Edible Halophytes with Functional Properties: In Vitro Protein Digestibility and Bioaccessibility and Intestinal Absorption of Minerals and Trace Elements from Australian Indigenous Halophytes.

Halophytes are considered emerging functional foods as they are high in protein, minerals, and trace elements, although studies investigating halophyte digestibility, bioaccessibility, and intestinal absorption are limited. Therefore, this study investigated the in vitro protein digestibility, bioaccessibility and intestinal absorption of minerals and trace elements in saltbush and samphire, two important Australian indigenous halophytes. The total amino acid contents of samphire and saltbush were 42.5 and 87.3 mg/g DW, and even though saltbush had a higher total protein content overall, the in vitro digestibility of samphire protein was higher than the saltbush protein. The in vitro bioaccessibility of Mg, Fe, and Zn was higher in freeze-dried halophyte powder compared to the halophyte test food, suggesting that the food matrix has a significant impact on mineral and trace element bioaccessibility. However, the samphire test food digesta had the highest intestinal Fe absorption rate, whereas the saltbush digesta exhibited the lowest (37.7 vs. 8.9 ng/mL ferritin). The present study provides crucial data about the digestive "fate" of halophyte protein, minerals, and trace elements and increases the understanding of these underutilized indigenous edible plants as future functional foods.

Post-prandial response in hepatopancreas and haemolymph of Penaeus monodon fed different diets. Omics insights into glycoconjugate metabolism, energy utilisation, chitin biosynthesis, immune function, and autophagy.

Raw materials or bioactive ingredients trigger mechanisms to assimilate nutrients and activate metabolic pathways that promote growth, immune function, or energy storage. Our understanding of these processes at a molecular level remains limited in aquaculture, especially in shrimp. Here, hepatopancreas proteomics and haemolymph metabolomics were used to investigate the post-prandial response of black tiger shrimps (Penaeus monodon) fed a conventional fishmeal diet (FM); a diet supplemented with the microbial biomass Novacq™ (NV); krill meal (KM); or, fasted (FS). Using FM as a control, a 2-fold change in abundance threshold was implemented to determine the significance of proteins and metabolites. NV fed shrimp showed preference for energy derived from carbohydrates indicated by a strong signature of glycoconjugate metabolism and activation of the amino- and nucleotide sugar metabolic pathway. KM activated the glyoxylate and dicarboxylate pathway that denoted shrimp preference for lipidic energy. KM also influenced energy generation by the TCA cycle inferred from higher abundance of the metabolites succinic semialdehyde, citric acid, isocitrate, alpha ketoglutarate and ATP and downregulation of the enzyme isocitrate dehydrogenase that catalyses oxidative decarboxylation of isocitrate. FS shrimp displayed down-regulation of oxidative phosphorylation and resorted to internal lipid reserves for energy homeostasis displaying a strong signature of autophagy. Pyrimidine metabolism was the preferred energy strategy in this group. Our study also provided evidence that during fasting or consumption of specific ingredients, shrimp share common pathways to meet their energy requirements, however, the intensity at which these pathways were impacted was diet dependent.

Nutritional and techno-functional properties of Australian Acacia seed flour: Effects of roasting on chemical composition, physicochemical properties, and in vitro digestibility and intestinal iron absorption.

Acacia seed (AS) is rich in protein and iron but contains protease inhibitors that can reduce protein digestibility (PD). The seeds are generally roasted prior to consumption, although no information on the PD of roasted AS is available. This study investigated the effect of roasting time (5, 7 and 9 min at 180 °C) on the chemical composition, physicochemical properties, and in vitro PD and intestinal iron absorption of three wild harvested Australian AS species, Acacia victoriae, A. coriacea and A. cowleana. Roasting A. victoriae and A. coriacea seeds for 7 min significantly increased PD in the seeds by 36 and 61 %, respectively. A 9-min roasting time was required to achieve 75 % reduction in trypsin inhibitor activity in A. coriacea seed, while a shorter roasting time (RT) was sufficient to achieve similar reduction rates in the other two Acacia species. Among the functional properties, water and oil absorption capacities were significantly enhanced as RT increased. The starch granules in 7- and 9-min roasted A. victoriae seed flour detached from the protein matrix while random coil increased in 7-min roasted A. victoriae and 9-min roasted A. coriacea and A. cowleana, thus, contributing to enhanced PD. Although the SDS-PAGE in 7- and 9-min roasted A. cowleana samples showed reductions in the intensity of bands for high molecular weight proteins, PD was not affected by RT. However, intestinal iron absorption was not significantly affected by roasting as compared to raw digesta samples. Compared to commercial roasted Acacia seed, the considerably shorter RT used in this study improved PD in the AS flour with less adverse effects on techno-functional properties.

