically glycosylated proteins in the vitreous humor of diabetic animals

One of the earliest pathologic changes of diabetes mellitus is increased nonenzymatic glycosylation (i.e., glycation) of proteins, which results in abnormal aggregation of collagen fibrils and production of superoxide radicals. These abnormalities may be responsible for the precocious senescence of connective tissue associated with the disease. We sought to determine whether glycation is increased in the vitreous humor of short-term diabetic cats (6 months' duration) and rabbits (2 months' duration), using a nitroblue tetrazolium colorimetric assay for fructosamine. Vitreous protein fructosamine concentration was significantly higher in diabetic cats and rabbits, compared with that in control (nondiabetic) animals. These results indicate that glycation is increased in the vitreous humor of short-term diabetic animals, and therefore may be one of the initial triggers for clinically apparent diabetic retinopathy.     

In vivo measurements of the viscoelasticity of the human vitreous humor.

A point of source of light against a dark background is perceived by the human retina as a point image enhanced by off-axis points (rays if the source is polychromatic) of light scattered from objects along the optic axis. As a consequence of movement of the vitreous humor, scattering centers imbedded there impart of this scattering pattern a corresponding movement. A method has been devised to give the vitreous humor reproducible initial conditions and to record the observed relaxation of the scattering pattern to its new rest position. The vitreous humor is found to be overdamped, and the heretofore unreported shear elastic modulus has been determined. A striking gravitational effect is revealed by comparing observations along a horizontal optic axis with ones along a vertical optic axis. For the former, gravitational torque is found to dominate the elastic torque. The reason nature has developed a slow-responding gravitational sensor in the vitreous humor is not clear.     

Biochemical sub-fractionation of the mammalian Golgi apparatus

We have exploited the breakdown of the Golgi apparatus that occurs during mitosis to isolate subfractions using immuno-affinity methods. Rat liver Golgi stacks were treated with mitotic cytosol from HeLa cells, and the fragments were then incubated with antibodies immobilized on magnetic beads. Antibodies against the cis -Golgi marker, GM130, bound membranes that were depleted in the trans -Golgi network marker, TGN38, whereas antibodies against the cytoplasmic tail of TGN38 did the reverse. A range of other Golgi enzymes, SNAREs and tethers were also tested and were found to bind to anti-GM130 antibodies to an extent that reflected their proximity to cis -cisternae as determined by other techniques. This method should provide a useful complement to the immuno-EM methods presently used to map the Golgi apparatus .     

Mathematical model of flow in the vitreous humor induced by saccadic eye rotations: effect of geometry

Saccadic eye rotations induce a flow in the vitreous humor of the eye. Any such flow is likely to have a significant influence on the dispersion of drugs injected into the vitreous chamber. The shape of this chamber deviates from a perfect sphere by up to 10–20% of the radius, which is predominantly due to an indentation caused by the lens. In this paper we investigate theoretically the effect of the domain shape upon the flow field generated by saccades by considering an idealized model. The posterior chamber geometry is assumed to be a sphere with a small indentation, undergoing prescribed small-amplitude sinusoidal torsional oscillations, and, as an initial step towards understanding the problem, we treat the vitreous humor as a Newtonian fluid filling the chamber. The latter assumption applies best in the case of a liquefied vitreous or a tamponade fluid introduced in the vitreous chamber after vitrectomy. We find the flow field in terms of vector spherical harmonics, focusing on the deviation from the flow that would be obtained in a perfect sphere. The flow induced by the departure of the domain geometry from the spherical shape has an oscillating component at leading order and a smaller-amplitude steady streaming flow. The oscillating component includes a circulation cell formed every half-period, which migrates from the indentation towards the center of the domain where it disappears. The steady component has two counter-rotating circulations in the anterior part of the domain. These findings are in good qualitative agreement with the experimental results of Stocchino et al. (Phys Med Biol 52:2021–2034, 2007). Our results predict a significant reduction in the expected time for drug dispersal across the eye compared with the situation in which there is no fluid flow present.     

