ABSTRACT
Recent studies have shown that dysregulation of transglutaminase 3 (TGM3) is related to the aggressive progression of several cancer types. Our study aimed to determine the function of TGM3 in cervical cancer (CC) tumorigenesis. Gene expression profiles GSE63514, GSE9750, GSE46857 and GSE67522 were obtained from the Gene Expression Omnibus (GEO) database. Overlapping differential expressed genes (DEGs) in CC were screened using GEO2R online tool and Venn diagram software. The Kaplan-Meier plotter was used to determine overall survival. TGM3 expression was analyzed based on GEO and The Cancer Genome Atlas (TCGA) databases, qRT-PCR and western blot analyses. Cell proliferation was evaluated by CCK-8 and EdU incorporation assays. The half-maximal inhibitory concentration (IC50) value of cisplatin and cell apoptosis was assessed by CCK-8 and TUNEL assays, respectively. P-glycoprotein (P-gp) expression and the changes of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway were examined using western blot analysis. We identified 3 overlapping DEGs, including TGM3, glutathione peroxidase 3 (GPX3), and alpha B-crystallin (CRYAB), which were downregulated in CC tissues. TGM3 expression was reduced in CC cells and related to the poor prognosis of CC patients. TGM3 overexpression retarded the proliferation, reduced IC50 value of cisplatin, accelerated cisplatin-induced apoptosis, and inhibited cisplatin-induced P-gp level in CC cells. Furthermore, TGM3 overexpression suppressed the PI3K/Akt pathway in CC cells. Moreover, treatment with 740Y-P, a PI3K activator, abolished the effect of TGM3 overexpression on proliferation and cisplatin resistance in CC cells. In conclusion, overexpression of TGM3 suppressed proliferation and cisplatin resistance in CC cells by blocking the PI3K/Akt pathway.
Subject(s)
Cisplatin , Peptide Fragments , Receptors, Platelet-Derived Growth Factor , Uterine Cervical Neoplasms , Female , Humans , Cisplatin/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Uterine Cervical Neoplasms/metabolism , Signal Transduction , Sincalide/pharmacology , Cell Line, Tumor , Cell Proliferation , Transglutaminases/metabolism , Transglutaminases/pharmacology , ApoptosisABSTRACT
Lactobacillus plantarum is a kind of probiotic that benefits the host by regulating the gut microbiota, but it is easily damaged when passing through the gastrointestinal tract, hindering its ability to reach the destination and reducing its utilization value. Encapsulation is a promising strategy for solving this problem. In this study, transglutaminase (TGase)-crosslinked gelatin (GE)/sodium hexametaphosphate (SHMP) hydrogels were used to encapsulate L. plantarum. The effects of TGase concentration and drying method on the physiochemical properties of the hydrogels were determined. The results showed that at a TGase concentration of 9 U/gGE, the hardness, chewiness, energy storage modulus, and apparent viscosity of the hydrogel encapsulation system were maximized. This concentration produced more high-energy isopeptide bonds, strengthening the interactions between molecules, forming a more stable three-dimensional network structure. The survival rate under the simulated gastrointestinal conditions and storage stability of L. plantarum were improved at this concentration. The thermal stability of the encapsulation system dried via microwave vacuum freeze drying (MFD) was slightly higher than that when dried via freeze drying (FD). The gel structure was more stable, and the activity of L. plantarum decreased more slowly during the storage period when dried using MFD. This research provides a theoretical basis for the development of encapsulation technology of probiotics.