Effects of incorporating processed Acacia seed as an emulsifying agent on the quality attributes of beef sausage.

In this study, partial or full replacement of 6% soy protein isolate (SPI) with 2, 4 and 6% roasted Acacia seed flour (ASRo) and Acacia seed protein concentrates (ASPC) in emulsified beef sausage were investigated. Emulsion stability and cooking loss were lower in samples formulated with ASPC at all levels and control samples compared to ASRo formulated samples. ASRo generated softer and less chewy sausages than ASPC. Cooked 2% ASPC sausages had similar L* and a* values as the control but with lower colour difference (ΔE) values that were similar to cooked 6% SPI sausages' values. An organized protein network structure was observed in the sausages formulated with ASPC at all levels and in the control samples. Therefore, ASPC, particularly at 2 and 4% inclusion, can be used as a functional ingredient to prepare emulsified beef sausages with good quality attributes.

Oral administration of dermatan sulphate reduces venous thrombus formation in vivo: potential use as a formulation for venous thromboembolism.

Dermatan sulphate (DS) is a sulphated polysaccharide that displays complexity in constituent sulphated disaccharides and interacts with proteins and signalling molecules to modulate numerous biological processes, including inhibition of the coagulation cascade and regulation of blood clotting and fibrinolysis. This study shows the antithrombotic and anticoagulant effects of DS prepared from bovine collagen waste liquor following oral and intravenous administrations in a deep vein thrombosis (DVT) rabbit model. In vitro, the prothrombin time, activated partial thromboplastin time, and thrombin citrated plasma clotting assays revealed that bovine DS had strong antithrombotic and anticoagulant effects comparable to low-molecular-weight heparin [Clexane® (enoxaparin sodium)]. In a DVT rabbit model, animals received intravenous and oral administrations of bovine DS and Clexane® providing further evidence that both agents had strong antithrombotic and anticoagulant effects by significantly reducing or preventing clot formation. Thromboelastography (TEG) assays revealed further that both bovine DS and Clexane® substantially prolonged the clotting time of recalcified citrated whole blood, but only bovine DS could retain clot strength suggesting that bovine DS had less effect on platelet-fibrin interactions. In conclusion, this is the first report that oral administration of DS from bovine collagen waste liquor reduces experimental venous thrombus formation warranting further research into bovine DS as an oral antithrombotic therapeutic.

In vitro Bioaccessibility and Intestinal Absorption of Selected Bioactive Compounds in Terminalia ferdinandiana.

Terminalia ferdinandiana (or Kakadu plum), a native Australian fruit with potential health benefits, contains bioactive compounds such as ellagic acid (EA), ascorbic acid (AA) and calcium, and antinutrients such as oxalic acid (OA). However, few is known about the biological fate of these compounds following ingestion; therefore, the aim of this study was to evaluate in vitro bioaccessibility and intestinal absorption of T. ferdinandiana compounds using the INFOGEST static digestion model and Caco-2-HT29-MTX-E12 intestinal absorption model. No significant changes (p > 0.05) were observed in total AA content throughout in vitro digestion, whereas bioaccessibility of EA, OA, and calcium increased significantly from 33, 72, and 67% in the gastric phase to 48, 98, and 90% in the intestinal phase, respectively. The intestinal absorption study revealed variable rates of movement across the cell barrier. Findings reveal novel and important insights for the prediction of in vivo bioavailability of selected T. ferdinandiana compounds.

