Measurements of reducing end groups on bovine vitreous-humour hyaluronic acid by reaction with [14C]cyanide.
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ABSTRACT:
SUBMITTER: Swann DA
PROVIDER: S-EPMC1153879 | biostudies-other | 1982 Dec
REPOSITORIES: biostudies-other
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Project description:The insoluble protein fraction was prepared from the central and posterior peripheral fraction of bovine vitreous humour. The collagen present in this fraction was solubilized by pepsin and fractionated by gel chromatography. Analysis of the solubilized collagen fractions showed that the alpha-chain component had an amino acid composition and yielded a series of CNBr-cleavage peptides that showed it was very similar to type II collagen obtained from articular cartilage. Bovine vitreous-humour collagen alpha-chains differed, however, from those of cartilage collagen in that they had a lower alanine content and differed in their susceptibility to cleavage by CNBr. Satisfactory cleavage was obtained after two CNBr treatments involving reduction and alkylation. In addition, significant quantities of other peptides constituents were present in the vitreous-humour collagen fractions, and the galactose and glucose content of the alpha-chain fraction was more than double that of the same fraction obtained from articular cartilage. Although the origin of the additional peptide constituents in the vitreous-humour collagen preparations is not known, the results obtained indicate that they are probably not derived from a distinct type of alpha-chain component but may be terminal peptides covalently linked to the alpha 1 type-II helical portions of the collagen. The differences in the chemical composition of the vitreous-humour collagen indicate that vitreous-humour fibres are composed of a special type-II collagen.
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Project description:The collagens of bovine vitreous-humour and nasal-septum cartilage have been extracted, fractionated and compared. Both tissues show the same heterogeneity of collagen types, consisting of type II, 1 alpha, 2 alpha, 3 alpha and C-PS collagens. The type II collagen of the vitreous humour was significantly more hydroxylated both in the lysine and proline residues than was that of cartilage. C-PS1 collagen, together with higher-Mr forms were present in the vitreous humour, but the higher-Mr forms were not seen in cartilage. Both C-PS1 and C-PS2 were present in vitreous humour and cartilage, but vitreous humour contained three times more of these collagens than did cartilage. Despite the difference in amount, the molar ratio C-PS1/C-PS2 was approx. 1 in both tissues, suggesting that they are components of a larger molecule. The 1 alpha, 2 alpha, 3 alpha collagens were present in the same concentration in both tissues. These three chains co-precipitated on dialysis against phosphate-buffered saline, pH 7.2, in a manner analogous to type V collagen.
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Project description:Native plant cellulose has an intrinsic supramolecular structure. Consequently, it can be isolated as nanocellulose species, which can be utilized as building blocks for renewable nanomaterials. The structure of cellulose also permits its end-wise modification, i.e., chemical reactions exclusively on one end of a cellulose chain or a nanocellulose particle. The premises for end-wise modification have been known for decades. Nevertheless, different approaches for the reactions have emerged only recently, because of formidable synthetic and analytical challenges associated with the issue, including the adverse reactivity of the cellulose reducing end and the low abundance of newly introduced functionalities. This Review gives a full account of the scientific underpinnings and challenges related to end-wise modification of cellulose nanocrystals. Furthermore, we present how the chemical modification of cellulose nanocrystal ends may be applied to directed assembly, resulting in numerous possibilities for the construction of new materials, such as responsive liquid crystal templates and composites with tailored interactions.
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Project description:Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 50. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and responsible for the majority of central vision impairment. Using non-biased microRNA arrays and individual TaqMan qPCRs, we profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and miR-152 in the vitreous humour which was reproducible in plasma. Moreover, miR-146a/miR-106b ratios discriminated patients with NV AMD with an area under the Receiver Operating Characteristic curve (ROC AUC) of 0,977 in vitreous humour and 0,915 in plasma suggesting potential for a blood-based diagnostic. Furthermore, using the AMD Gene Consortium (AGC) we mapped a NV AMD-associated SNP (rs1063320) in a binding site for miR-152-3p in the HLA-G gene. The relationship between our detected miRNAs and NV AMD related genes was also investigated using gene sets derived from the Ingenuity Pathway Analysis (IPA). To our knowledge, our study is the first to correlate vitreal and plasma miRNA signatures with NV AMD, highlighting potential future worth as biomarkers and providing insight on NV AMD pathogenesis.
