Synthesis of sulfated glycosaminoglycans by the three cell types of the rabbit cornea in culture.

Rabbit corneal cells were cultivated for 21 days and then exposed to Na235SO4, a precursor of sulfated glycosaminoglycans (GAG). All 3 cell types of the cornea, the fibroblasts, the epithelial as well as the endothelial cells, synthesize GAG. The fractionation-patterns of the epithelial and endotherlial GAG are almost identical and differ clearly from the one of fibroblastic GAG.     

Piroxicam-induced analgesia: evidence for a central component which is not opioid mediated.

Piroxicam is a nonsteroidal anti-inflammatory drug with a potent analgesic effect. In order to establish whether the analgesic action of Piroxicam has a central component, we studied the effect of the drug on the nociceptive orbicularis oculi reflexes evoked by electrical stimulation of the cornea and supraorbital nerve in healthy subjects. Piroxicam significantly suppressed the corneal reflex and R3 component of the blink reflex by 28% (p < 0.05) and 50% (p < 0.01), respectively. This effect was not reversed by the i.v. injection of naloxone. Beta-endorphin levels did not change. Piroxicam administration induces distinct inhibitory changes in nociceptive reflexes, which suggests that the analgesic action of the drug has a central component. The ineffectiveness of naloxone, and the lack of beta-endorphin changes, indicate that this central action is independent of the opioid system; other pain regulatory systems are probably involved.     

Piroxicam-induced analgesia: Evidence for a central component which is not opioid mediated

Piroxicam is a nonsteroidal anti-inflammatory drug with a potent analgesic effect. In order to establish whether the analgesic action of Piroxicam has a central component, we studied the effect of the drug on the nociceptive orbicularis oculi reflexes evoked by electrical stimulation of the cornea and supraorbital nerve in healthy subjects. Piroxicam significantly suppressed the corneal reflex and R3 component of the blink reflex by 28% (p<0.05) and 50% (p<0.01), respectively. This effect was not reversed by the i.v. injection of naloxone. Beta-endorphin levels did not change. Piroxicam administration induces distinct inhibitory changes in nociceptive reflexes, which suggests that the analgesic action of the drug has a central component. The ineffectiveness of naloxone, and the lack of beta-endorphin changes, indicate that this central action is independent of the opioid system; other pain regulatory systems are probably involved.     

Synthesis of sulfated glycosaminoglycans by the three cell types of the rabbit cornea in culture

Summary Rabbit corneal cells were cultivated for 21 days and then exposed to Na₂ ³⁵SO₄, a precursor of sulfated glycosaminoglycans (GAG). All 3 cell types of the cornea, the fibroblasts, the epithelial as well as the endothelial cells, synthesize GAG. The fractionation-patterns of the epithelial and endothelial GAG are almost identical and differ clearly from the one of fibrolastic GAG.Supported by SNSF, grant No. 3.534.71.     

Cornea regeneration in the Pacific giant octopus,Octopus dofleini,and the common octopus,O. vulgaris

Summary Cornea regeneration in a Pacific giant octopus,Octopus dofleini, occurred within 10 days after the injury was observed. Surgical removal of the cornea in 2 common octopi,O. vulgaris experimentally duplicated this cornea regeneration within a 10-day period. It is, therefore, concluded that besides sucking discs, arms, and nerve fibres, octopi can also regenerated corneal tissue.The authors would like to thank U. E. Friese, B. Borowski, P. Burn, J.W. Atz, G.J. Nelson, H. Feinberg, P. Fenimore, C. Roper, M. Nixon, M. Segalla, G. Clark, D.O. Calligaro, J.M. Anderson and R.J. Koestler for assistance and advice. Illustrations were drawn by G.D. This work was supported by the New York Aquarium of the New York Zoological Society, while G.D. was at the New York Aquarium.     

Activation of innate immunity by lysozyme fibrils is critically dependent on cross-β sheet structure

Inflammation occurs in many amyloidoses, but its underlying mechanisms remain enigmatic. Here we show that amyloid fibrils of human lysozyme, which are associated with severe systemic amyloidoses, induce the secretion of pro-inflammatory cytokines through activation of the NLRP3 (NLR, pyrin domain containing 3) inflammasome and the Toll-like receptor 2, two innate immune receptors that may be involved in immune responses associated to amyloidoses. More importantly, our data clearly suggest that the induction of inflammatory responses by amyloid fibrils is linked to their intrinsic structure, because the monomeric form and a non-fibrillar type of lysozyme aggregates are both unable to trigger cytokine secretion. These lysozyme species lack the so-called cross-β structure, a characteristic structural motif common to all amyloid fibrils irrespective of their origin. Since fibrils of other bacterial and endogenous proteins have been shown to trigger immunological responses, our observations suggest that the cross-β structural signature might be recognized as a generic danger signal by the immune system.     