Impact of polyphenol-rich extracts of Terminalia ferdinandiana fruits and seeds on viability of human intestinal and liver cells in vitro.

Terminalia ferdinandiana (Kakadu plum) is a native Australian fruit consumed by Indigenous Australians for centuries. Commercial interest in T. ferdinandiana has increased in recent years due to its high vitamin C content, however, food safety assessments are lacking. To explore the safety of extracts prepared from T. ferdinandiana using different solvents, in vitro cell viability of undifferentiated and differentiated Caco-2, HT29-MTX-E12, and HepG2 cells was measured using the CyQUANT® NF Cell Proliferation Assay. Changes to cell viability produced IC50 values between 3650 and 14400 µg/mL for all extracts and cell lines tested with HepG2 cells impacted the most by T. ferdinandiana extracts, followed by HT29-MTX-E12 cells, and undifferentiated and differentiated Caco-2 cells. Different solvents also produced extracts with variable effects on cell viability that were dependent on tissue source, however, extracts from seedcoats appeared to impact cell viability less than fruit extracts. The IC50 values for ellagic acid, an abundant phytochemical in T. ferdinandiana, varied from 1190 to 2390 µg/mL across different cells and were significantly lower than extract IC50 values. Findings from this study will help to inform future safety studies, select which solvents to use when preparing T. ferdinandiana extracts, and decide whether fruit flesh should be separated from seeds during extract preparation.

Interactions Between Phytochemicals and Minerals in Terminalia ferdinandiana and Implications for Mineral Bioavailability.

Oxalic and phytic acid are phytochemicals considered to be anti-nutritional factors as they are predominantly found as oxalates and phytates bound to minerals like calcium and potassium. Studies have associated excessive oxalate consumption with increased urinary excretion of oxalate (hyperoxaluria) and calcium oxalate kidney stone formation, and excessive phytate consumption with decreased bioaccessibility and bioavailability of certain minerals and reduced utilization of dietary protein. However, other studies suggest that dietary consumption of phytate may be beneficial and inhibit formation of calcium oxalate kidney stones. In light of these conflicting reports, dietary intake of oxalate and phytate enriched plants should be considered in relation to potential health outcomes following consumption. Terminalia ferdinandiana is one such plant and is investigated here with respect to oxalate, phytate, and mineral contents. Assessment of oxalate and phytate contents in T. ferdinandiana fruit, leaf, and seedcoat tissues through hydrolysis into acid forms revealed oxalic acid contents ranging from 327 to 1,420 mg/100 g on a dry weight (DW) basis whilst phytic acid contents ranged from 8.44 to 121.72 mg/100 g DW. Calcium content in the different tissues ranged from 131 to 1,343 mg/100 g. There was no correlation between oxalic acid and calcium, however a significant, positive correlation was observed between phytic acid and calcium (r = 0.9917; p < 0.001), indicating that tissues rich in phytic acid also contain higher levels of calcium. The high content of phytic acid in comparison to oxalic acid in T. ferdinandiana fruit found in this study and the dietary significance of this in terms of calcium bioavailability, needs to be investigated further.

Antioxidant Rich Extracts of Terminalia ferdinandiana Inhibit the Growth of Foodborne Bacteria.

Terminalia ferdinandiana (Kakadu plum) is a native Australian plant containing phytochemicals with antioxidant capacity. In the search for alternatives to synthetic preservatives, antioxidants from plants and herbs are increasingly being investigated for the preservation of food. In this study, extracts were prepared from Terminalia ferdinandiana fruit, leaves, seedcoats, and bark using different solvents. Hydrolysable and condensed tannin contents in the extracts were determined, as well as antioxidant capacity, by measuring the total phenolic content (TPC) and free radical scavenging activity using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. Total phenolic content was higher in the fruits and barks with methanol extracts, containing the highest TPC, hydrolysable tannins, and DPPH-free radical scavenging capacity (12.2 ± 2.8 g/100 g dry weight (DW), 55 ± 2 mg/100 g DW, and 93% respectively). Saponins and condensed tannins were highest in bark extracts (7.0 ± 0.2 and 6.5 ± 0.7 g/100 g DW). The antimicrobial activity of extracts from fruit and leaves showed larger zones of inhibition, compared to seedcoats and barks, against the foodborne bacteria Listeria monocytogenes, Bacillus cereus, Methicillin resistant Staphylococcus aureus, and clinical isolates of Pseudomonas aeruginosa. The minimum inhibitory concentration and minimum bactericidal concentration in response to the different extracts ranged from 1.0 to 3.0 mg/mL. Scanning electron microscopy images of the treated bacteria showed morphological changes, leading to cell death. These results suggest that antioxidant rich extracts of Terminalia ferdinandiana fruits and leaves have potential applications as natural antimicrobials in food preservation.

