Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Early Amyloid-like Oligomers and Their Implication in alpha-Synuclein Aggregation

Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of alpha-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote alpha-synuclein aggregation. Taking into account the toxicity of alpha-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.

Urinary glycosaminoglycans in horse osteoarthritis. Effects of chondroitin sulfate and glucosamine

Our objectives were to characterize the urinary excretion of glycosaminoglycans (GAGs) in horse osteoarthritis, and to investigate the effects of chondroitin sulfate (CS) and glucosamine (GlcN) upon the disease. Urinary GAGs were measured in 47 athletic horses, 20 healthy and 27 with osteoarthritis. The effects of CS and GlcN were investigated in mild osteoarthritis. In comparison to normal, urinary GAGs were increased in osteoarthritis, including mild osteoarthritis affecting only one joint. Treatment with CS + GlcN led to a long lasting increase in the urinary CS and keratan sulfate (KS), and significant improvement in flexion test of tarsocrural and metacarpophalangeal joints was observed. In conclusion, urinary CS and KS seems to reflect the turnover rates of cartilage matrix proteoglycans, and the measurement of these compounds could provide objective means of evaluating and monitoring joint diseases. (C) 2011 Elsevier Ltd. All rights reserved.

Synovial fluid chondroitin sulphate indicates abnormal joint metabolism in asymptomatic osteochondritic horses

Reasons for performing study: Alternative methods to evaluate the joint condition in asymptomatic osteochondrosis dissecans (OCD) and other joint diseases may be useful. Objectives: To investigate possible changes in synovial fluid composition that may lead to joint conditions in asymptomatic OCD, in mature horses. Methods: Animals aged >2 years, of different breeds, with OCD in the intermediate ridge of distal tibia, symptomatic or not, were studied. Synovial fluid samples (10 healthy; 11 asymptomatic OCD; 25 symptomatic OCD) were collected by arthroscopy from 29 horses. Glycosaminoglycans (GAGs) were analysed by a combination of agarose gel electrophoresis and enzymatic degradation with specific GAG lyases. The viscosity, white blood cell (WBC) count, protein concentration and hyaluronic acid (HA) molecular weight were also determined. Results: The method used here to analyse synovial fluid GAGs is reliable, reproducible and specific. The main synovial fluid GAGs are HA and chondroitin sulphate (CS), 93% and 7% respectively in normal horses. In symptomatic OCD, the concentrations of both increased (expressed as GAG/urea ratios), but CS increased more. The CS increased also in asymptomatic OCD. An inflammatory reaction was suggested by the increased WBC counts in OCD. The molecular weight of the synovial fluid HA was reduced in OCD, explaining the lower viscosity observed. Conclusions: The increased CS in synovial fluid of OCD joints in mature horses suggests that the synovial fluid CS and the WBC count are good markers of the joint conditions, allowing the identification of pathological phase in joint diseases. Potential relevance: The analysis of synovial fluid GAGs shows that cartilage damage occurs even in asymptomatic OCD, implying that arthroscopic removal of osteochondral fragments should be performed even in asymptomatic OCD.

Cerebral White Matter Oxidation and Nitrosylation in Young Rodents With Kaolin-Induced Hydrocephalus