Subject(s)
Lactobacillus plantarum , Probiotics , Gelatin/pharmacology , Microbial Viability , Transglutaminases/pharmacology , Hydrogels/pharmacology , Freeze Drying , Probiotics/chemistryABSTRACT
Myopia, a prevalent refractive error disorder worldwide, is characterized by the elongation of the eye, leading to visual abnormalities. Understanding the genetic factors involved in myopia is crucial for developing therapeutic and preventive measures. Unfortunately, only a limited number of genes with well-defined functionality have been associated with myopia. In this study, we found that the homozygous TGM2-deleted gene in mice protected against the development of myopia by slowing down the elongation of the eye. The effectiveness of gene knockdown was confirmed by achieving a 60 percent reduction in TGM-2 transcript levels through the use of TGM-2-specific small interfering RNA (siRNA) in human scleral fibroblasts (SFs). Furthermore, treating normal mouse SFs with various transglutaminase inhibitors led to the down-regulation of TGM-2 expression, with the most significant reduction observed with specific TGM-2 inhibitors. Additionally, the study found that the pharmacological blockade of muscarinic receptors also slowed the progression of myopia in mice, and this effect was accompanied by a decrease in TGM-2 enzyme expression. Specifically, mice with homozygous mAChR5, mAChR1, and/or mAChR4 and knockout mice exhibited higher levels of TGM-2 mRNA compared to mice with homozygous mAChR2 and three knockout mice (fold changes of 5.8, 2.9, 2.4, -2.2, and -4.7, respectively; p < 0.05). These findings strongly suggest that both TGM-2 and muscarinic receptors play central roles in the development of myopia, and blocking these factors could potentially be useful in interfering with the progression of this condition. In conclusion, targeting TGM-2 may have a beneficial effect regarding myopia, and this may also be at least partially be the mechanism of anti-muscarinic drugs in myopia. Further studies should investigate the interaction between TGM-2 and muscarinic receptors, as well as the changes in other extracellular matrix genes associated with growth during the development of myopia.
Subject(s)
Myopia , Receptors, Muscarinic , Animals , Humans , Mice , Receptors, Muscarinic/metabolism , Myopia/drug therapy , Myopia/genetics , Myopia/metabolism , Sclera/metabolism , Transglutaminases/genetics , Transglutaminases/metabolism , Transglutaminases/pharmacology , Mice, KnockoutABSTRACT
Gelation properties of myofibrillar protein (MP)/wheat gluten (WG) induced by glutamine transaminase (TGase) were studied. Results showed that the inclusion of transglutaminase increased the gel strength, water-holding capacity (WHC), and nonfreezable water (Wnf) of MP/WG mixture. Circular dichroism (CD) analysis showed that the ß-sheet and random coil content of the MP/WG treated with TGase addition increased by 12.1% and 3.7%, while the α-helix and ß-turn content decreased by 14.2% and 1.8%. Rheological measurements showed that TGase induced higher energy storage modulus value during the MP/WG gel heating-cooling cycle. the hydrogen bond and hydrophobic interaction content of the MP/WG gels increased by 80 and 120 ug/L, and the disulfide bond decreased by 200 ug/L, with TGase addition was increased from 0 to 120 U/g protein. Scanning electron microscope (SEM) showed that MP/WG gel with TGase had uniform and dense network structure. PRACTICAL APPLICATION: The properties of myofibrillar/wheat gluten gel induced by TGase crosslinking was studied. The gel structure and water holding capacity of MP/WG were improved by the cross-linking of TGase. The study of the gel properties of MP/WG induced by TGase crosslinking also can provide a theoretical basis for analyzing the effect of TGase on the application of gluten protein in complex meat emulsion system.