Antioxidant-Rich Extracts of Terminalia ferdinandiana Interfere with Estimation of Cell Viability.

The impact of plant extracts and phytochemicals on in vitro cell viability is usually assessed by employing cell viability assays dependent upon the activity of dehydrogenase enzymes. The CellTiter 96® AQueous One Solution Cell Proliferation Assay (CellTiter) was used to measure cell viability in response to antioxidant-rich extracts of Terminalia ferdinandiana fruits. Conflicting results were obtained from this assay whereby higher concentrations of extracts significantly increased cell viability compared to lower concentrations. Intrinsic reductive potential was observed in a cell-free system when extracts were added directly to the CellTiter assay reagent. To confirm this effect in a similar cell proliferation assay, we employed the CellTiter-Blue® Cell Viability Assay and again observed increased viability with increased concentrations of the extracts and direct reduction of the assay reagent by the extracts in cell-free systems. In the search for a cell proliferation assay that would not be directly affected by the plant extracts, we identified the CyQUANT® NF Cell Proliferation Assay that is based on the estimation of DNA content in viable cells. Cell viability decreased with increasing concentrations of the extracts. Accordingly, the results of the present study indicated that cell viability assays reliant upon dehydrogenase activity may lead to false positive results when testing antioxidant-rich plant extracts with intrinsic reductive potential, and alternative cell viability assays should be used to measure the cell viability.

Release of Indospicine from Contaminated Camel Meat following Cooking and Simulated Gastrointestinal Digestion: Implications for Human Consumption.

Indospicine, a hepatotoxic arginine analog, occurs in leguminous plants of the Indigofera genus and accumulates in the tissues of grazing animals that consume these plants. Furthermore, indospicine has caused toxicity in dogs following consumption of indospicine-contaminated meat; however, the potential impact on human health is unknown. The present study was designed to determine the effect of simulated human gastrointestinal digestion on the release and degradation of indospicine from contaminated camel meat following microwave cooking. Results showed no significant (p > 0.05) indospicine degradation during cooking or in vitro digestion. However, approximately 70% indospicine was released from the meat matrix into the liquid digesta during the gastric phase (in the presence of pepsin) and increased to >90% in the intestinal phase (with pancreatic enzymes). Following human consumption of contaminated meat, this soluble and more bioaccessible fraction of intact indospicine could be readily available for absorption by the small intestine, potentially circulating indospicine throughout the human body to tissues where it could accumulate and cause detrimental toxic effects.

Indospicine cytotoxicity and transport in human cell lines.

Indospicine, a non-proteinogenic analogue of arginine, occurs only in Indigofera plant species and accumulates in the tissues of animals grazing on Indigofera. Canine deaths have resulted from the consumption of indospicine-contaminated meat but only limited information is available regarding indospicine toxicity in humans. In this study three human cell lines, Caco-2 (colorectal adenocarcinoma), HT29-MTX-E12 (colorectal adenocarcinoma) and HepG2 (hepatocellular carcinoma), were used to investigate the cytotoxicity of indospicine and its metabolite 2-aminopimelic acid in comparison to arginine. Indospicine and 2-aminopimelic acid were more cytotoxic than arginine, displaying the highest toxicity in HepG2 liver cells. Intestinal transport in vitro also revealed a 2-fold higher transport rate of indospicine compared to arginine. The sensitivity of HepG2 cells to indospicine is consistent with observed canine hepatotoxicity, and considering the higher in vitro transport of indospicine across an intestinal barrier, it is possible that similar ill effects could be seen in humans consuming contaminated meat.

