Guinea Pig Liver Research Articles

An in situ and in vitro investigation for the transglutaminase potential in tissue engineering

Transglutaminases (TGases) constitute a family of enzymes that stabilize protein assemblies by gamma-glutamyl-epsilon-lysine crosslinks. The role of tissue transglutaminase (TGase 2) in several pathophysiologies, wound healing applications, biomaterials functionalization, and drug delivery systems provides grounds for its use in tissue engineering. Herein, we initially studied the endogenous TGase activity and expression under normal (skin, duodenum, colon, and small bowel) and pathophysiological (keloid scar) conditions on cadaveric human tissues. Successful inhibition was achieved using low concentrations of BOC-DON-QIV-OMe (0.1 mM and 1 mM for normal skin and keloid scar, respectively), iodoacetamide (0.1 mM and 1 mM for normal skin and keloid scar, respectively), and cystamine dihydrochloride (1 mM and 10 mM for normal skin and keloid scar, respectively), whilst di-BOC-cystamine was found ineffective even at 100 mM concentration. Secondly, the addition of exogenous guinea pig liver transglutaminase (gpTGase) onto the inhibited tissues and collagen scaffolds was studied, and results presented advocate its use as potential tissue adhesive and drug delivery tool. However, the investigation of its crosslinking extent using second harmonic generation microscopy and differentially scanning calorimetry revealed rather poor stabilization function. Overall, our study indicates that TGase 2 has a role as a biological glue to consolidate various micro-structural components of tissues and biomaterials.

Changes in liver weight are independent of hepatic lipid content in guinea pigs fed a high‐cholesterol low‐carbohydrate diet

We have used high cholesterol (HC) diets to study mechanisms of atherosclerosis in the guinea pig and shown that low carbohydrate diets (LCD) mitigate aortic cholesterol accumulation and inflammatory expression. We have also shown that HC diets induce fatty liver in guinea pigs. We evaluated whether LCD could improve hepatic steatosis in guinea pigs. Male Hartley guinea pigs were fed a HC diet (0.25g/100g) and randomly assigned (n=10/group) to consume either a LCD or high‐carbohydrate diet (HCD) for 12 wk. Plasma and whole livers were collected for lipid analysis. Plasma LDL cholesterol was lower in animals fed the LCD (P < 0.01) diet while plasma triglycerides (TG) were higher (P < 0.05). There were no differences in plasma free fatty acids or plasma glucose between groups. However, glucagon‐like peptide‐1 (GLP‐1) was lower in the LCD group (P < 0.01). Liver weight was 40% higher in the LCD compared to the HCD group. Interestingly despite the increased liver weight in LCD there were no differences in hepatic TG, cholesterol or non‐esterified fatty acids between groups. Histological analysis performed by hematoxylin and eosin staining confirmed there were no differences in hepatic lipid accumulation or steatosis between groups. Collectively these results suggest that in the presence of a HC‐LCD, the increase in liver mass is unrelated to lipid accumulation. An increased number of hepatocytes through an accelerated regenerative process may be responsible for these observations in the LCD group.

Photolabeling of Tissue Transglutaminase Reveals the Binding Mode of Potent Cinnamoyl Inhibitors

We have recently developed a new class of cinnamoyl derivatives as potent tissue transglutaminase (TG2) inhibitors. Herein, we report the synthesis of a diazirine derivative of these inhibitors and its application to the photolabeling of its binding site on guinea pig liver transglutaminase. Two novel homology models were generated for this commonly studied TG2, which differ in the conformational state they represent. Tryptic digest and mass spectrometric analysis of the photolabeling experiment showed that only residue Cys230 was labeled, and our homology models were used to visualize these results. This visualization suggested that Cys230 is somewhat more solvent-exposed in the "closed" conformation of TG2, compared to the "open" conformation. Docking experiments suggested binding modes consistent with the labeling pattern that would block access to the tunnel leading to the active site, consistent with the observed mode of inhibition. However, while these modeling simulations favored the closed conformation as the target of our cinnamoyl inhibitors, native PAGE experiments indicated the open conformation of the enzyme in fact predominates in the presence of our photolabeling derivative. These results are important for understanding the binding modes of TG2 inhibitors in general and will be critical for the structure-based design of future inhibitors.

Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity

Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca 2+-dependent protein cross-linking enzymes. Based on the transamidation activity of TGase 2, a novel colorimetric assay has been developed using covalently coupled spermine to carboxy-substituted polystyrene plates and biotinylated pepT26, an excellent acyl-donor substrate, highly specific for TGase 2. The assay is based on the incorporation of the gamma-carboxamide of glutamine of pepT26 into the immobilized spermine. The amount of biotinylated pepT26 bound to the plate, as measured by the activity of streptavidin-peroxidase, is directly proportional to the TGase activity. The colorimetric procedure showed a good correlation ( r = 0.995) with the commonly used radiometric filter paper method for TGase2, and provides linear dose–response curves over a wide range of hrTGase2 concentrations (2.5–40 μU/ml). In addition, the assay shows higher sensitivity when compared with our previous TG-colorimetric test (more than 50-fold increase) and other existing assays. PepT26 displays strong reactivity with TGase 2, and no reactivity with TGases 1, 3, and FXIII. The procedure constitutes a rapid, TG2-specific, sensitive, and nonisotopic method for the measurement of TGase 2 activity in as low as 4 ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25 μg of guinea pig liver extracts.

Electrochemical immunosensor for the diagnosis of celiac disease

A novel electrochemical immunosensing strategy for the detection of antibodies to tissue transglutaminase (tTG) in human serum is presented. The proposed immunosensor consists of the immobilization by physical adsorption of tTG from guinea pig liver on graphite–epoxy composite (GEC) electrodes. After the reaction with the human serum (containing the specific antibodies in the case of celiac disease), the electrode was incubated with different kinds of secondary labeled antibodies, namely, horseradish peroxidase (HRP)-conjugated goat antibodies to human whole immunoglobulins (Igs), to human IgG, and finally to human IgA. Among the different classes of antibodies in human serum toward tTG, the best results were achieved when anti-tTG IgA antibodies were investigated. In total, 10 positive and 10 negative serum samples were processed, obtaining a sensitivity of 70% and a specificity of 100% compared with the commercial enzyme-linked immunosorbent assay (ELISA) method performed in a hospital laboratory. This strategy offers great promise for a simple, cost-effective, and user-friendly analytical method that allows point-of-care diagnosis of celiac disease.

Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments

TGases (transglutaminases) are a class of calcium-dependent enzymes that catalyse the interactions between acyl acceptor glutamyl residues and amine donors, potentially making cross-links between proteins. To assess the activity of apple (Malus domestica) pollen TGase on the functional properties of actin and tubulin, TGase was prepared from apple pollen by hydrophobic- interaction chromatography and assayed on actin and tubulin purified from the same cell type. The enzyme catalysed the incorporation of putrescine into the cytoskeleton monomers. When tested on actin filaments, pollen TGase induced the formation of high-molecular-mass aggregates of actin. Use of fluorescein-cadaverine showed that the labelled polyamine was incorporated into actin by pollen TGase, similar to with guinea pig liver TGase. The pollen TGase also reduced the enzyme activity and the binding of myosin to TGase-treated actin filaments. Polymerization of tubulin in the presence of pollen TGase also yielded the formation of high-molecular-mass aggregates. Furthermore, the pollen TGase also affected the binding of kinesin to microtubules and reduced the motility of microtubules along kinesin-coated slides. These results indicate that the pollen TGase can control different properties of the pollen tube cytoskeleton (including the ability of actin and tubulin to assemble and their interaction with motor proteins) and consequently regulate the development of pollen tubes.

Tissue Distribution of Mevalonate Pyrophosphate Decarboxylase in Guinea Pig

We previously reported that mevalonate pyrophosphate decarboxylase (MPD) is located in the cytosol and that MPD level in the liver is higher than in other rat tissues. In the present study, we further investigated the tissue distribution of MPD in guinea pigs by immunoblotting using anti-rat MPD antiserum. When immunoblot analysis was carried out using guinea pig brain, the antiserum reacted with 46-kDa protein as well as a substance with the same molecular weight of MPD in mice. Protein of 46-kDa detected in guinea pig liver treated with 0.1% pravastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor indicating a liver-specific effect, was increased 3-fold as compared with nontreated guinea pigs; however, 46-kDa protein in the brain treated with pravastatin was similar to that treated without pravastatin. When the subcellular distribution of MPD in the brain, liver, kidney, and testis, was examined by cell fractionation, MPD was mostly detected in the cytosol fraction of all tissues. From these data, the 46-kDa protein was identified as MPD. Next, when the tissue distribution of MPD was examined, MPD in the liver was higher than in other tissues. The relative amount of MPD in guinea pig kidney was higher than in rats and similar to in mice, as MPD in the liver of the same species was taken as 1. Furthermore, the correlation coefficient between guinea pigs and rats or mice in the tissue distribution of MPD was 0.69 or 0.72, respectively. These data indicate a relationship in tissue distribution between guinea pigs and rats or mice, although the tissue-specific regulator of MPD between species somewhat differed.