Subject(s)
Gels/chemistry , Glutens/chemistry , Myofibrils/metabolism , Rheology , Transglutaminases/pharmacology , Triticum/chemistry , Glutens/drug effects , Glutens/metabolism , Hydrophobic and Hydrophilic Interactions , Myofibrils/drug effects , Triticum/drug effects , Triticum/metabolismABSTRACT
Tofu prepared by conventional methods often has a bitter taste and poor water-holding capacity (WHC). To improve the quality of the product, alternative processes must be developed. Herein, the effect of ultrasound pretreatment on the properties of soymilk and tofu gel derived thereof were investigated. Treatment of soymilk with ultrasound gave rise to a reduction in the particle size and an enhancement in the surface hydrophobicity, whereby optimum values were obtained after 15 min treatment. Subsequently, microbial transglutaminase (MTG) was added to ultrasound-treated soymilk to promote the soy protein crosslinking. The gel strength, WHC, and nonfreezable water content of MTG-catalyzed tofu gel obtained from treated soymilk increased with the extension of the ultrasound pretreatment time, whereas the free sulfhydryl content decreased because of the formation of disulfide bonds. Fourier transform infrared spectroscopy demonstrated variations in the secondary structure of MTG-catalyzed tofu gel. Furthermore, soymilk's exposure to high-intensity ultrasound pretreatment led to a tofu gel with a dense, homogenous, and stable network structure, as evidenced by scanning electron microscopy. Therefore, this study answers for the theoretical support of the industrial production of MTG-catalyzed tofu gel from ultrasound-treated soymilk. PRACTICAL APPLICATION: High-intensity ultrasound pretreatment improved the texture properties of MTG-catalyzed tofu gel. The resulting MTG-catalyzed tofu gel has potential application in industrial production.
Subject(s)
Gels/chemistry , Soy Foods/analysis , Soy Milk/chemistry , Soybean Proteins/chemistry , Transglutaminases/pharmacology , Ultrasonic Waves , Bacteria/enzymology , Catalysis , Gels/metabolism , Gels/radiation effects , Spectroscopy, Fourier Transform InfraredABSTRACT
Celiac disease (CD) is a chronic immune-mediated disease in which gluten ingestion leads to damage of the small intestinal mucosa in genetically susceptible individuals. The enteropathy is mainly induced by the production of IFN-γ from intestinal CD4+T cells that recognise gliadin peptides following deamidation by tissue transglutaminase. The only available therapy is a strict, lifelong gluten-free diet (GFD). This diet is strongly demanding for patients, which justifies the search for alternative strategies. The enzyme approach is one promising strategy to address this issue. In particular, transamidation of wheat gliadin by microbial transglutaminase (mTG) was fully effective at inhibiting gliadin-specific IFN-γ secretion in intestinal T cells from CD patients. Furthermore, transamidated gliadin induced higher levels of the anti-inflammatory IL-10 than native gliadin in different in vitro models. These data suggest that a more balanced immune response could be induced by mTG-treated gliadin in the small intestine of celiac patients. Furthermore, the highlighted biological property of mTG-treated gliadin could be exploited to induce tolerance to native gliadin in at-risk individuals.
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , Celiac Disease/drug therapy , Gliadin/metabolism , Intestinal Mucosa/drug effects , Transglutaminases , Triticum , Bacteria/enzymology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Flour , Humans , Interleukin-10/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Transglutaminases/pharmacology , Transglutaminases/therapeutic use , Triticum/drug effects , Triticum/metabolismABSTRACT
Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.