Hydrocephalus is associated with reduced blood flow in periventricular white matter. To investigate hypoxic and oxidative damage in the brains of rats with hydrocephalus, kaolin was injected into the cisterna magna of newborn 7- and 21-day-old Sprague-Dawley rats, and ventricle size was assessed by magnetic resonance imaging at 7, 21, and 42 days of age. In-situ evidence of hypoxia in periventricular capillaries and glial cells was shown by pimonidazole hydrochloride binding. Biochemical assay of thiobarbituric acid reaction and immunohistochemical detection of malondialdehyde and 4-hydroxy-2-nonenal indicated the presence of lipid peroxidation in white matter. Biochemical assay of nitrite indicated increased nitric oxide production. Nitrotyrosine immunohistochemistry showed nitrosylated proteins in white matter reactive microglia and astrocytes. Activities of the antioxidant enzymes catalase and glutathione peroxidase were not increased, and altered hypoxia-inducible factor 1 alpha was not detected by quantitative reverse transcription-polymerase chain reaction. Cerebral vascular endothelial growth factor expression determined by quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay was not changed, but vascular endothelial growth factor immunoreactivity was increased in reactive astrocytes of hydrocephalic white matter. To determine if nitric oxide synthase is involved in the pathogenesis, we induced hydrocephalus in 7-day-old wild-type and neuronal nitric oxide synthase-deficient mice. At 7 days, the wild-type and mutant mice exhibited equally severe ventriculomegaly and no behavioral differences, although increased glial fibrillary acidic protein was less in the mutant mice. We conclude that hypoxia, via peroxidation and nitrosylation, contributes to brain changes in young rodents with hydrocephalus and that compensatory mechanisms are negligible.

High Viscosity of Imidazolium Ionic Liquids with the Hydrogen Sulfate Anion: A Raman Spectroscopy Study

Ionic liquids based on 1-alkyl-3-methylimidazolium cations and the hydrogen sulfate (or bisulfate) anion, HSO4-, are much more viscous than ionic liquids with alkyl sulfates, RSO4-. The structural origin of the high viscosity of HSO4- ionic liquids is unraveled from detailed comparison of the anion Raman bands in 1-ethyl-3-methylimidazolium hydrogen sulfate and 1-butyl-3-methylimidazolium hydrogen sulfate with available data for simple HSO(4)(-) salts in crystalline phase, molten phase, and aqueous solution. Two Raman bands at 1046 and 1010 cm(-1) have been assigned as symmetric stretching modes nu(s)(S = O) of HSO4-, the latter being characteristic of chains of hydrogen-bonded anions. The intensity of this component increases in the supercooled liquid phase. For comparison purposes, Raman spectra of 1-ethyl-3-methylimidazolium ethyl sulfate and 1-butyl-3-methylimidazolium methyl sulfate have been also obtained. There is no indication of difference in the strength of hydrogen bond interactions of imidazolium cations with HSO4- or RSO4- anions. Raman spectra at high pressures, up to 2.6 GPa, are also discussed. Raman spectroscopy provides evidence that hydrogen-bonded anions resulting in anion-anion interaction is the reason for the high viscosity of imidazolium ionic liquids with HSO4-. If the ionic liquid is exposed to moisture, these structures are disrupted upon absorption of water from the atmosphere.

Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.

Glycosaminoglycans modulate C6 glioma cell adhesion to extracellular matrix components and alter cell proliferation and cell migration

Abstract Background Adhesion to extracellular matrix (ECM) components has been implicated in the proliferative and invasive properties of tumor cells. We investigated the ability of C6 glioma cells to attach to ECM components in vitro and described the regulatory role of glycosaminoglycans (GAGs) on their adhesion to the substrate, proliferation and migration. Results ECM proteins (type IV collagen, laminin and fibronectin) stimulate rat C6 glioma cell line adhesion in vitro, in a dose-dependent manner. The higher adhesion values were achieved with type IV collagen. Exogenous heparin or chondroitin sulfate impaired, in a dose-dependent manner the attachment of C6 glioma cell line to laminin and fibronectin, but not to type IV collagen. Dextran sulfate did not affect C6 adhesion to any ECM protein analyzed, indicating a specific role of GAGs in mediating glioma adhesion to laminin and fibronectin. GAGs and dextran sulfate did not induce C6 glioma detachment from any tested substrate suggesting specific effect in the initial step of cell adhesion. Furthermore, heparin and chondroitin sulfate impaired C6 cells proliferation on fibronectin, but not on type IV collagen or laminin. In contrast, both GAGs stimulate the glioma migration on laminin without effect on type IV collagen or fibronectin. Conclusion The results suggest that GAGs and proteoglycans regulate glioma cell adhesion to ECM proteins in specific manner leading to cell proliferation or cell migration, according to the ECM composition, thus modulating tumor cell properties.

