Gender-related differences in the formation of skatole metabolites by specific CYP450 in porcine hepatic S9 fractions.

Higher accumulation of skatole in the fat of male pigs compared with female pigs might be due to gender-related differences in the rate of skatole degradation. In the present study, skatole metabolites and cytochrome P450 (CYP450) isoforms involved in skatole metabolism were for the first time investigated in hepatic S9 fractions from six male and four female pigs (crossbred Landrace×Yorkshire dams and Duroc boar). Surprisingly, the rates of production of major skatole metabolites were similar in male and female pigs. The most abundant metabolite of skatole was 3-hydroxy-3-methyloxindole (HMOI) followed by 3-methyloxindole and indole-3-carbinol in both male and female S9 fractions. Concentrations of formed HMOI and 3-methyloxindole did not differ between the genders (P=0.124 for HMOI, and P=0.575 for 3-methyloxindole). Indole-3-carbinol formation was higher in S9 fractions from the females compared with male pigs (P=0.0001). Enzyme kinetic parameters were similar for both genders (P>0.05). In both male and female pigs, ellipticine, diallyl sulphide (DAS) and quercetin inhibited HMOI formation, confirming the involvement of CYP1A1 and CYP2E1. The formation of 3-methyloxindole was reduced in the presence of the CYP2E1 inhibitor DAS, and formation of indole-3-carbinol was reduced in the presence of CYP1A1 and CYP2A19 inhibitors. We found only minor differences in skatole metabolism between male and female pigs, particularly the involvement of CYP2C and CYP3A in indole-3-carbinol formation in female but not in male pigs. This is a very essential finding, suggesting the involvement of larger number of CYP450 isoforms in female pigs. On the other hand, indole-3-carbinol is a minor skatole metabolite, and the physiological significance of CYP2C and CYP3A involvement in its formation in female pigs, but not in male pigs, needs to be elucidated. Our results, however, should be interpreted with caution because of the low number of animals and possibility of breed and age effects on skatole metabolism.

Novel aspects of health promoting compounds in meat.

Meat is an integral part of the human diet. Besides essential amino acids and nutritive factors of high quality and availability, meat provides often overlooked components of importance for human health. These are amino acids and bioactive compounds that may be very important in i) preventing muscle wasting diseases, such as in sarcopenia, ii) reducing food and caloric intake to prevent metabolic syndrome, iii) blood pressure homeostasis via ACE-inhibitory components from connective tissue, and iv) maintaining functional gut environment through meat-derived nucleotides and nucleosides. In addition, meat could be an important source of phytanic acid, conjugated linoleic acids and antioxidants. Further, it becomes increasingly apparent that design of in vitro meat will be possible, and that this development may lead to improved health benefits from commercially viable and sustainable meat products.

Expression and activities of hepatic cytochrome P450 (CYP1A, CYP2A and CYP2E1) in entire and castrated male pigs.

This study aimed to provide further insights into the mechanism of in vivo regulation of cytochrome P450 (CYP450) 1A, 2A and 2E1 activities in pigs with different levels of testicular steroids. Hepatic mRNA and protein expression and enzymatic activity of CYP1A, CYP2A and CYP2E1 were compared between entire male and castrated pigs. Castration was performed either surgically or immunologically. The pigs were divided into four groups. In the first group, piglets were surgically castrated without anaesthesia. Immunological castration was performed by vaccination with Improvac® (Pfizer Ltd). Vaccinated pigs were subdivided into two groups according to the vaccination regimen: early and standard vaccination. Pigs in the early vaccination group were vaccinated when aged 11 and 15 weeks. Pigs in the standard vaccination group were vaccinated when aged 17 and 21 weeks. In the control group, pigs remained intact throughout the study. Hepatic CYP450 mRNA expression, measured by real-time RT-PCR, differed significantly between groups for all isoforms measured: CYP1A2 (P = 0.002), 2A (P = 0.000) and 2E1 (P = 0.002). Lower CYP450 mRNA in entire male pigs suggests suppression of CYP1A2, CYP2A and CYP2E1 by testicular steroids at the transcriptional level. However, this suppression was not always reflected in decreased protein expression and activities of these isoforms, suggesting that at least some CYP450s (e.g. CYP2E1) are regulated by a post-transcriptional mechanism.

In vitro cytochrome P450 2E1 and 2A activities in the presence of testicular steroids.