Subject(s)
Antibodies/pharmacology , Immunoconjugates/chemistry , Oligonucleotides, Antisense/immunology , RNA, Small Interfering/immunology , Antibodies/chemistry , Antibodies/immunology , Azides/chemistry , Cell Lineage/drug effects , Cycloaddition Reaction , Cyclooctanes/chemistry , Drug Delivery Systems , Endosomes/drug effects , Hepatocytes/drug effects , Hepatocytes/immunology , Humans , Immunoconjugates/immunology , Immunoconjugates/pharmacology , Liver/drug effects , Liver/immunology , Oligonucleotides, Antisense/antagonists & inhibitors , Oligonucleotides, Antisense/chemistry , Peptides/chemistry , Peptides/pharmacology , RNA, Small Interfering/antagonists & inhibitors , RNA, Small Interfering/chemistry , Transglutaminases/chemistry , Transglutaminases/immunology , Transglutaminases/pharmacologyABSTRACT
The effects of whey protein addition and transglutaminase treatment, alone and in combination, on the physical and sensory properties of reduced-fat ice cream were investigated. Adding whey protein with or without enzyme treatment decreased melting rate, overrun, and hardness of the reduced-fat ice cream; however, the enzyme-treated sample had a higher melting rate and overrun and softer texture. Whey protein-fortified samples showed higher melting resistance, but lower overrun and firmer texture compared with the enzyme-treated sample without added whey protein. Whey protein addition with or without transglutaminase treatment caused an increase in apparent viscosity and a decrease in flow index of the reduced-fat ice cream; nevertheless, the flow behavior of full-fat sample was most similar to the enzyme-treated reduced-fat sample with no added whey protein. Descriptive sensory analyses showed that neither whey protein addition nor transglutaminase treatment significantly influenced the flavor and odor of reduced-fat ice cream, but they both noticeably improved the color and texture of the final product. The results of this study suggest that whey protein addition with transglutaminase treatment improves the physical and sensory properties of reduced-fat ice cream more favorably than does whey protein addition or transglutaminase treatment alone.
Subject(s)
Food Technology , Ice Cream/analysis , Transglutaminases/pharmacology , Whey Proteins/pharmacology , Animals , TasteABSTRACT
With the utilization of enzymes including endoxylanase, glucose oxidase (GOX) and transglutaminase (TG), and emulsifiers comprising sodium stearoyl lactate (SSL) and soy lecithin, the microstructural, textural, and sensory properties of whole-wheat noodle (WWN) were modified. The development time and stability of whole-wheat dough (WWD) were enhanced by TG due to the formation of a more compact gluten network, and by SSL resulting from the enhanced gluten strength. Microstructure graphs by scanning electron microscopy (SEM) verified that TG and SSL promoted the connectivity of gluten network and the coverage of starch granules in WWN. TG increased the hardness and elasticity of cooked WWN, while two emulsifiers increased the noodle cohesiveness. Additionally, TG and SSL improved the sensory properties of noodle such as bite, springiness, and mouth-feel. The results suggest that TG and SSL are effective ingredients in enhancing the gluten strength of WWD and improving the qualities of WWN.
Subject(s)
Emulsifying Agents/pharmacology , Flour/analysis , Transglutaminases/pharmacology , Triticum/chemistry , Triticum/ultrastructure , Cooking/methods , Food Handling/methods , Glutens/analysis , Humans , Microscopy, Electron, Scanning/methods , Taste/drug effects , Taste/physiology , Triticum/drug effectsABSTRACT
Endostatin (EST), an antiangiogenic factor, is enriched in aging kidneys. EST is also an interactive partner of transglutaminase 2 (TG2), an enzyme that cross-links extracellular matrix proteins. Here we tested whether EST and TG2 play a role in the fibrosis of aging. In wild-type mice, aging kidneys exhibited a 2- to 4-fold increase in TG2 paralleled by increased cross-linked extracellular matrix proteins and fibrosis. Mice transgenic to express EST showed renal fibrosis at a young age. One-month delivery of EST via minipumps to young mice showed increased renal fibrosis that became more robust when superimposed on folic acid-induced nephropathy. Upregulated TG2 and impaired renal function were apparent with EST delivery combined with folic acid-induced nephropathy. Subcapsular injection of TG2 and/or EST into kidneys of young mice not only induced interstitial fibrosis, but also increased the proportion of senescent cells. Thus, kidney fibrosis in aging may represent a natural outcome of upregulated EST and TG2, but more likely it appears to be a result of cumulative stresses occurring on the background of synergistically acting geronic (aging) proteins, EST and TG2.
