Effects of Thermal Modifications on the Rheological Characteristics and Protein Quality of Three Species of Fish of Varying Collagen Content

School of Industrial Fisheries, Cochin University of Science and Technology

Influence of collagen content on the texture andstorage quality of fish mince basedproducts

Objectives of the present study are to find out the proximate composition of 20 commercially important tropical fish species on the west coast of India. To determine the collagen content in these commercially important fish species and fractionation of collagen into acid soluble collagen (ASC) and hot water soluble (insoluble) collagen (ISC). To classify fishes according to its collagen content and To study the different storage characteristics in the mince based product—surimi, from different species of fishes. The researcher tries to find out a suitable collagen source to incorporate in surimi. and studies the different storage qualities in the mince based product, surimi at different levels of collagen in different species of fishes. The optimum collagen level to get desirable texture and storage quality for mince based product. The researcher aims to develop some products from surimi with desirable level of collagen. And compare the products prepared from surimi of lesser collagen content fish containing desirable level of collagen with surimi prepared with high collagen content fish without collagen. This study gains in importance as there is littleinformation on the collagen content of different species of fishes in India. So far no attempt was made to classify fishes according to its collagen content.

Study of chemical properties and evaluation of collagen in mantle, epidermal connective tissue and tentacle of Indian Squid, Loligo duvauceli Orbigny

The chemical composition and evaluation of Indian squid (Loligo duvauceli) mantle, epidermal connective tissue and tentacle is investigated in this current study. It is observed that squid mantle contains 22.2% total protein; 63.5% of the total protein is myofibrillar protein. The unique property of squid myofibrillar protein is its water solubility. Squid mantle contains 12.0% total collagen. Epidermal connective tissue has highest amounts of total collagen (17.8%). SDS-PAGE of total collagen identified high molecular weight α-, β- and γ- sub-chains. Amino acid profile analysis indicates that mantle and tentacle contain essential amino acids. Arginine forms a major portion of mantle collagen (272.5 g/100 g N). Isoleucine, glutamic acid and lysine are other amino acids that are found in significantly high amounts in the mantle. Sulphur containing cystine is deficit in mantle collagen. Papain digest of mantle and epidermal connective tissue is rich in uronic acid, while papain digest, collagenase digest and urea digest of epidermal connective tissue has significant amounts of sialic acid (25.2, 33.2 and 99.8 μmol /100 g, respectively). PAS staining of papain digest, collagenase digest and urea digest also identify the association of hexoses with low molecular weight collagen fragments. Histochemical sectioning also emphasized the localized distribution of collagen in epidermal and dermal region and very sparse fibres traverse the myotome bundles

Biochemical Characterization and Bio-evaluation of Collagen and Collagen Peptides Extracted and Purified from Fish Skin: In vitro and In vivo Studies on Antiarthritic and Wound Healing Properties

In fish processing plants, there is huge amount of skin that is left as the waste. When this skin is taken and processed into fish collagen, it will save large amount of money that is used for extraction of collagen from other animal s.Fish collagen can be used as an alternative to replace mammalian collagen, especially collagen extracted from bovine, when we consider the outbreak of bovine spongiform encephalopathy (BSE), transmissible spongiform encephalopathy (TSE) and the foot - and-mouth disease (FMD) issues. BSE and TSE are progressive neurological disorders affecting cattles caused by proteinacious infectious particles called prions.The study aims in producing collagen that has been extracted from fish skin to replace other animal collagen so as to overcome the problem of other animal collagen issues. Also the study utilized the abandoned fish waste produced by fish processing industry since bone, skin, fin and scales of fish can be a useful source of collagen.

Regenerative capabilities of the rat seventh nerve using suture and collagen splint techniques: a comparative study

This paper discusses the effectiveness of repair procedures on the rat facial nerve using the current clinical surgical procedure of suturing nerve ends and a proposed technique utilizing splints made of collagen.

Ex Vivo Construction of an Artificial Ocular Surface by Combination of Corneal Limbal Epithelial Cells and a Compressed Collagen Scaffold Containing Keratocytes

We have investigated the use of a laminin coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane. Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibres within the compressed gel were dense, homogeneous and similar in structure to those within denuded amniotic membrane. Limbal epithelial cells were successfully expanded upon the compressed collagen resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology and cell stratification. Similar protein expression profiles for cytokeratin 3 and cytokeratin 14 and no significant difference in cytokeratin 12 mRNA expression levels by real time PCR were also observed. This study provides the first line of evidence that a laminin coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification and differentiation to a degree that is comparable to the leading conventional scaffold, denuded amniotic membrane.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study