Cytochrome P450 2E1 (CYP2E1) and 2A (CYP2A) are the main enzymes involved in the metabolism of skatole in pigs. In this study, physiological concentrations of androstenone, 17ß-oestradiol and testosterone were tested for their ability to regulate CYP2E1 and CYP2A activity in liver microsomes isolated from entire male and female pigs as well as in microsomes from Saccharomyces cerevisiae expressing either human recombinant CYP2E1 or CYP2A6. We found that physiological concentrations of androstenone and oestradiol had the ability to inhibit CYP2E1 activity. The magnitude of this inhibition (approximately 30%) was similar in recombinant human CYP2E1 and microsomes from entire male pigs. This inhibition was only seen when adding the steroid to the assay 15 min before the substrate. Interestingly, CYP2E1 activity in the microsomes from female pigs was not affected. None of the investigated steroids modified the activity of recombinant human CYP2A6. However, CYP2A activity was slightly increased in the microsomes from female pigs in the presence of oestradiol, but the magnitude of this increase was very low (below 10%) and probably irrelevant. Overall, these results indicate that physiological concentrations of androstenone and oestradiol have a potential to inhibit CYP2E1 activities in vitro, and that this inhibition is gender-specific. Further studies are needed to investigate the biochemical mechanisms underlying those differences between the genders.

Gender-related differences in cytochrome P450 in porcine liver--implication for activity, expression and inhibition by testicular steroids.

In pigs, the hepatic cytochrome P450 (CYP) 1A2, 2A and 2E1 activity is important in the regulation of skatole accumulation in adipose tissue. This study investigated gender-related differences in CYP1A2, 2A and 2E1 dependent activity, protein and mRNA expression. This study also investigated the gonadal steroid dependent inhibition of CYP activity in relation to gender and dietary composition. Microsomes were prepared from the liver of female and entire male pigs (Landrace × Yorkshire sire and Duroc boars) reared under similar conditions and slaughtered at an age of 164 days. A group of entire male pigs fed dried chicory root for 16 days prior to slaughter were included in the study. CYP activities were assessed by the use of probe substrates, whilst mRNA and protein expression were analysed by RT-PCR and Western blotting. Furthermore inhibition of CYP dependent activity by gonadal steroids was assessed in vitro. Microsomes from female pigs had greater CYP1A2 and 2A activity, as well as mRNA expression compared to entire male pigs. The antibodies used did not detected differences in protein expression. In vitro inhibition by 17ß-oestradiol, oestrone, androstenone and 3ß-OH androstenol of CYP2E1 activity in microsomes from entire male pigs as well as inhibition of CYP1A activity in chicory fed entire male pigs was observed. Apart from that no effect of steroids was shown. In conclusion, female pigs show greater CYP activity and mRNA expression. Including chicory in the diet for 16 days changed the gonadal steroid dependent inhibition of CYP activity in entire male pigs.

IgE cross reactivity between reindeer and bovine milk beta-lactoglobulins in cow's milk allergic patients.

BACKGROUND: Allergic reactions to cow's milk are common in small children. One of the main protein allergens found in cow's milk is beta-lactoglobulin (beta-Lg). Reindeer and bovine milk both contain related beta-Lg proteins, but the allergenicity of reindeer beta-Lg has not previously been studied. The purpose of this study was to analyze the immunological cross-reactivity of IgE antibodies from children with cow's milk allergy to reindeer and bovine beta-Lg. METHODS: Sera from 17 children and a serum pool of 4 patients with elevated cow's milk-specific IgE were investigated. Beta-Lg from bovine and reindeer milk was isolated in native form and an enzyme-linked immunosorbent inhibition assay was developed. Bovine beta-Lg was used as a capturing antigen and the inhibiting effects of reindeer and bovine beta-Lg on the IgE binding were measured. RESULTS: Cross-reactivity patterns of bovine milk beta-Lg specific IgE to reindeer beta-Lg varied among patients. In general, reindeer beta-Lg showed significantly lower inhibition (mean 43%) of IgE binding to the capturing antigen than did bovine beta-Lg (mean 89%). In some patients, even high concentrations of reindeer beta-Lg only partly eliminated the IgE binding to bovine beta-Lg. CONCLUSIONS: The partial cross-reactivity of human anti-bovine IgE with reindeer beta-Lg suggests that it lacks important bovine epitopes and those that are recognized are only weakly bound.

Bet v 1 homologues in strawberry identified as IgE-binding proteins and presumptive allergens.