Subject(s)
Aging/metabolism , Collagen Type XVIII/metabolism , Endostatins/metabolism , GTP-Binding Proteins/metabolism , Kidney Diseases/pathology , Kidney/pathology , Transglutaminases/metabolism , Animals , Cells, Cultured , Cellular Senescence/drug effects , Collagen Type XVIII/genetics , Collagen Type XVIII/pharmacology , Endostatins/genetics , Endostatins/pharmacology , Endothelial Cells , Extracellular Matrix Proteins , Fibrosis , Folic Acid/toxicity , GTP-Binding Proteins/genetics , GTP-Binding Proteins/pharmacology , Kidney/drug effects , Kidney/metabolism , Kidney Diseases/chemically induced , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/genetics , Transglutaminases/pharmacology , Up-RegulationABSTRACT
Salt reduction in food is a challenging task. The food processing sector has adopted taste enhancers to replace salt partially. In this study, a flavour enhancer formulation (liquid seasoning) was produced using enzymatically hydrolysed poultry proteins isolate (PPI). The PPI obtained through the isoelectric solubilisation precipitation process (ISP) was hydrolysed with Alcalase and glycated with glucosamine (GlcN) at moderate temperatures (37/50°C) in the presence or absence of transglutaminase (TGase). The glycated hydrolysates showed reduced fluorescence advanced glycated end-products (AGE) and a reduced amount of alpha-dicarbonyl compounds (α-DC). An untrained consumer panel ranked the meat protein hydrolysate seasoning saltier than the salty standard seasoning solution (p<0.05) regardless of GlcN glycation (both tested at 0.3M Na(+)). GlcN treatments showed a tendency (p=0.0593) to increase savouriness. Free glutamic acid and free aspartic acid found in the PPI hydrolysate likely increased the salty perception.
Subject(s)
Dietary Proteins/chemistry , Food Handling , Glucosamine/pharmacology , Taste , Transglutaminases/pharmacology , Dietary Proteins/analysis , Glycation End Products, Advanced/chemistry , Glycosylation , Hydrolysis , MeatABSTRACT
OBJECTIVE: To study the interactive effects of Type II glutamine transaminase (TG2) and bone morphogenetic protein-9 (BMP-9) in the induction of osteogenesis in mice mesenchymal stem cells (MSCs) C3H10T1/2 model. MATERIALS AND METHODS: Batches of MSCs C3H10T1/2, divided into two groups, were treated with BMP-9 (control group) or BMP-9 and TG2 (experimental group) under oxygen deficient conditions. The secreted alkaline phosphatase (SEAP) chemiluminescence and the histochemical staining methods were used to detect the alkaline phosphatase (ALP) expression. The alizarin red S staining was used to detect the calcium salt precipitation and the caspase-3 protein expression was monitored using Western blot. Flow cytometry was employed to identify cell cycle, and trypan blue exclusion method to count the living cells and monitor cell proliferation. RESULTS: The levels of ALP expression in the experimental group were much higher than that of the control group. The level of expression of advanced caspase-3 protein was significantly lower (p < 0.05) in the experimental group than in the control group. The highest fraction of cells in the experimental group was in the phase M while cells in the control group were in the interphase. Moreover, cell number in the experimental group was significantly increased (p < 0.05) relatively to the control group. CONCLUSIONS: BMP-9 interacts with TG2 in osteogenesis of MSCs C3H10T1/2 cells. Further studies are needed to understand the exact mechanism of BMP9/PG2 interactions in osteogenesis.