Von Willebrand factor antigen is not an accurate marker of rat and guinea pig liver endothelial cells

To determine whether von Willebrand Factor (vWF) is a valid marker of liver endothelial cells, we determined vWF immunoreactivity in rat and guinea pig liver sections and in smears of elutriated nonparenchymal cells isolated from these two species. In frozen sections, positive staining for vWF was seen only in the endothelium lining large hepatic vessels in both species, and no immunoactivity was detected in the sinusoids. On the other hand, immunohistochemical staining for vimentin (a marker of mesenchymal cells) showed positive reaction throughout the vascular and sinusoidal endothelial cells in both the rat and guinea pig liver. In fractions of elutriated rat and guinea pig nonparenchymal liver cells, which included almost exclusively liver endothelial cells, only 25-40% of the cells displayed a positive reaction for vWF. However, when these same fractions were stained for vimentin, 70-95% of the cells exhibited immunoreactivity. Most of the vWF-negative cells were not red and white blood cells, biliary epithelial and Kupffer cells, and hepatocytes, and had ultrastructural features of sinusoidal endothelial cells. We conclude that in both the rat and guinea pig, liver sinusoidal endothelial cells do not exhibit vWF immunoreactivity. Thus, in routine immunohistochemical assays, vWF is not an accurate marker of rat and guinea pig liver endothelial cells. Vimentin is more appropriate for this purpose, provided that other mesenchymal cells are separated or independently identified.

Na/K-ATPase assay in the intact guinea pig liver submitted to in situ perfusion

We describe an assay for the enzyme Na/K-ATPase in intact guinea pig livers perfused through the portal vein with modified Hank’s solution. The model uses the measurement of non-radioactive rubidium ion incorporation by liver cells, both in the absence and in the presence of the specific Na/K-ATPase inhibitor ouabain, followed by a rinsing procedure with cold saline. The concentration of Rb + in acid-digested liver lobes was measured by atomic emission spectrometry and Na/K pump activity was calculated by the difference between the incorporation of Rb + in the absence and in the presence of ouabain. The optimal conditions for Rb + incorporation were: perfusion flow rate, 3 ml/min per liver; perfusion time at 37 °C, 60 min; rinsing time with cold saline, 5–10 min; and concentration of ouabain, 3 mM. The calculated ouabain IC 50 was 100 μM. The major advantage of this model is the possibility of testing experimental drugs affecting this enzyme in conditions close to those in the intact organ.

Inhibitory effects of flavonoids on aldehyde oxidase activity

Flavonoids are an important group of natural compounds that can interfere with the activity of some enzymes. In this study, effects of various flavonoids on aldehyde oxidase (AO) activity were evaluated in vitro. AO was partially purified from guinea pig liver. The effects of 12 flavonoids from three subclasses of flavon-3-ol, flavan-3-ol and flavanone on the oxidation of vanillin and phenanthridine as substrates of AO and xanthine as a substrate of xanthine oxidase (XO) were investigated spectrophotometrically. Among the 12 flavonoids, myricetin and quercetin were the most potent inhibitors of both AO and XO. In general, the oxidation of vanillin was more inhibited by flavonoids than that of phenanthridine. Almost all of the flavonoids inhibited AO activity more potently than XO, which was more evident with non-planner flavanols. A planner structure seems to be essential for a potent inhibitory effect and any substitution by sugar moieties reduces the inhibitory effects. This study could provide a new insight into AO natural inhibitors with potential to lead to some food-drug interactions.

In vitro metabolism of norbormide in rat, mouse and guinea pig liver preparations

Differences between species in response to norbormide (NRB) may arise through differential pharmacodynamic and/or pharmacokinetic properties. We hypothesise that species-selectivity is at least partly determined by differences in metabolism based on in vitro data generated in liver preparations from rats, mice and guinea pigs. HPLC separation and LC/MS identification revealed that NRB undergoes metabolism primarily to hydroxylated form that was tentatively identified in both rat and non-rat species with NADPH as the preferred cofactor. However, the metabolic profile and the rate are different between species. Gender differences are also reported in the metabolic rate in rats and we postulate that this may be responsible for different toxic sensitivities seen between sexes. Using this knowledge, we aim to develop pharmacological tool(s) for use in designing a new class of drugs that can be targeted in a tissue-selective manner. Further in vivo pharmacokinetic with receptor affinity studies are warranted.