BACKGROUND: No strawberry allergen has so far been identified and characterized. METHODS: Serum samples were collected from patients with a suggestive case history of adverse reactions to strawberry and other fruits. Extracts from fresh and frozen strawberries were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and mass spectrometry. Patient blood samples were analysed for inhibition of IgE binding and basophil degranulation. RESULTS: Several IgE-binding proteins could be detected. In more than half of the patient sera, a 20/18-kDa doublet band was observed in Western blotting. These two bands were excised and analysed by mass spectrometry showing the presence of proteins belonging to the Bet v 1 family of allergens. Inhibition of the IgE binding to the 20/18-kDa doublet was obtained by addition of two recombinantly expressed allergens belonging to the Bet v 1 family (Bet v 1 and Mal d 1) and strawberry protein extract. In a cell-based assay of patient blood samples, basophil degranulation could be induced by strawberry protein extract and by Bet v 1 and Mal d 1. CONCLUSIONS: We conclude that strawberry homologues to Bet v 1 may be allergens of importance for adverse reactions to strawberry.

Approach to estimation of infrared point-target range.

The detection range for a blackbody point target is considered. An integrating photon detector is assumed, and the signal-to-noise ratio is expressed by use of photon-flux quantities. An equation for range, valid for the case of a background-limited photodetector, is formulated, and the solution to this equation is dependent on two parameters only: the extinction coefficient representing the atmospheric attenuation and one other parameter that includes other relevant system parameters in a simple way. This means that range as a function of different parameters can be conveniently summarized in one graph. Relations between different parameters are also discussed.

The social aspects of food biotechnology: a European view.

The application of the modern biotechnology to food, notably through the use of GM, has raised concern amongst the European public. Values that underlie this public concern about food biotechnology, include perceptions of: trust, choice, need, and care for a sustainable society and natural balance. Recommendations are advocated for addressing these social aspects, in terms of improving consumer choice, promoting greater public involvement in decision making and achieving a sustainable society. A model of risk analysis for genetically modified organisms (GMOs) and genetically modified food that incorporates this social dimension, through the integration of risk analysis with a social impact analysis is proposed, in order to build greater popular trust into the decision making processes.

Loss of DCC expression and glioma progression.

The deleted in colorectal cancer (DCC) gene, a candidate tumor suppressor gene on chromosome 18q21, encodes a neural cell adhesion molecule family protein that is most highly expressed in the nervous system. To address the hypothesis that DCC may play a role in glioma development and/or progression, we examined DCC expression by immunohistochemistry in 57 resected human astrocytic tumors. Overall, low-grade astrocytomas were predominantly DCC positive (15 of 16, or 94%), whereas high-grade tumors significantly less often expressed the DCC protein (27 of 41, or 66%; P = 0.03). We were able to directly assess the relationship between DCC expression and tumor progression in 15 patients who initially presented with a low-grade astrocytoma and subsequently recurred with a glioblastoma. Within this panel of paired lesions from the same patient, 14 of 15 (93%) low-grade tumors expressed the DCC protein, whereas only 7 of 15 (47%) corresponding glioblastomas were DCC positive. We also observed that secondary glioblastomas resulting from malignant progression of low-grade astrocytomas were more often DCC negative (8 of 15, or 53%) compared with primary or de novo glioblastomas (6 of 26, or 23%; P = 0.05). These findings implicate DCC inactivation in glioma progression and also demonstrate that DCC expression is preferentially, but not exclusively, lost in the genetic pathway to secondary glioblastoma multiforme.

DCC expression is altered by multiple mechanisms in brain tumours.

The DCC (deleted in colorectal cancer) candidate tumor suppressor gene spans greater than 1350 kilobases at chromosomes 18q21.1 and encodes a transmembrane protein of unknown function. Although DCC is expressed in a number of adult tissues, its expression is highest in the brain and we have, therefore, undertaken studies to determine if DCC inactivation might contribute to tumors arising there. Decreased or absent DCC protein expression was noted in more than 50% of the thirty brain tumors studied. Although specific mutations in the DCC gene were not identified, a variety of mechanisms appeared to contribute to the altered DCC expression, including allelic loss, aberrant splicing of transcripts and allele-specific loss of transcripts. In total, the data suggest that DCC inactivation may be important in brain tumor pathogenesis.

Malondialdehyde concentration in plasma is inversely correlated to the proportion of linoleic acid in serum lipoprotein lipids.