Subject(s)
GTP-Binding Proteins/metabolism , Growth Differentiation Factor 2/metabolism , Mesenchymal Stem Cells/metabolism , Osteogenesis/physiology , Transglutaminases/metabolism , Alkaline Phosphatase/metabolism , Animals , Caspase 3/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Proliferation/drug effects , Cell Proliferation/physiology , Cells, Cultured , GTP-Binding Proteins/pharmacology , Growth Differentiation Factor 2/pharmacology , Mesenchymal Stem Cells/drug effects , Mice , Mice, Inbred C3H , Osteogenesis/drug effects , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/pharmacologyABSTRACT
The effect of transglutaminase (TGase) on the properties of tilapia scale gelatin films in the presence of soy protein isolate (SPI) was investigated. When 3% TGase was added into gelatin films, the total soluble matter and protein solubility of films were decreased from 89.36% and 92.78% to 35.83% and 40.05%, respectively, and the decline was promoted by adding 5% SPI. The strength of the films was increased by adding 1% TGase irrespective of SPI addition, but decreased when the TGase concentration was further raised. No obvious colour change was observed in the films with or without TGase and SPI. Based on the results of SDS-PAGE, DSC and SEM, it was revealed that the movement of low molecular weight hydrophilic protein was depressed by the cross-linking network structure induced by TGase and SPI during film drying, indicating that adding SPI is essential to improve the thermal stability and water resistance properties of TGase-induced gelatin films.
Subject(s)
Gelatin/chemistry , Soybean Proteins/chemistry , Tilapia/metabolism , Transglutaminases/pharmacology , Animals , Calorimetry, Differential Scanning , Electrophoresis, Polyacrylamide Gel , Gelatin/ultrastructure , Solubility , Tensile Strength , WaterABSTRACT
BACKGROUND: Microbial transglutaminase (MTGase) is an enzyme utilized in the food industry in many areas. In this study, the suppression effect of MTGase at various levels (0, 2, 5, 10 g kg(-1)) on the formation of biogenic amines in mackerel was determined during refrigerated storage of 8 days. RESULTS: Mince added with 2 g kg(-1) MTGase showed the lowest formation of putrescine, cadaverine and tyramine throughout the storage. Histamine exceeded the consumable limit (500 mg kg(-1) ) after the 4th day, except for that containing 2 g kg(-1) MTGase. The formation of total volatile basic nitrogen and total free amino acid content were retarded and the pH value was unaltered by addition of MTGase. With increasing MTGase levels, the growth of total psychrophilic bacteria, mould, yeast and coliform bacteria was retarded. The sensory scores of mackerel mince increased as MTGase concentrations increased. CONCLUSION: MTGase plays a role in maintaining the quality of mackerel mince during refrigerated storage. As a result of the present study, a new use for MTGase in the food industry is revealed. It will contribute especially in the field of development of products for consumers with allergic sensitivity.
Subject(s)
Biogenic Amines/biosynthesis , Food Handling/methods , Food Microbiology , Food Preservation/methods , Perciformes , Seafood/analysis , Transglutaminases/metabolism , Amino Acids/metabolism , Animals , Bacteria/drug effects , Cold Temperature , Colony Count, Microbial , Fungi/drug effects , Humans , Nitrogen/metabolism , Refrigeration , Seafood/microbiology , Seafood/standards , Taste , Transglutaminases/pharmacologyABSTRACT
The growing social and economic consequences of pollution derived from plastics are focusing attention on the need to produce novel bioprocesses for enhancing food shelf-life. As a consequence, in recent years the use of edible films for food packaging is generating a huge scientific interest. In this work we report the production of an edible hydrocolloid film made by using Citrus pectin and the protein phaseolin crosslinked by microbial transglutaminase, an enzyme able to covalently modify proteins by formation of isopeptide bonds between glutamine and lysine residues. The films were characterized and their morphology was evaluated by both atomic force microscopy and scanning electron microscopy. Mechanical properties and barrier properties to CO2, O2 and water vapor have demonstrated that these films possess technological features comparable to those possessed by commercial plastics. It is worth noting that these characteristics are maintained even following storage of the films at 4°C or -20°C, suggesting that our bioplastics can be tailored to protect food at low temperature. Moreover, gastric and duodenal digestion studies conducted under the same conditions found in the human digestion system have demonstrated that transglutaminase-containing films are regularly digested encouraging an application of the proposed materials as food coatings.