Reversible and Competitive Cinnamoyl Triazole Inhibitors of Tissue Transglutaminase

A series of 15 cinnamoyl triazole derivatives was prepared by Cu(I)-catalyzed azide/alkyne [3+2]-cycloaddition reactions and examined as inhibitors of guinea-pig liver transglutaminase. Several compounds exhibited activity as reversible inhibitors that were competitive with acyl donor transglutaminase substrates. For example, triazole 4d has a K(i) value of 174 nM and represents one of the most potent reversible transglutaminase inhibitors reported to date.

Serologic markers of celiac disease in psoriatic patients

Aetiopathogenesis of psoriasis is complex and not yet well known. In recent years, it has been observed that psoriasis can coexist with clinically asymptomatic celiac disease and a gluten-free diet helps to obtain remission, even in patients with very chronic lesions. The aim of our work was to investigate how often the positive titres of antibodies characteristic for celiac disease occur in psoriatics' serum in exacerbation in comparison with controls. Serum samples from 67 patients with intensified psoriatic lesions were investigated. Serum from healthy people at a comparable age and with no familial predisposition to psoriasis and celiac disease was the control material. Antibodies against human tissue transglutaminase (recombinant antigen), against tissue transglutaminase isolated from guinea pig's liver and against gliadin were determined by enzyme-linked immunosorbent assay technique. Anti-endomysial antibodies were determined by indirect immunofluorescence method. Patients with psoriasis have significantly higher mean concentrations of antibodies against tissue transglutaminase (human recombinant and guinea pig-derived antigen) and against gliadin for IgA. IgA antibodies against tissue transglutaminase (both antigens) and gliadin positively correlate with psoriasis activity. No anti-endomysial antibodies for IgA were found in any serum. Our results seem to imply an association between psoriasis and asymptomatic celiac disease/gluten intolerance. High percentage of positive results to guinea pig-derived tTG could be due to cellular activity of tissue transglutaminase in psoriasis.

Identification of Factor-XIIIa-Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100-kDa propeptide, called the propolypeptide of von Willebrand factor (pp-vWF), is generated during biosynthesis. We found that pp-vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311–123161. As such, it could form cross-linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp-vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861–867]. In this work, we identified the Gln residues in pp-vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme-reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti-dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp-vWF, only four could be modified in the factor-XIIIa-catalyzed reaction.

Cinnamoyl Inhibitors of Tissue Transglutaminase

Transglutaminases (TGases) catalyze the intermolecular cross-linking of certain proteins and tissue TGases (TG2) are involved in diverse biological processes. Unregulated, high TGase activities have been implicated in several physiological disorders, but few reversible inhibitors of TG2 have been reported. Herein, we report the synthesis of a series of novel trans-cinammoyl derivatives, discovered to be potent inhibitors of guinea pig liver transglutaminase. The most effective inhibitors evaluated can be sorted into two subclasses: substituted cinnamoyl benzotriazolyl amides and the 3-(substituted cinnamoyl)pyridines, referred to more commonly as azachalcones. Kinetic evaluation of both of these subclasses revealed that they display reversible inhibition and are competitive with acyl donor TGase substrates at IC50 values as low as 18 microM. An analysis of structure-activity relationships within these series of inhibitors permitted the identification of potentially important binding interactions. Further testing of some of the most potent inhibitors demonstrated their selectivity for TG2 and their potential for further development.

Phytosterols and pectin added to a high‐saturated fat diet do not show hypocholesterolemic activity in female guinea pigs

AbstractThis paper presents the results of a study whose aim was to test the effects of several doses of pectin and phytosterols on the sterol content in plasma, liver and feces of guinea pigs, when added to a high‐saturated fat diet. The treatments followed a 3×3 factorial design, with three levels of pectin (0, 3.67 and 6.93%) and three levels of phytosterols (0, 1.37 and 2.45%). Seventy‐two female Dunkin Hartley guinea pigs were randomly assigned to the treatment groups (eight animals per group). The duration of the treatment was 4 weeks. No differences were found in plasma cholesterol concentrations, while in liver we saw a reduction in cholesterol concentration after phytosterol feeding. Moreover, we found no pectin effects. Plant sterol concentration increased in plasma and liver after phytosterol ingestion, with the highest concentrations being obtained with the intermediate pectin dose. Our results suggest that a high‐saturated diet may impair the cholesterol‐lowering properties of plant sterols and pectin.