Anti-Coagulant and Anti-Thrombotic Properties of Blacklip Abalone (Haliotis rubra): In Vitro and Animal Studies.

Sulphated polysaccharides with anti-thrombotic and anti-coagulant activities have been found in various marine biota. In this study, a previously characterised anti-thrombotic and anti-coagulant extract from blacklip abalone was fractionated by anion exchange chromatography (AEC), pooled (on a sulphated polysaccharide basis) and administered to Wistar rats via oral gavage (N = 8) for assessment as an oral therapeutic. To ensure that the preparation had anti-coagulant activity prior to oral administration, it was assessed in rat blood by thromboelastography (TEG) significantly increasing reaction (R) time (or time until clot formation). Following in vitro confirmation of anti-coagulant activity, 40 mg of the preparation was orally administered to rats with blood samples collected at 2, 4, and 6 h post-gavage. Assessment of all blood samples by TEG showed some prolongation of R time from 355 to 380 s after 4 h. Dosing of the post-gavage blood samples with the abalone preparation to confirm anti-thrombotic activity in vitro revealed residual anti-coagulant activity, further suggesting that oral administration did increase anti-coagulant potential in the collected blood but that bioavailability was low. Assessment of tissues and haematological parameters showed no obvious harmful effects of the abalone preparation in animals. In summary, even though oral administration of fractionated and pooled blacklip abalone extract to rats delayed clotting after 4 h, bioavailability of the preparation appeared to be low and may be more appropriate for intravenous administration as an anti-thrombotic or anti-coagulant therapeutic.

In vitro anti-thrombotic and anti-coagulant properties of blacklip abalone (Haliotis rubra) viscera hydrolysate.

Abalone viscera contain sulphated polysaccharides with anti-thrombotic and anti-coagulant activities. In this study, a hydrolysate was prepared from blacklip abalone (Haliotis rubra) viscera using papain and bromelain and fractionated using ion exchange and size exclusion chromatography. Hydrolysates and fractions were investigated for in vitro thrombin inhibition mediated through heparin cofactor II (HCII) as well as anti-coagulant activity in plasma and whole blood. On the basis of sulphated polysaccharide concentration, the hydrolysate inhibited thrombin through HCII with an inhibitor concentration at 50% (IC50) of 16.5 µg/mL compared with 2.1 µg/mL for standard heparin. Fractionation concentrated HCII-mediated thrombin inhibition down to an IC50 of 1.8 µg/mL and improved anti-coagulant activities by significantly delaying clotting time. This study confirmed the presence of anti-thrombotic and anti-coagulant molecules in blacklip abalone viscera and demonstrated that these activities can be enriched with a simple chromatography regime. Blacklip abalone viscera warrant further investigation as a source of nutraceutical or functional food ingredients. Graphical abstract Schematic showing preparation of bioactive extracts and fractions from blacklip abalone.

In vitro Anti-Thrombotic Activity of Extracts from Blacklip Abalone (Haliotis rubra) Processing Waste.

Waste generated from the processing of marine organisms for food represents an underutilized resource that has the potential to provide bioactive molecules with pharmaceutical applications. Some of these molecules have known anti-thrombotic and anti-coagulant activities and are being investigated as alternatives to common anti-thrombotic drugs, like heparin and warfarin that have serious side effects. In the current study, extracts prepared from blacklip abalone (Haliotis rubra) processing waste, using food grade enzymes papain and bromelain, were found to contain sulphated polysaccharide with anti-thrombotic activity. Extracts were found to be enriched with sulphated polysaccharides and assessed for anti-thrombotic activity in vitro through heparin cofactor-II (HCII)-mediated inhibition of thrombin. More than 60% thrombin inhibition was observed in response to 100 µg/mL sulphated polysaccharides. Anti-thrombotic potential was further assessed as anti-coagulant activity in plasma and blood, using prothrombin time (PT), activated partial thromboplastin time (aPTT), and thromboelastography (TEG). All abalone extracts had significant activity compared with saline control. Anion exchange chromatography was used to separate extracts into fractions with enhanced anti-thrombotic activity, improving HCII-mediated thrombin inhibition, PT and aPTT almost 2-fold. Overall this study identifies an alternative source of anti-thrombotic molecules that can be easily processed offering alternatives to current anti-thrombotic agents like heparin.