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Project description:Collagen from pig vitreous humour was fractionated into a soluble and an insoluble fraction by centrifugation. Most of the collagen II in the soluble fraction was present as pN-collagen II (procollagen II without the C-terminal propeptide), besides smaller quantities of procollagen II, collagen II and two as yet unidentified alpha-chains of collagen II. Other collagen types may be present only in trace amounts. Collagen II of the insoluble fraction, which is mostly deposited in fibrillar aggregates, consists of both pN-collagen II and collagen II. To determine the possible role of collagen II precursors in the formation of the extracellular matrix of the vitreous humour these collagen molecules were purified and in vitro fibrillogenesis was used to demonstrate that pN-collagen II could form fibrils in mixtures with collagen II. These fibrils have a reduced mass per unit length depending on the content of pN-collagen in the mixture. Cross-sections of the newly formed fibrillar aggregates revealed a flattened shape. The incomplete processing of the precursors of collagen II may be part of regulatory mechanisms possibly controlling the formation of a translucent scaffold as is required in the vitreous humour.
| S-EPMC1136601 | biostudies-other
Project description:Multiple ophthalmic pathologies, such as retinal detachment and diabetic retinopathy, require the removal and replacement of the vitreous humor. Clinical tamponades such as silicone oil and fluorinated gases are utilized but limited due to complications and toxicity. Therefore, there is a need for biocompatible, stable, vitreous humor substitutes. In this study, enzymatically crosslinked silk-hyaluronic acid (HA) hydrogels formed using horseradish peroxidase and H2O2 were characterized for use as vitreous humor substitutes. The composite network structure was characterized with dynamic light scattering. In addition, the rheological, optical, and swelling properties of hydrogels with varying silk to HA ratios and crosslinking densities controlled via H2O2 were determined over time. Hydrogels had refractive indexes of 1.336 and were clear with 75-91% light transmission. Hydrogel shear storage modulus ranged between ~6 and 240 Pa where increased H2O2 increased the modulus. After 1 month of aging, there were no changes in modulus for hydrogels with lower silk ratios, while those with higher silk ratios exhibited a significant increase in modulus. Decreasing H2O2 concentration in the reactions led to increased hydrogel volume during swelling, with higher silk ratios returning to their original size after 15 days. Dynamic light scattering results show three diffusive modes, revealing the possible structures of the hydrogel composite and are consistent with the mechanical properties and swelling results. The normalized intraocular pressure of ex vivo porcine eyes after injecting hydrogels were comparable with those treated with silicone oil showing the potential clinical utility of the hydrogels as vitreous substitutes. The versatility of the silk-HA hydrogel system, the tunable swelling properties, and the stability of hydrogels with lower silk ratios show the benefit of utilizing silk-HA hydrogels as vitreous substitutes.
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Project description:To reveal the expression profiles of transfer RNA-derived small RNA (tsRNA)s and microRNA (miRNA)s in the vitreous humour of proliferative diabetic retinopathy (PDR).
2022-06-10 | GSE199852 | GEO
Project description:PurposeTo study alginate- and hyaluronic acid-based hydrogels in vitro as vitreous substitutes.MethodsBiopolymeric hydrogels based on high-molecular alginate (0.5% and 1.0%) and hyaluronic acid (1.0% and Healaflow) were compared with extracted human vitreous bodies and silicone oil (SIL-5000) regarding their optical properties (refractive index, transmission) and viscoelastic characteristics (storage modulus G', loss modulus G″). The cytotoxic (metabolic activity, apoptosis) and antiproliferative profiles were determined using cultured human fibroblasts, ARPE-19, and photoreceptor cells. The hydrogel systems were applied to human fetal retinal pigment epithelial cells cultured for two months until maximum transepithelial electrical resistance (TEER) to investigate the effect of the gel matrices on tight junctions using TEER measurements and immunostainings against the tight junction protein ZO-1.ResultsTested alginate- and hyaluronic acid-based hydrogels resembled the natural refractive index of human vitreous bodies (1.3356-1.3360) in contrast to SIL-5000 (1.4034) and showed high optical transparency (>90%) within the visible light region. The biopolymeric hydrogels exhibited viscoelastic properties similar to juvenile vitreous bodies with G'>G″ adjustable via different gelation times, contrary to SIL-5000 (G'<G″). The metabolic activity, apoptosis and tight junctions of all tested ocular cells were unaffected by the alginate- and hyaluronic acid-based vitreous substitutes.ConclusionsThe present in vitro study demonstrates good optical, viscoelastic, and biocompatible properties of alginate- and hyaluronic acid-based hydrogels required for their use as vitreous substitutes.Translational relevanceBiopolymer-based hydrogels represent a promising vitreous replacement strategy to treat vitreoretinal diseases.