Epidemiological data suggest that those who consume a diet rich in quercetin-containing foods may have a reduced risk of CVD. Furthermore, in vitro and ex vivo studies have observed the inhibition of collagen-induced platelet activation by quercetin. The aim of the present study was to investigate the possible inhibitory effects of quercetin ingestion from a dietary source on collagen-stimulated platelet aggregation and signalling. A double-blind randomised cross-over pilot study was undertaken. Subjects ingested a soup containing either a high or a low amount of quercetin. Plasma quercetin concentrations and platelet aggregation and signalling were assessed after soup ingestion. The high-quercetin soup contained 69 mg total quercetin compared with the low-quercetin soup containing 5 mg total quercetin. Plasma quercetin concentrations were significantly higher after high-quercetin soup ingestion than after low-quercetin soup ingestion and peaked at 2.59 (SEM 0.42) mu mol/l. Collagen-stimulated (0.5 mu g/ml) platelet aggregation was inhibited after ingestion of the high-quercetin soup in a time-dependent manner. Collagen-stimulated tyrosine phosphorylation of a key component of the collagen-signalling pathway via glycoprotein VI, Syk, was significantly inhibited by ingestion of the high-quercetin soup. The inhibition of Syk tyrosine phosphorylation was correlated with the area under the curve for the high-quercetin plasma profile. In conclusion, the ingestion of quercetin from a dietary source of onion soup could inhibit some aspects of collagen-stimulated platelet aggregation and signalling ex vivo. This further substantiates the epidemiological data suggesting that those who preferentially consume high amounts of quercetin-containing foods have a reduced risk of thrombosis and potential CVD risk.

Platelet adhesion to collagen and collagen-related peptide under flow. Roles of the alpha(2)beta(1) integrin, GPVI, and Src tyrosine kinases

Objective - Platelet stimulation by collagen and collagen-related peptides (CRPs) is associated with activation of protein tyrosine kinases. In the present study, we investigated the role of Src family tyrosine kinases in the initial adhesion events of human platelets to collagen and cross-linked CRP. Methods and Results - Under arterial flow conditions, a glycoprotein VI - specific substrate, cross-linked CRP, caused rapid (<2 second) platelet retention and protein tyrosine phosphorylation that were markedly decreased by the Src family kinase inhibitor pyrozolopyrimidine (PP2) or by aggregation inhibitor GRGDSP. CRP-induced platelet retention was transient, and 90% of single platelets or aggregates detached within seconds. PP2, although having no effect on RGD peptide-binding to CRP, completely blocked aggregation and tyrosine phosphorylation of Syk and phospholipase Cγ2 (PLCγ2). In contrast, PP2 weakly (<30%) suppressed firm adhesion to collagen mediated primarily by the alpha(2)beta(1) integrin. Although PP2 prevented activation of Syk and PLCgamma2 in collagen-adherent platelets, tyrosine phosphorylation of several unidentified protein bands persisted, as did autophosphorylation of pp125(FAK). Conclusions - These findings indicate that activation of Src-tyrosine kinases Syk and PLCgamma2 is not required for the initial stable attachment of human platelets to collagen and for FAK autophosphorylation. However, Src-tyrosine kinases are critical for glycoprotein VI - mediated signaling leading to platelet aggregation.

Peroxynitrite-modified collagen-II induces p38/ERK and NF-kappa B-dependent synthesis of prostaglandin E-2 and nitric oxide in chondrogenically differentiated mesenchyrnal progenitor cells

Objective: Peroxynitrite (ONOO-) is formed in the inflamed and degenerating human joint. Peroxynitrite-modified collagen-II (PMC-II) was recently discovered in the serum of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Therefore we investigated the cellular effects of PMC-II on human mesenchymal progenitor cells (MPCs) as a model of cartilage and cartilage repair cells in the inflamed and degenerating joint. Design: MPCs were isolated from the trabecular bone of patients undergoing reconstructive surgery and were differentiated into a chondrogenic lineage. Cells were exposed to PMC-II and levels of the proinflammatory mediators nitric oxide (NO) and prostaglandin E-2 (PGE(2)) measured. Levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), phosphorylated mitogen activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kappa B) activation were measured by enzyme linked immunosorbent assay (ELISA) together with specific MAPK and NF-kappa B inhibitors. Results: PMC-II induced NO and PGE(2) synthesis through upregulation of iNOS and COX-2 proteins. PMC-II also lead to the phosphorylation of MAPKs, extracellularly regulated kinase 1/2 (ERK1/2) and p38 [but not c-Jun NH2-terminal kinase (JNK1/2)] and the activation of proinflammatory transcription factor NF-kappa B. Inhibitors of p38, ERK1/2 and NF-kappa B prevented PMC-II induced NO and PGE(2) synthesis, NOS and COX-2 protein expression and NF-kappa B activation. Conclusion: iNOS, COX-2, NF-KB and MAPK are known to be activated in the joints of patients with OA and RA. PMC-II induced iNOS and COX-2 synthesis through p38, ERK1/2 and NF-KB dependent pathways suggesting a previously unidentified pathway for the synthesis of the proinflammatory mediators, NO and PGE(2), further suggesting that inhibitors of these pathways may be therapeutic in the inflamed and degenerating human joint. (c) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans

Background: Quercetin, a flavonoid present in the human diet, which is found in high levels in onions, apples, tea and wine, has been shown previously to inhibit platelet aggregation and signaling in vitro. Consequently, it has been proposed that quercetin may contribute to the protective effects against cardiovascular disease of a diet rich in fruit and vegetables. Objectives: A pilot human dietary intervention study was designed to investigate the relationship between the ingestion of dietary quercetin and platelet function. Methods: Human subjects ingested either 150 mg or 300 mg quercetin-4'-O-beta-D-glucoside Supplement to determine the systemic availability of quercetin. Platelets were isolated from subjects to analyse collagen-stimulated cell signaling and aggregation. Results: Plasma quercetin concentrations peaked at 4.66 mum (+/-0.77) and 9.72mum (+/-1.38) 30min after ingestion of 150-mg and 300-mg doses of quercefin-4'-O-beta-D-glucoside, respectively, demonstrating that quercetin was bioavailable, with plasma concentrations attained in the range known to affect platelet function in vitro. Platelet aggregation was inhibited 30 and 120 min after ingestion of both doses of quercetin-4'-O-beta-D-glucoside. Correspondingly, collagen-stimulated tyrosine phosphorylation of total platelet proteins was inhibited. This was accorripanied by reduced tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2, components of the platelet glycoprotein VI collagen receptor signaling pathway. Conclusions: This study provides new evidence of the relatively high systemic availability of quercetin in the form of quercetin-4'-O-beta-D-glucoside by supplementation, and implicates quercetin as a dietary inhibitor of platelet cell signaling and thrombus formation.

Quercetin inhibits collagen-stimulated platelet activation through inhibition of multiple components of the glycoprotein VI signaling pathway

Background: The regulation of platelet function by pharmacological agents that modulate platelet signaling haspharmacolo proven a successful approach to the prevention of thrombosis. A variety of molecules present in the diet have been shown to inhibit platelet activation, including the antioxidant quercetin. Objectives: In this report we investigate the molecular mechanisms through which quercetin inhibits collagen-stimulated platelet aggregation. Methods: The effect of quercetin on platelet aggregation, intracellular calcium release, whole cell tyrosine phosphorylation and intracellular signaling events including tyrosine phosphorylation and kinase activity of proteins involved in the collagen-stimulated glycoprotein (GP) signaling pathway were investigated. Results: We report that quercetin inhibits collagen-stimulated whole cell protein tyrosine phosphorylation and intracellular mobilization of calcium, in a concentration-dependent manner. Quercetin was also found to inhibit various events in signaling generated by the collagen receptor GPVI. This includes collagen-stimulated tyrosine phosphorylation of the Fc receptor gamma-chain, Syk, LAT and phospholipase Cgamma2. Inhibition of phosphorylation of the Fc receptor gamma-chain suggests that quercetin inhibits early signaling events following stimulation of platelets with collagen. The activity of the kinases that phosphorylate the Fc receptor gamma-chain, Fyn and Lyn, as well as the tyrosine kinase Syk and phosphoinositide 3-kinase was also inhibited by quercetin in a concentration-dependent manner, both in whole cells and in isolation. Conclusions: The present results provide a molecular basis for the inhibition by quercetin of collagen-stimulated platelet activation, through inhibition of multiple components of the GPVI signaling pathway, and may begin to explain the proposed health benefits of high quercetin intake.

A structural basis for the inhibition of collagen-stimulated platelet function by quercetin and structurally related flavonoids

Background and purpose: Molecular mechanisms underlying the links between dietary intake of flavonoids and reduced cardiovascular disease risk are only partially understood. Key events in the pathogenesis of cardiovascular disease, particularly thrombosis, are inhibited by these polyphenolic compounds via mechanisms such as inhibition of platelet activation and associated signal transduction, attenuation of generation of reactive oxygen species, enhancement of nitric oxide production and binding to thromboxane A2 receptors. In vivo, effects of flavonoids are mediated by their metabolites, but the effects and modes of action of these compounds are not well-characterized. A good understanding of flavonoid structure–activity relationships with regard to platelet function is also lacking. Experimental approach: Inhibitory potencies of structurally distinct flavonoids (quercetin, apigenin and catechin) and plasma metabolites (tamarixetin, quercetin-3′-sulphate and quercetin-3-glucuronide) for collagen-stimulated platelet aggregation and 5-hydroxytryptamine secretion were measured in human platelets. Tyrosine phosphorylation of total protein, Syk and PLCγ2 (immunoprecipitation and Western blot analyses), and Fyn kinase activity were also measured in platelets. Internalization of flavonoids and metabolites in a megakaryocytic cell line (MEG-01 cells) was studied by fluorescence confocal microscopy. Key results: The inhibitory mechanisms of these compounds included blocking Fyn kinase activity and the tyrosine phosphorylation of Syk and PLCγ2 following internalization. Principal functional groups attributed to potent inhibition were a planar, C-4 carbonyl substituted and C-3 hydroxylated C ring in addition to a B ring catechol moiety. Conclusions and implications: The structure–activity relationship for flavonoids on platelet function presented here may be exploited to design selective inhibitors of cell signalling.

Fibrillar superstructure from extended nanotapes formed by a collagen-stimulating peptide

The nanostructure of a peptide amphiphile in commercial use in anti-wrinkle creams is investigated. The peptide contains a matrikine, collagen-stimulating, pentapeptide sequence. Selfassembly into giant nanotapes is observed and the internal structure was found to comprise bilayers parallel to the flat tape surfaces.