NLRP3 inflammasome activation in macrophage cell lines by prion protein fibrils as the source of IL-1β and neuronal toxicity

Prion diseases are fatal transmissible neurodegenerative diseases, characterized by aggregation of the pathological form of prion protein, spongiform degeneration, and neuronal loss, and activation of astrocytes and microglia. Microglia can clear prion plaques, but on the other hand cause neuronal death via release of neurotoxic species. Elevated expression of the proinflammatory cytokine IL-1β has been observed in brains affected by several prion diseases, and IL-1R-deficiency significantly prolonged the onset of the neurodegeneration in mice. We show that microglial cells stimulated by prion protein (PrP) fibrils induced neuronal toxicity. Microglia and macrophages release IL-1β upon stimulation by PrP fibrils, which depends on the NLRP3 inflammasome. Activation of NLRP3 inflammasome by PrP fibrils requires depletion of intracellular K⁺, and requires phagocytosis of PrP fibrils and consecutive lysosome destabilization. Among the well-defined molecular forms of PrP, the strongest NLRP3 activation was observed by fibrils, followed by aggregates, while neither native monomeric nor oligomeric PrP were able to activate the NLRP3 inflammasome. Our results together with previous studies on IL-1R-deficient mice suggest the IL-1 signaling pathway as the perspective target for the therapy of prion disease.     

Amphibious vision in Coryphoblennius galerita L. (Perciformes).

The morphology of the ophthalmic cornea in the blenniid fish Coryphoblennius galerita (Teleostei) shows adaptation to the amphibious life. Amphibious vision is provided by a flattened area within the cornea. Eyes of other, non-amphibious blenniids are compared with those of C. galerita.     

Amphibious vision inCoryphoblennius galerita L. (Perciformes)

The morphology of the ophthalmic cornea in the blenniid fishCoryphoblennius galerita (Teleostei) shows adaptation to the amphibious life. Amphibious vision is provided by a flattened area within the cornea. Eyes of other, non-amphibious blenniids are compared with those ofC. galerita.     

Retention of topical liposomal formulations on the cornea

The ability of liposomes composed of different kinds of phospholipid materials to adhere to the surface of the cornea was studied in the rabbit. The liposomes were labelled with tracer amounts of an I125-labelled phosphatidylethanolamine derivative and were instilled in 10 microliters drops onto the cornea. The retention of radioactivity was monitored. The results show that liposomes containing positively charged phospholipids are better retained than an albumin control. Thus, it may be possible to develop a drug delivery liposome system which would permit long-term sustained release of ophthalmic drugs onto the cornea.     

Transformation of amyloid β(1–40) oligomers into fibrils is characterized by a major change in secondary structure

Alzheimer’s disease (AD) is a neurodegenerative disorder occurring in the elderly. It is widely accepted that the amyloid beta peptide (Aβ) aggregation and especially the oligomeric states rather than fibrils are involved in AD onset. We used infrared spectroscopy to provide structural information on the entire aggregation pathway of Aβ(1–40), starting from monomeric Aβ to the end of the process, fibrils. Our structural study suggests that conversion of oligomers into fibrils results from a transition from antiparallel to parallel β-sheet. These structural changes are described in terms of H-bonding rupture/formation, β-strands reorientation and β-sheet elongation. As antiparallel β-sheet structure is also observed for other amyloidogenic proteins forming oligomers, reorganization of the β-sheet implicating a reorientation of β-strands could be a generic mechanism determining the kinetics of protein misfolding. Elucidation of the process driving aggregation, including structural transitions, could be essential in a search for therapies inhibiting aggregation or disrupting aggregates.     

Immunoglobulin light chains, glycosaminoglycans, and amyloid

Immunoglobulin light chains are the precursor proteins for fibrils that are formed during primary amyloidosis and in amyloidosis associated with multiple myeloma. As found for the approximately 20 currently described forms of focal, localized, or systemic amyloidoses, light chain-related fibrils extracted from physiological deposits are invariably associated with glycosaminoglycans, predominantly heparan sulfate. Other amyloid-related proteins are either structurally normal, such as beta2-microglobulin and islet amyloid polypeptide, fragments of normal proteins such as serum amyloid A protein or the precursor protein of the beta peptide involved in Alzheimer's disease, or are inherited forms of single amino acid variants of a normal protein such as found in the familial forms of amyloid associated with transthyretin. In contrast, the primary structures of light chains involved in fibril formation exhibit extensive mutational diversity rendering some proteins highly amyloidogenic and others non-pathological. The interactions between light chains and glycosaminoglycans are also affected by amino acid variation and may influence the clinical course of disease by enhancing fibril stability and contributing to resistance to protease degradation. Relatively little is currently known about the mechanisms by which glycosaminoglycans interact with light chains and light-chain fibrils. It is probable that future studies of this uniquely diverse family of proteins will continue to shed light on the processes of amyloidosis, and contribute as well to a greater understanding of the normal physiological roles of glycosaminoglycans.     

RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease

Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The biology of RAGE is driven by the settings in which these ligands accumulate, such as diabetes, inflammation, neurodegenerative disorders and tumors. In this review, we discuss the context of each of these classes of ligands, including advance glycation end-products, amyloid beta peptide and the family of beta sheet fibrils, S100/calgranulins and amphoterin. Implications for the role of these ligands interacting with RAGE in homeostasis and disease will be considered.     

Retention of topical liposomal formulations on the cornea

Summary The ability of liposomes composed of different kinds of phospholipid materials to adhere to the surface of the cornea was studied in the rabbit. The liposomes were labelled with tracer amounts of an I¹²⁵-labelled phosphatidylethanolamine derivative and were instilled in 10 l drops onto the cornea. The retention of radioactivity was monitored. The results show that liposomes containing positively charged phospholipids are better retained than an albumin control. Thus, it may be possible to develop a drug delivery, liposome system which would permit long-term sustained release of ophthalmic drugs onto the cornea.     

Stabilisation of collagen by betel nut polyphenols as a mechanism in oral submucous fibrosis

Treatment of reconstituted collagen fibrils and pieces of rat dermis with the crude extract, purified tannins or (+)-catechin from betel nut (Areca catechu) increases their resistance to both human and bacterial collagenases in a concentration-dependent manner. These tanning agents may stabilise collagen in vivo following damage to the oral epithelium, and promote the sub-epithelial fibrosis which occurs in betel nut chewers.     

Stabilisation of collagen by betel nut polyphenols as a mechanism in oral submucous fibrosis

Summary Treatment of reconstituted collagen fibrils and pieces of rat dermis with the crude extract, purified tannins or (+)-catechin from betel nut (Areca catechu) increases their resistance to both human and bacterial collagenases in a concentration-dependent manner. These tanning agents may stabilise collagen in vivo following damage to the oral epithelium and promote the sub-epithelial fibrosis which occurs in betel nut chewers.     

Structure of rat tail tendon collagen examined by atomic force microscope

The Atomic Force Microscope (AFM) was used to inspect collagen fibrils deposited on mica sheets at different fibrillogenesis times. Collagen was obtained from rat tail tendon fibers. Various fibril forms were observed, together with the characteristic periodic intra-fibril structure (D-bands). The fibril thickness, width, D-band periodicity and depth were measured and the statistical distribution of these parameters at 1, 2, 5, 10 and 15 days of in vitro fibril formation time was calculated. The fibrils showed an increasing size with time, but the band interval measure remained stable. The band depth, after an initial increase, exhibited a relative steadiness. The results indicate that AFM offers, at low resolution, images qualitatively similar to those obtained with electron microscopy, but with less manipulation of the sample. A quantitative evaluation of collagen structural features in the nanometer scale is made possible by AFM.     

On the structural definition of amyloid fibrils and other polypeptide aggregates

Amyloid fibrils occur inside the human body, associated with ageing or a group of diseases that includes, amongst others, Alzheimer’s disease, atherosclerosis and type II diabetes. Many natural polypeptide chains are able to form amyloid fibrils in vivo or in vitro, and this ability has been suggested to represent an inherent consequence of the chemical structure of the polypeptide chain. Recent literature has provided a wealth of information about the structure of aggregates, precipitates, amyloid fibrils and other types of fibrillar polypeptide assemblies. However, the biophysical meaning associated with these terms can differ considerably depending on the context of their usage. This overview presents a structural comparison of amyloid fibrils and other types of polypeptide assemblies and defines amyloid fibrils, based on structural considerations, as fibrillar polypeptide aggregates with a cross-β conformation. Received 1 March 2007; received after revision 15 March 2007; accepted 25 April 2007     

Biological activity and pathological implications of misfolded proteins

The physiological metabolism of proteins guarantees that different cellular compartments contain the appropriate concentration of proteins to perform their biological functions and, after a variable period of wear and tear, mediates their natural catabolism. The equilibrium between protein synthesis and catabolism ensures an effective turnover, but hereditary or acquired abnormalities of protein structure can provoke a premature loss of biological function, an accelerated catabolism and diseases caused by the loss of an irreplaceable function. In certain proteins, abnormal structure and metabolism are associated with a strong tendency to self-aggregation into a polymeric fibrillar structure, and in these cases the disease is not principally caused by the loss of an irreplaceable function but by the action of this new biological entity. Amyloid fibrils are an apparently inert, insoluble, mainly extracellular protein polymer that kills the cell without tissue necrosis but by activation of the apoptotic mechanism. We analyzed the data reported so far on the structural and functional properties of four prototypic proteins with well-known biological functions (lysozyme, transthyretin, β2-microglobulin and apolipoprotein AI) that are able to create amyloid fibrils under certain conditions, with the perspective of evaluating whether the achievement of biological function favors or inhibits the process of fibril formation. Furthermore, studying the biological functions carried out by amyloid fibrils reveals new types of protein-protein interactions in the transmission of messages to cells and may provide new ideas for effective therapeutic strategies. Received 9 November 1998; received after revision 15 January 1999; accepted 15 January 1999