Mitotic division of the mammalian Golgi apparatus

Successful cell reproduction requires faithful duplication and proper segregation of cellular contents, including not only the genome but also intracellular organelles. Since the Golgi apparatus is an essential organelle of the secretory pathway, its accurate inheritance is therefore of importance to sustain cellular function. Regulation of Golgi division and its coordination with cell cycle progression involves a series of sequential events that are subjected to a precise spatiotemporal control. Here, we summarize the current knowledge about the underlying mechanisms, the molecular players and the biological relevance of this process, particularly in mammalian cells, and discuss the unsolved problems and future perspectives opened by the recent studies.     

Modular organization of the mammalian Golgi apparatus

The Golgi apparatus is essential for post-translational modifications and sorting of proteins in the secretory pathway. In addition, it further performs a broad range of specialized functions. This functional diversity is achieved by combining basic morphological modules of cisternae into higher ordered structures. Linking cisternae into stacks that are further connected through tubules into a continuous Golgi ribbon greatly increases its efficiency and expands its repertoire of functions. During cell division, the different modules of the Golgi are inherited by different mechanisms to maintain its functional and morphological composition.     

Mitotic disassembly of the mammalian Golgi apparatus

A cell-free system that mimics mitotic fragmentation of Golgi stacks has provided a working model for the disassembly process. Two distinct pathways, one COP-dependent and one COP-independent, act on Golgi stacks to give rise to two types of end products: transport vesicles and larger, more heterogeneous vesicles and tubules. We suggest that both mitotic end products result from enhanced fission of Golgi membranes under conditions where membrane fusion is generally inhibited.     

The normal fine structure and aging changes of the human zonular fibers

Summary The normal zonular fibrils of the human eye do not differ from the fibrils of the zonula Zinnii of the rat. Furthermore, there is no difference between the single zonular fibrils and the fibrils of the vitreous body in rat and man. The average diameter of the human zonular fibrils is 109 Å. They are transversely striated at intervals with a periodicity of 70–150 Å. Over periods of mostly 400–440 å, but also of 500–640 Å were observed. At a few places over periods similar to those of the long spacing-type packing were found. Like in the rat eye, the zonular fibrils of the human eye must be regarded as a special form of collagen.An indirect proof for the collageneous nature of the zonular fibrils is the occurence in advanced age of degenerative alterations which are exclusively observed in connective tissue (hyalinization, elastoid degeneration).Supported by the Deutsche Forschungsgemeinschaft. Acknowledgement: I wish to thank Dr. H. Faßl, Institut für Medizinische Statistik und Dokumentation der Universität Mainz, for the statistical analysis.     

Reduced viability of vascular endothelial cells by high concentration of ascorbic acid in vitreous humor

Normal mammalian vitreous humor maintains its avascularity after regression of hyaloid vessels. Neovascularization in adults is only detected under pathological conditions which suggests that antiangiogenic factors are present in the vitreous humor. To elucidate the mechanism of vitreal angiogenic inhibition, we investigated the effect of vitreous humor on cultured vascular endothelial cells. When bovine aortic endothelial cells were cultured in the presence of bovine vitreous humor in medium, a decrease in cell viability was observed within 24 h. Ascorbic acid from vitreous humor has been identified as a cell death inducing factor with high performance liquid chromatography (HPLC) and molecular mass analysis. Ascorbic acid reduced endothelial cell viability at concentrations normally present in vitreous humor. This effect was completely inhibited by antioxidants, N-acetylcysteine and catalase. Amongst the ascorbic acid derivatives tested, ascorbic acid 2-phosphate did not induce cell death, suggesting that the production of ascorbyl radical is required for induction of cell death. Furthermore, capillary formation in three-dimensional collagen gel cultures characteristic of vascular endothelial cells were disrupted in the presence of ascorbic acid. Since ascorbic acid is highly concentrated in ocular tissues, especially in vitreous humor, it may function as a neovascularization inhibitor.