Regulation of hepatic lipids and antioxidants by dietary carbohydrate restriction and cholesterol in guinea pigs

Carbohydrate restriction (CR) improves parameters of metabolic syndrome and inflammation, but effects on hepatic lipids and antioxidants are less clear. To test the hypothesis that CR favorably affects hepatic oxidative stress and lipids, we assessed hepatic lipids and antioxidants in retired breeder guinea pigs fed a low (L‐) or high (H‐) carbohydrate (CARB) diet with low (0.04%) or high (0.25%) cholesterol (CHOL) for 12 wk (n = 10/dietary treatment). Liver weight was increased by H‐CHOL (p<0.001) and L‐CARB (p<0.05). Guinea pigs fed H‐CHOL had increased (p<0.001) hepatic triglyceride and cholesterol while CARB did not affect hepatic lipids. LCARB increased (p<0.001) hepatic and plasma α‐tocopherol (α‐T) by 1.8 and 4‐times, respectively, whereas H‐CHOL decreased hepatic (p=0.06) and plasma (p<0.05) α‐T by 24% and 54%. Hepatic glutathione ratio (GSH/GSSG) decreased 30% with L‐CARB (p<0.01) and 29% with H‐CHOL (p<0.001), and was due to higher GSSG (p<0.01) and unchanged GSH. GSSG was inversely related (r = ‐0.370; p<0.05) to hepatic α‐T. Hepatic ascorbic acid and uric acid were unaffected by CARB and CHOL. These data suggest that L‐CARB and H‐CHOL result in an oxidative shift of hepatic thiol redox status while L‐CARB improved α‐T status in plasma and liver in aged guinea pigs.

Isolation of liver aldehyde oxidase containing fractions from different animals and determination of kinetic parameters for benzaldehyde

Aldehyde oxidase activity containing fractions from rabbit, guinea pig, rat and mouse livers were obtained by heat treatment and ammonium sulfate precipitation. Aldehyde oxidase activity was observed in rabbit and guinea pig livers, while aldehyde oxidase activity was absent in rat and mouse liver fractions. Enzyme kinetic parameters, Km and Vmax, were determined for the oxidation of benzaldehyde to benzoic acid by rabbit and guinea pig liver fractions, by spectrophotometric method, with potassium ferricyanide as the electron acceptor. The Km values obtained for both animal liver fractions were in the range of 10.3-19.1 μM.

Effects of phenol, glycerin and acetic acid on the liver of guinea pigs

To investigate the histolytic action of a solution composed of phenol, glycerin and acetic acid for irresectable hepatic metastasis. Thirty-two (n=32) guinea pigs were randomly distributed into two groups of 16 animals. The animals in group 1 (experimental) and group 2 (control) were redistributed in two subgroups of eight animals each, according to the day of sacrifice (24 hours and four weeks after injection). All the animals were submitted to median laparotomy, which was followed by the injection of solution E and saline into the livers of subjects in both the experimental and control groups, respectively. The animals were evaluated for biochemical and anatomopathological (liver) alterations after 24 hours and four weeks of the experiment. It was observed that solution E produced necrosis limited to the injected area and that hepatic tissue recovery occurred after four weeks with the formation of a small necrosis area. No biochemical parameters were altered either in the experimental or in the control group. In view of the obtained results, the possibility of using the proposed solution can be considered in cases of irresectable metastasis.

Protective role of pinacidil against adrenaline-induced myocardium injury in guinea pig liver mitochondria

AbstractWe investigated the role of the ATP-sensitive potassium channel opener pinacidil and blocker glibenclamide on guinea pig liver mitochondrial function, and a possible significance of pinacidil in the pharmacological treatment during myocardium dystrophy. First, a series of experiments was performed to determine the effect of pinacidil and glibenclamide on mitochondrial oxygen consumption. We found that pinacidil increased the rate of mitochondrial respiration for FAD-generated substrate (succinate oxidation), but was most effective for α-ketoglutarate oxidation with enhancement of respiratory control ratio. Oxidation of FAD-generated substrate inhibited efficiency of phosphorylation for α-ketoglutarate oxidation in pinacidil-treated animals. Glibenclamide decreased the rate of respiration with the lowest value of efficiency of phosphorylation, especially for α-ketoglutarate oxidation. A second series of experiments was performed to determine the effects of pinacidil and glibenclamide on oxidative phosphorylation during adrenaline-induced myocardium dystrophy. The increase in respiratory control ratio and efficiency of phosphorylation for α-ketoglutarate oxidation was greater than for succinate oxidation in mitochondria of pinacidil-pretreated animals during myocardium dystrophy. Inhibitory analysis with malonate suggested that endogenous succinate increased oxidation of NADH-generated substrates in mitochondria. Pinacidil is mainly involved in the adrenaline-induced alterations of mitochondrial function due to elevation of phosphorylation efficiency for α-ketoglutarate oxidation and a decreased level of lipid peroxidation.