It has been suggested that the susceptibility of low density lipoprotein (LDL) to oxidative stress depends on the balance between its contents of polyunsaturated fatty acids and antioxidants. In a healthy reference population (n = 103), the plasma concentration of malondialdehyde (MDA) (mean 0.86, range 0.50-1.27 mumol/l) was positively correlated to the serum concentrations of LDL cholesterol (r = 0.31, P = 0.001), very low density lipoprotein triglycerides (r = 0.25, P = 0.009) and apolipoprotein B (r = 0.23, P = 0.03), and negatively correlated to lipid corrected alpha tocopherol in serum (r = -0.22, P = 0.02) and lipoprotein(a) (Lp(a)) (r = -0.26, P = 0.01). Plasma MDA was negatively correlated to the content of linoleic acid in the serum lipoprotein phospholipids (r = -0.35, P = 0.0008). In a stepwise regression analysis 12% of the variation in plasma MDA was explained by variations in the content of linoleic acid and 27% after addition of Lp(a) and abdominal sagittal diameter. The significant negative relation between plasma MDA and the amount of linoleic acid in the lipoprotein lipids indicates that other factors, e.g. the availability of anti-oxidants and the lipoprotein metabolism, may be of greater importance for intravascular lipid peroxidation than the proportion of polyunsaturated fatty acids in the lipoprotein lipids.

Studies of the deleted in colorectal cancer gene in normal and neoplastic tissues.

Chromosome 18q is among the chromosomal regions thought to harbor a tumor suppressor gene(s) that is frequently inactivated during the development of several cancer types, particularly those of the gastrointestinal tract. Moreover, preliminary data suggest that colorectal cancers with 18q LOH have a more aggressive clinical behavior than those cancers without 18q LOH. A candidate tumor suppressor gene from 18q, termed DCC for deleted in colorectal cancer, has been identified. The DCC gene is contained within the common region of LOH on 18q, its expression is markedly decreased or absent in colorectal cancers and cell lines, and a subset of colorectal cancers have been shown to have somatic mutations within the DCC gene. Thus, DCC represents the most promising candidate tumor suppressor gene from 18q. At present, however, many questions remain regarding the mechanisms underlying the inactivation of DCC in the majority of colorectal cancers. In addition, although studies of 18q LOH and DCC gene expression in other cancer types suggest that DCC inactivation may contribute to the pathogenesis of other tumor types, few studies have provided definitive data to demonstrate that DCC inactivation is a critical genetic event in these tumors. Moreover, little is known about the function of DCC in the regulation of normal cell growth and tumor suppression. The predicted structural similarity of DCC to the N-CAM family of cell-surface proteins suggests that it may function through cell-cell and/or cell-extracellular matrix interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of storage on ice and various freezing treatments on enzyme leakage in muscle tissue of rainbow trout (Oncorhynchus mykiss).

In order to study the biochemical changes in fish muscle during ice storage and freezing-thawing processes, the activities of certain marker enzymes in the cell interstitial fluid from muscle tissue of rainbow trout (Oncorhynchus mykiss) were measured. The enzymes analysed were: lysosomal alpha-glucosidase (E.C. 3.2.1.20), beta-N-glucosaminidase (E.C. 3.2.1.30) and acid phosphatase (E.C. 3.1.3.2). The activity in centrifuged tissue fluid (CTF) was compared with the activity in total homogenate. When ice storage was varied between 3 and 14 days, it did not affect enzyme leakage into the CTF significantly. However, there was a distinct difference between fresh fish and fish iced even for only 1 day, which gave increased leakage of marker enzymes. When the ice-stored samples were subject to a freezing-thawing cycle they showed a marked increase in enzyme activity in the press juice. When the freezing process was varied so as to achieve different freezing rates, the slowest freezing rate caused the highest enzyme leakage.

Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity.

The heme oxygenase inhibitor, tin protoporphyrin, is being studied for the prevention of neonatal jaundice. This potential drug, however, is also a photosensitizer that could cause serious and unknown side effects when administered to newborns. Therefore, we have developed in vitro and in vivo procedures for the screening and further characterization of potentially safe heme oxygenase inhibitors. The ideal inhibitor: 1) contains a biocompatible metal, 2) is not degraded in tissues, 3) is a highly potent inhibitor of heme oxygenase, and 4) does not participate in photochemical reactions. Proto- and mesoporphyrin derivatives with the tin, zinc, manganese, chromium, nickel, and magnesium were screened in vitro for suitability. Chromium protoporphyrin and mesoporphyrin were further studied in vitro and in vivo and were found to meet the ideal criteria. Chromium mesoporphyrin appeared to be the most potent in vitro inhibitor of adult Wistar rat tissue heme oxygenase. Four mumol of chromium protoporphyrin or chromium mesoporphyrin/kg body weight, administered intraperitoneally to adult male Wistar rats given a heme load through intraperitoneal administration of 30 mumol heme/kg body weight, caused significant suppression of hemolysis-induced increase in carbon monoxide production to 72 and 44% of control, respectively, 5.5 h after treatment. At t = 6 h, the tissue heme oxygenase activity, measured in vitro, was significantly reduced to 33 and < 5% in liver and to 22 and < 5% in spleen after the administration of chromium protoporphyrin and mesoporphyrin, respectively, but was not reduced in brain. The results show that there exist effective metalloporphyrin heme oxygenase inhibitors without photosensitizing properties.