Subject(s)
Bacterial Proteins/pharmacology , Citrus/chemistry , Colloids/chemical synthesis , Cross-Linking Reagents/pharmacology , Food Packaging/instrumentation , Pectins/chemistry , Plant Proteins/chemistry , Transglutaminases/pharmacology , Cryopreservation , Digestion , Food Preservation , Glycerol/chemistry , Humans , Materials Testing , Microscopy, Atomic Force , Microscopy, Electron, Scanning , Pectins/isolation & purification , Plant Proteins/drug effects , Pliability , Tensile StrengthABSTRACT
The potential of using the unwashed mince of farmed meagre as raw material for the preparation of heat-induced gel products was assessed taking into account the effect of age (small size <1 kg meagre vs commercial size > 2kg meagre), lower salt levels (1.0%, w/w, vs 2.5%, w/w), and microbial transglutaminase (MTGase) incorporation (0.0%, w/w, vs 0.5%, w/w). Heat-induced gel products from > 2 kg fish were of superior quality. Salt reduction from 2.5% to 1.0% (w/w) was detrimental for textural quality, particularly, of gels prepared from >2 kg meagre mince. MTGase addition improved texture. Moreover, MTGase incorporation led to a greater importance of non-covalent hydrophobic bonding.
Subject(s)
Fish Products/analysis , Fishes , Food Quality , Gels/standards , Sodium Chloride/pharmacology , Transglutaminases/pharmacology , Age Factors , Analysis of Variance , Animals , Aquaculture/methods , Chemical Phenomena , Electrophoresis, Polyacrylamide Gel/methods , Food Handling/methods , Gels/analysis , Hot Temperature , Hydrophobic and Hydrophilic InteractionsABSTRACT
Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n = 27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n = 31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n = 11) and control subjects (n = 11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease.
Subject(s)
CD4 Antigens/immunology , Chromogranin A/immunology , Diabetes Mellitus, Type 1/genetics , Peptides/immunology , Adolescent , Adult , Autoantigens/genetics , Autoantigens/immunology , CD4 Antigens/genetics , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Child , Chromogranin A/genetics , Chromogranin A/pharmacology , Diabetes Mellitus, Type 1/immunology , Female , Gene Expression , HLA-DQ Antigens/genetics , HLA-DQ Antigens/immunology , Histocompatibility Testing , Humans , Male , Peptides/genetics , Peptides/pharmacology , Primary Cell Culture , Transglutaminases/metabolism , Transglutaminases/pharmacologyABSTRACT
Small artery remodeling involves matrix reorganization, but may also encompass changed smooth muscle cell biomechanical properties. Here we study the temporal relationship between such contractile plasticity and matrix remodeling in small rat mesenteric arteries subjected to 1 or 3 days of altered flow or acute interventions on matrix structure; cross-linking by transglutaminase and matrix digestion by elastase. Diameter-tension relations were made in the passive state and upon full activation (125 mM K+ and 10â»5 M norepinephrine). In low flow (LF), inward matrix remodeling occurred after 1 day, when the distended diameter at full dilation (D100) was reduced from 351±15µm to 299±14µm (SEM, n=8, p<0.05). The optimal diameter for force development (D(opt)) was reduced after 3 days, from 291±10µm to 247±5µm (LF, p<0.05). As a result, a mismatch of D(opt)/D100 existed after 1 day of LF, which normalized after 3 days. Dynamics of contraction were studied following quick isometric release by 0.2âD100; tension recovery was faster in anatomically smaller vessels following normal flow. This association was partly lost after 1 day of LF, while after 3 days the vessels became not only smaller but also faster, re-establishing this association. High flow vessels demonstrated similar contractile plasticity. Active diameter-tension relations at low distension did not change following transglutaminase or elastase. However, at high distension, any alteration in passive tension coincided with an opposite change in active tension. These data demonstrate an intrinsic interaction between passive and active biomechanics that occurs instantaneously during matrix remodeling at high distensions while contractile plasticity lags matrix remodeling after flow interventions.