Current and potential uses of bioactive molecules from marine processing waste.

Food industries produce huge amounts of processing waste that are often disposed of incurring expenses and impacting upon the environment. For these and other reasons, food processing waste streams, in particular marine processing waste streams, are gaining popularity amongst pharmaceutical, cosmetic and nutraceutical industries as sources of bioactive molecules. In the last 30 years, there has been a gradual increase in processed marine products with a concomitant increase in waste streams that include viscera, heads, skins, fins, bones, trimmings and shellfish waste. In 2010, these waste streams equated to approximately 24 million tonnes of mostly unused resources. Marine processing waste streams not only represent an abundant resource, they are also enriched with structurally diverse molecules that possess a broad panel of bioactivities including anti-oxidant, anti-coagulant, anti-thrombotic, anti-cancer and immune-stimulatory activities. Retrieval and characterisation of bioactive molecules from marine processing waste also contributes valuable information to the vast field of marine natural product discovery. This review summarises the current use of bioactive molecules from marine processing waste in different products and industries. Moreover, this review summarises new research into processing waste streams and the potential for adoption by industries in the creation of new products containing marine processing waste bioactives.

Marine-Based Nutraceuticals: An Innovative Trend in the Food and Supplement Industries.

Recent trends in functional foods and supplements have demonstrated that bioactive molecules play a major therapeutic role in human disease. Nutritionists and biomedical and food scientists are working together to discover new bioactive molecules that have increased potency and therapeutic benefits. Marine life constitutes almost 80% of the world biota with thousands of bioactive compounds and secondary metabolites derived from marine invertebrates such as tunicates, sponges, molluscs, bryozoans, sea slugs and many other marine organisms. These bioactive molecules and secondary metabolites possess antibiotic, antiparasitic, antiviral, anti-inflammatory, antifibrotic and anticancer activities. They are also inhibitors or activators of critical enzymes and transcription factors, competitors of transporters and sequestrants that modulate various physiological pathways. The current review summaries the widely available marine-based nutraceuticals and recent research carried out for the purposes of isolation, identification and characterization of marine-derived bioactive compounds with various therapeutic potentials.

In vitro transport and satiety of a beta-lactoglobulin dipeptide and beta-casomorphin-7 and its metabolites.

Understanding the digestive behaviour and biological activities of dairy proteins may help to develop model dairy products with targeted health outcomes including increased satiety and healthy weight maintenance. Caseins and whey proteins constitute over 95% of milk proteins with consumption of these proteins associated with increased satiety and a decreased prevalence of metabolic disorders. To investigate the in vitro digestive behaviour and satiety of dairy proteins at the intestinal epithelium, the in vitro transport and hydrolysis of 500-2000 µM ß-casomorphin-7 (YPFPGPI or ß-CM7) and a ß-lactoglobulin (ß-Lg) dipeptide (YL) was measured using Caco-2 cell monolayers grown on transwells as a model of the intestinal epithelium. Transport of YL was concentration dependent and ranged from 0.37-5.26 × 10(-6) cm s(-1), whereas transport of ß-CM7 was only detected at 2000 µM and was significantly lower at 0.13 × 10(-6) cm s(-1). Rapid hydrolysis of ß-CM7 in the apical chamber by the Caco-2 cells produced three peptide metabolites: YP, GPI and FPGPI. All of these metabolites were detected in the basolateral chamber after 30 min with both the YP and GPI peptides transporting at a higher rate than intact ß-CM7. In vitro satiety was indicated by the secretion of cholecystokinin [26-33] (CCK-8) and glucagon-like peptide 1 (GLP-17-36NH2) in the STC-1 enteroendocrine cell model. CCK-8 secretion was highest in response to ß-CM7 followed by the ß-CM7 metabolite FPGPI. CCK-8 secretion however was not significantly stimulated by the tri- or dipeptides. Secretion of GLP-1 was not significantly stimulated by ß-CM7 or YL. These in vitro results suggest that dairy peptide size enhances CCK-8 secretion, whilst limiting transport across Caco-2 monolayers.