Fenton reagents may not initiate lipid peroxidation in an emulsified linoleic acid model system.

This study includes two parts. First, the Fe2+ autooxidation and chelation processes in the presence of the chelators ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA) were studied by measuring UV light absorbance alterations. Competition for Fe3+ between chelators and water or phosphate buffer (PB) ions was confirmed. The addition of EDTA or DTPA to Fe3+ in water or PB only slowly turned the water/PB-Fe3+ complexes to EDTA-Fe3+ or DTPA-Fe3+ complexes. In the second part of this study, the initiation mechanisms of Tween 20 emulsified linoleic acid peroxidation under stimulation by chelator-Fe-O2 complexes were studied by measuring changes in UV light absorbance following diene conjugation. Fe3+ in the presence of EDTA or DTPA did not stimulate diene conjugation. Fe2+ (0.10 mM) and EDTA (0.11 mM) stimulated diene conjugation of the linoleic acid emulsion, but only after apparent Fe2+ autooxidation. Fe2+ and DTPA, as well as premixed DTPA-Fe2+ complex, resulted in very fast diene conjugation in a wide range of concentrations. A nonlinear, mainly square root relation between Fe2+ concentration and peroxidation rate was noted. Superoxide dismutase (SOD), catalase, and mannitol did not prevent the lipid peroxidation. H2O2 substantially decreased the DTPA-Fe2+ stimulated, otherwise rapid, diene conjugation but slightly enhanced the slower one stimulated by EDTA-Fe2+. Without ambient oxygen, Fenton reagents did not result in .H abstraction-related diene conjugation. The findings suggest that .OH resulting from Fenton reagents may not be the main cause for the initiation of peroxidation in this model system. Furthermore, a study with different combinations of Fe2+ and Fe3+ did not support the Fe2+/Fe3+ (1:1) optimum ratio hypothesis. We therefore conclude that perferryl ions or chelator-Fe-O2 complexes may be responsible for the first-chain initiation of lipid peroxidation, at least in this model system.

Inhibition of the Antibacterial Lactoperoxidase-Thiocyanate-Hydrogen Peroxide System by Heat-Treated Milk.

The antibacterial system, lactoperoxidase-H2O2-SCN- was affected by the presence of heated milk or skim milk reconstituted from powders having received severe heat treatment. This inhibitory effect was related to the increase in exposed sulfhydryl groups and to the redistribution of protein between micellar and whey phases. Chromatographic analyses of heat-treated milk showed that the inhibitory factor was associated with the casein micelle fraction. The inhibition, however, was overcome by addition of unheated skim milk.

Casein micelle size and composition related to the enzymatic coagulation process.

Chymosin (EC 3.4.23.4) and rennet, the latter containing about 85% chymosin and 15% pepsin, have been compared according to their coagulation properties with native micelles of different sizes or monomeric caseins as substrate. The casein micelles were separated on columns of controlled-pore glass (CPG-10/3000), which fractionates particles of up to 300 nm diameter. The results show that the coagulation time varies with the micelle size. The effect, which is more pronounced with chymosin than with rennet, appears to be related to the availability of kappa-casein. Therefore the largest micelles, with a lower kappa-casein content, showed longer coagulation times than medium size micelles. In the region of the smallest micelles this time increases again, probably due to an increased beta-casein content. Addition of monomeric kappa-casein decreased the coagulation time with both rennet and chymosin, but alpha s1-and beta-casein had the opposite effect. When isolated monomeric caseins were treated alone with rennet or chymosin, kappa-casein caused turbidity, but alpha s1-and beta-casein did not. Centrifugation experiments with micelles after monomeric casein addition showed that a limited amount of the added casein was able to join the micelle. This was confirmed by chromatographic studies.

CPG-chromatography studies of the stability of casein micelles.

Casein micelles fractionated on controlled pore glass (CPG-10/3000) were shown to be stable by recycling experiments. Only minor effects on the size distribution of the casein micelles were found after heating skim-milk to 100 degrees C for 10 min, or freeze-drying skim-milk at -70 degrees C followed by resuspension in the synthetic milk serum of Jenness & Koops (1962). The heating caused some whey proteins (beta-lactoglobulin) to enter the micelle fractions while the freeze-drying caused some of the largest micelles to disrupt. In colloidal calcium phosphate-free skim-milk prepared according to Pyne & McGann (1960) all the micelles appeared to dissociate into monomeric caseins.