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Project description:In order to elucidate involvement of cyclic AMP and intracellular Ca2+,[Ca2+]i, in the modulation of aqueous humour formation (AHF), we studied the effects of terbutaline, forskolin and 8-Br-cAMP in the isolated bovine eye. We also studied the interaction of cAMP on calcium signaling in cultured ciliary epithelial (CE) cells. Drug effects on AHF were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the fura-2 fluorescence ratio technique. Terbutaline (100 nmol-100 M), forskolin (30 nM-100 M) or 8-Br-cAMP (100 nM- 10 μM), administered in the arterial perfusate produced significant reductions in AHF. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT-5720). ATP (100 M) caused a rapid, transient (peak) increase in [Ca2+]i followed by a sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8-Br-cAMP significantly reduced the peak calcium response to ATP. The sustained plateau phase of the response, on the other hand, was augmented by each of the agents. KT-5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. These data indicate: (a) that AHF in the bovine eye can be manipulated through cyclic AMP, operating via protein kinase A, (b) that protein kinase A can affect [Ca2+]i homeostasis, (c) that calcium release from the intracellular store, not the entry, affects AHF, and (d) that interaction of [Ca2+]i with cAMP plays a role in modulating AH secretion.
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Project description:Intravitreal injection of biodegradable nanoparticles (NP) holds promise for gene therapy and drug delivery to the back of the eye. In some cases, including gene therapy, NP need to diffuse rapidly from the site of injection in order to reach targeted cell types in the back of the eye, whereas in other cases it may be preferred for the particles to remain at the injection site and slowly release drugs that may then diffuse to the site of action. We studied the movements of polystyrene (PS) NP of various sizes and surface chemistries in fresh bovine vitreous. PS NP as large as 510nm rapidly penetrated the vitreous gel when coated with polyethylene glycol (PEG), whereas the movements of NP 1190nm in diameter or larger were highly restricted regardless of surface chemistry owing to steric obstruction. PS NP coated with primary amine groups (NH2) possessed positively charged surfaces at the pH of bovine vitreous (pH=7.2), and were immobilized within the vitreous gel. In comparison, PS NP coated with COOH (possessing negatively charged surfaces) in the size range of 100-200nm and at particle concentrations below 0.0025% (w/v) readily diffused through the vitreous meshwork; at higher concentrations (~0.1% w/v), these nanoparticles aggregated within vitreous. Based on the mobility of different sized PEGylated PS NP (PS-PEG), we estimated the average mesh size of fresh bovine vitreous to be ~550±50nm. The bovine vitreous behaved as an impermeable elastic barrier to objects sized 1190nm and larger, but as a highly permeable viscoelastic liquid to non-adhesive objects smaller than 510nm in diameter. Guided by these studies, we next sought to examine the transport of drug- and DNA-loaded nanoparticles in bovine vitreous. Biodegradable NP with a diameter of 227nm, composed of a poly(lactic-co-glycolic acid) (PLGA)-based core coated with poly(vinyl alcohol) rapidly penetrated vitreous. Rod-shaped, highly-compacted CK30PEG10k/DNA with PEG coating (neutral surface charge; hydrodynamic diameter ~60nm) also diffused rapidly within vitreous. These findings will help guide the development of nanoparticle-based therapeutics for the treatment of vision-threatening ocular diseases.
| S-EPMC3693951 | biostudies-literature