Subject(s)
Mesenteric Arteries/physiology , Animals , Biomechanical Phenomena , Cross-Linking Reagents/pharmacology , Male , Mesenteric Arteries/drug effects , Muscle Contraction/physiology , Muscle, Smooth, Vascular/physiology , Pancreatic Elastase/pharmacology , Rats , Rats, Wistar , Regional Blood Flow , Transglutaminases/pharmacology , Vascular Resistance/physiology , Vasoconstriction/physiologyABSTRACT
PURPOSE: Molar accounting of bioactive fluids can expose new regulatory mechanisms in the growing proteomic focus on epithelial biology. Essential for the viability of the surface epithelium of the eye and for normal vision is the thin, but protein-rich, tear film in which the small tear glycoprotein lacritin appears to play a prominent prosecretory, cytoprotective, and mitogenic role. Although optimal bioactive levels in cell culture are 1 to 10 nM over a biphasic dose optimum, ELISA suggests a sustained tear lacritin concentration in the midmicromolar range in healthy adults. Here we identify a reconciling mechanism. METHODS: Monoclonal anti-lacritin 1F5 antibody was generated, and applied together with a new anti-C-terminal polyclonal antibody to tear and tissue Western blotting. In vitro tissue transglutaminase (Tgm2) cross-linking was monitored and characterized by mass spectrometry. RESULTS: Blotting for lacritin in human tears or saliva surprisingly detected immunoreactive material with a higher molecular weight and prominence equal or exceeding the â¼23 to 25 kDa band of monomeric glycosylated lacritin. Exogenous Tgm2 initiated lacritin cross-linking within 1 minute and was complete by 90 minutes-even with as little as 0.1 nM lacritin, and involved the donors lysine 82 and 85 and the acceptor glutamine 106 in the syndecan-1 binding domain. Lacritin spiked into lacritin-depleted tears formed multimers, in keeping with â¼0.6 µM TGM2 in tears. Cross-linking was absent when Tgm2 was inactive, and cross-linked lacritin, unlike recombinant monomer, bound syndecan-1 poorly. CONCLUSIONS: Since syndecan-1 binding is necessary for lacritin mitogenic and cytoprotective activities, TGM2 cross-linking negatively regulates lacritin bioactivity.
Subject(s)
Cross-Linking Reagents/pharmacology , Eye Proteins/analysis , GTP-Binding Proteins/pharmacology , Glycoproteins/analysis , Tears/chemistry , Transglutaminases/pharmacology , Antibodies, Monoclonal , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Eye Proteins/chemistry , Glycoproteins/chemistry , Humans , Mass Spectrometry , Protein Glutamine gamma Glutamyltransferase 2 , Saliva/chemistryABSTRACT
Biodegradable and edible protein films have received considerable research interest due to the increase in environment protection consciousness. However, protein films always have poor mechanical properties and water-resistant ability compared with synthetic films. Thus, the effects of transglutaminase on sorption characteristics including adsorption kinetics and sorption isotherm, mechanical properties, moisture content, total soluble matter and surface hydrophobicity (S0) of gelatin films were analyzed in this article. Relative humidity and plasticizer content significantly affected the moisture equilibration time. The lower the relative humidity and plasticizer content, the shorter the moisture equilibration time. Transglutaminase treatment significantly decreased moisture adsorption rate and balance moisture content of gelatin films. The sorption isotherm data were mathematically fitted to the GAB model. Transglutaminase treatment increased the tensile strength value and the mean contact angle by 8.4-25.6% and 2.1-2.3°, respectively; while decreasing the moisture content value by 2.6-9%. These results show that transglutaminase treatment could be an effective and practical method to modify moisture sorption characteristics, moisture content and surface hydrophobicity of gelatin films, which suggests that the water-barrier ability of gelatin films can be improved by transglutaminase treatment.