RNF14 is a regulator of mitochondrial and immune function in muscle.

BACKGROUND: Muscle development and remodelling, mitochondrial physiology and inflammation are thought to be inter-related and to have implications for metabolism in both health and disease. However, our understanding of their molecular control is incomplete. RESULTS: In this study we have confirmed that the ring finger 14 protein (RNF14), a poorly understood transcriptional regulator, influences the expression of both mitochondrial and immune-related genes. The prediction was based on a combination of network connectivity and differential connectivity in cattle (a non-model organism) and mice data sets, with a focus on skeletal muscle. They assigned similar probability to mammalian RNF14 playing a regulatory role in mitochondrial and immune gene expression. To try and resolve this apparent ambiguity we performed a genome-wide microarray expression analysis on mouse C2C12 myoblasts transiently transfected with two Rnf14 transcript variants that encode 2 naturally occurring but different RNF14 protein isoforms. The effect of both constructs was significantly different to the control samples (untransfected cells and cells transfected with an empty vector). Cluster analyses revealed that transfection with the two Rnf14 constructs yielded discrete expression signatures from each other, but in both cases a substantial set of genes annotated as encoding proteins related to immune function were perturbed. These included cytokines and interferon regulatory factors. Additionally, transfection of the longer transcript variant 1 coordinately increased the expression of 12 (of the total 13) mitochondrial proteins encoded by the mitochondrial genome, 3 of which were significant in isolated pair-wise comparisons (Mt-coxII, Mt-nd2 and mt-nd4l). This apparent additional mitochondrial function may be attributable to the RWD protein domain that is present only in the longer RNF14 isoform. CONCLUSIONS: RNF14 influences the expression of both mitochondrial and immune related genes in a skeletal muscle context, and has likely implications for the inter-relationship between bioenergetic status and inflammation.

Antithrombin activity and disaccharide composition of dermatan sulfate from different bovine tissues.

Dermatan sulfate is a glycosaminoglycan that selectively inhibits the action of thrombin through interaction with heparin cofactor II. Unlike heparin it does not interact with other coagulation factors and is able to inhibit thrombin associated with clots. This property has made dermatan sulfate an attractive candidate as an antithrombotic drug. Previous studies have showed that dermatan sulfate derived from porcine/bovine intestinal mucosa/skin or marine invertebrates is capable of stimulating heparin cofactor II-mediated thrombin inhibition in vitro. This biological activity is reported for the first time in this study using dermatan sulfate derived from mammalian tissues other than intestinal mucosa or skin. Ten different bovine tissues including the aorta, diaphragm, eyes, large and small intestine, esophagus, skin, tendon, tongue, and tongue skin were used to prepare dermatan sulfate-enriched fractions by anion exchange chromatography and acetone precipitation. Heparin cofactor II/dermatan sulfate-mediated thrombin inhibition measured in vitro revealed activity comparable to or higher than the commercial standard with 2-fold differences observed between some tissues. Analysis of the extracted dermatan sulfate using fluorophore-assisted carbohydrate electrophoresis revealed significant differences in the relative percentage of all the mono-sulfated disaccharides, in particular the predominant mammalian disaccharide uronic acid-->N-acetyl-D-galactosamine-4-O-sulfate, confirming previous reports regarding variations in sulfation in dermatan sulfate from different tissues. Overall, these findings demonstrate that dermatan sulfate extracted from a range of bovine tissues exhibits in vitro antithrombin activity equivalent to or higher than that observed for porcine intestinal mucosa, identifying additional sources of dermatan sulfate as potential antithrombotic agents.