A clinical comparison between DisCoVisc and 2% hydroxypropylmethylcellulose in phacoemulsification: a fellow eye study

OBJECTIVE: This study sought to compare the effects and outcomes of two ophthalmic viscosurgical devices, 1.6% hyaluronic acid/4.0% chondroitin sulfate and 2.0% hydroxypropylmethylcellulose, during phacoemulsification. METHODS: This prospective, randomized clinical trial comprised 78 eyes (39 patients) that received phacoemulsification performed by the same surgeon using a standardized technique. Patients were randomly assigned to receive either 1.6% hyaluronic acid/4.0% chondroitin sulfate or 2.0% hydroxypropylmethylcellulose on the first eye. The other eye was treated later and received the other viscoelastic agent. Preoperative and postoperative examinations (5, 24 and 48 hours; 7 and 14 days; 3 and 6 months) included measurements of the total volume of the ophthalmic viscosurgical device, ultrasound and washout times to completely remove the ophthalmic viscosurgical device, intraocular pressure, central corneal thickness and best-corrected visual acuity. The corneal endothelial cell count was measured at baseline and at six months postoperatively. ClinicalTrials.gov: NCT01387620. RESULTS: There were no statistically significant differences between groups in terms of cataract density or ultrasound time. However, it took longer to remove 2.0% hydroxypropylmethylcellulose than 1.6% hyaluronic acid/ 4.0% chondroitin sulfate, and the amount of viscoelastic material used was greater in the 2.0% hydroxypropylmethylcellulose group. In addition, the best-corrected visual acuity was significantly better in the hyaluronic acid/ chondroitin sulfate group, but this preferable outcome was only observed at 24 hours after the operation. There were no statistically significant differences between the two ophthalmic viscosurgical devices regarding the central corneal thickness or intraocular pressure measurements at any point in time. The corneal endothelial cell count was significantly higher in the hyaluronic acid/chondroitin sulfate group. CONCLUSION: The ophthalmic viscosurgical device consisting of 1.6% hyaluronic acid/4.0% chondroitin sulfate was more efficient during phacoemulsification and was easier to remove after IOL implantation than 2.0% hydroxypropylmethylcellulose. In addition, the corneal endothelial cell count was significantly higher following the use of hyaluronic acid/chondroitin sulfate than with hydroxypropylmethylcellulose, which promoted an improved level of corneal endothelium protection.

MyD88 signaling is directly involved in the development of murine placental Malaria

Malaria is a widespread infectious disease caused by the parasite Plasmodium. During pregnancy, malaria infection leads to a range of complications that can affect both the mother and fetus, including stillbirth, infant mortality, and low birth weight. In this study, we utilized a mouse model of placental malaria (PM) infection to determine the importance of the protein MyD88 in the host immune response to Plasmodium during pregnancy. Initially, we demonstrated that Plasmodium berghei NK65GFP adhered to placental tissue via chondroitin sulfate A and induced PM in mice with a C57BL/6 genetic background. To evaluate the involvement of MyD88 in the pathology of PM, we performed a histopathological analysis of placentas obtained from MyD88(-/-) and wild-type (WT) mice following infection on the 19th gestational day. Our data demonstrated that the detrimental placental alterations observed in the infected mice were correlated with the expression of MyD88. Moreover, in the absence of this protein, production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) was significantly reduced in the infected mice. More importantly, in contrast to fetuses from infected WT mice, which exhibited a reduction in body weight, the fetuses from infected MyD88(-/-) mice did not display significant weight loss compared to their noninfected littermates. In addition, we observed a decrement of maternal care associated with malaria infection, which was attenuated in the MyD88-deficient mice. Collectively, the results of this study illustrate the pivotal importance of the MyD88 signaling pathway in the pathogenesis of placental malaria, thus presenting new possibilities for targeting MyD88 in therapeutic interventions.