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We have disrupted expression of the mitochondrial Friedreich ataxia protein frataxin specifically in murine hepatocytes to generate mice with impaired mitochondrial function and decreased oxidative phosphorylation. These animals have a reduced life span and develop multiple hepatic tumors. Livers also show increased oxidative stress, impaired respiration and reduced ATP levels paralleled by reduced activity of iron-sulfur cluster (Fe/S) containing proteins (ISP), which all leads to increased hepatocyte turnover by promoting both apoptosis and proliferation. Accordingly, phosphorylation of the stress-inducible p38 MAP kinase was found to be specifically impaired following disruption of frataxin. Taken together, these findings indicate that frataxin may act as a mitochondrial tumor suppressor protein in mammals
Spectrofluorimetric studies have revealed that aflatoxin B-1 (AFB(1)) interacts with signal recognition particle (SRP), which acts as an escort for polyribosomes with signal peptides to be transported and bound to the cytoplasmic face of the endoplasmic reticulum (ER). We further report that the binding of AFB(1) to SRP is selective as it only binds to two (SRP9 and 14) out of its three constituent polypeptides studied. Binding of AFB(1) to proteins is known to alter their conformations. Interactions of AFB(1) with SRP polypeptides may generate structural and functional alterations in this particle and hinder secretory protein synthesis. Copyright (C) 2004 John Wiley Sons, Ltd
Appropriate animal models such as preruminant calves are necessary to study the complex physiological functions of carotenoids and to relate them to possible health effects in humans. In this study, the bioavailability and metabolism of lycopene from 2 dietary supplements were compared. LycoVit (R) containing synthetic lycopene and Lyc-O- Mato (R) containing natural tomato oleoresin were administered to 2 groups of preruminant calves (each n = 8) for 14 d in daily doses of 15 mg of lycopene. Plasma was analyzed for carotenoids before the intervention period, directly after, and each day for 5 d after the end of the intervention. All-trans and 5-cis lycopene, and 3 lycopene metabolites not previously found in calf plasma were detected. These metabolites contributed 52% of the total lycopene content measured at the end of the intervention period. Based on spectroscopic data, they might be hydrogenation products, which are formed from all-trans and/or 5-cis lycopene. In the LycoVit group, total lycopene concentrations were similar to 300% higher (286 +/- 89 nmol/L) than in the Lyc-O-Mato group (72 33 nmol/L) (P < 0.001). This indicates that, unlike in humans, lycopene from LycoVit and Lyc-O-Mato does not have equal bioavailabilities in preruminant calves. Therefore, the preruminant calf may not be a suitable animal model with which to study the biological and physiological effects of lycopene
Xanthohumol (XN) is the principal prenylated flavonoid of the hop plant and has recently gained considerable interest due to its potential cancer-chemopreventive effects. However, the metabolism of XN has not yet been investigated in detail. Therefore, we studied the in vitro phase 11 metabolism of XN using nine human recombinant UDP- glucuronosyltransferases (UGT) and five sulfotransferases (SULT). The identification of the metabolites formed was elucidated using HPLC with diode array detection as well as HPLC/API-ES MS. XN was efficiently glucuronidated by UGT 1A8, 1A9, and 1A10; further important UGTs were UGT 1A1, 1A7, and 2B7. With respect to the sulfation reaction, SULT 1A1*2, 1A2, and 1E1 were the most active SULT forms. UGT 1A3, 1A4, and 1A6 as well as SULT 1A3 and 2A1 were of minor importance for the conjugation of XN. Three mono-glucuronides as well as three mono-sulfates were identified. Considering the tissue distribution of the tested UGT and SULT enzyme forms, these findings suggest a prominent role for the glucuronidation and sulfation of XN in the liver as well as in the gastrointestinal tract
Bromelain was allowed to react with phenolic compounds. The activity and selected physico-chemical properties of the resulting derivatives were characterized. In vitro experiments showed that the proteolytic activity of bromelain was inhibited. Bromelain also serves as a food protein, because food stuffs based on pineapple contain relatively high concentrations of bromelain. In vitro digestion of bromelain derivatives with the main proteolytic enzymes of the gastrointestinal tract was also adversely affected. A covalent attachment of the phenolic compounds was identified at the tryptophan, free amino (lysines and N-terminal) and thiol groups of bromelain. A decrease in solubility of the derivatives was observed. The isoelectric point was shifted to lower pH values and high molecular weight fractions were identified. All effects observed depended on the reactivity of the phenolic substances. Two supplementary food products containing both bromelain and quercetin were also tested in terms of their proteolytic activity and digestibility
The intention of this study was to increase the knowledge on the composition and structure of coffee bean proteins and the changes induced in them especially with regard to their interactions with the phenolic compounds also present. For this purpose green coffee beans were extracted by means of standard methanol extraction to quantify the chlorogenic acid content. Different solubilisation buffers were applied to extract the protein fractions with or without prior fat removal. The protein samples thus obtained were analysed by different methods (RP-HPLC, SDS-PAGE and SELDI-TOF- MS). Preliminary model studies were performed to characterize the interactions between the isolated green coffee protein fractions and chlorogenic acid (the major phenolic compound in coffee beans) with the intention of fulfilling the ultimate goal of characterizing such reactions in roasted coffee. The results show that the content of chlorogenic bound covalently to the protein increases. A reaction with the nucleophilic protein side chains (tryptophan, cystein and lysine) was recorded. Cross-inked protein polymers were also detected, whereby the a-chain was found to be more reactive. These reactions effect the solubility of the coffee bean proteins, the latter in turn becoming more acidic in nature. The secondary structure was affected only slightly as determined by circular dichroism. The in-vitro tryptic digestibility was also influenced, where again the cc-chain seems to be more susceptible. The observed polymerisation due to derivatisation by chorogenic acid declines the digestion. Similar digestion behaviour was also observed during tryptic hydrolysis of roasted coffee compared to that of green coffee, roasting allowing more stronger denaturation caused by the accompanying Maillard reaction. The derivatised green coffee bean proteins were found to have moderate antioxidative capacity
In the context of this study, the noncovalent binding of selected phenolic compounds (chlorogenic, ferulic, and gallic acids, quercetin, rutin, and isocluercetin) to different proteins (human serum albumin, bovine serum albumin, soy glycinin, and lysozyme) was studied with direct (Hummel- Dreyer/size exclusion chromatography) and/or indirect methods (fluorescence absorbance properties of the binding components). In the latter case, the measurement of the phenol binding was achieved by exploiting the intrinsic fluorescence emission properties of cluercetin as a probe. From the data obtained, the binding constants and the number of binding sites were calculated. The binding parameters were influenced by different factors, where, e.g., increasing temperature and ionic strength as well as decreasing pH cause a diminished binding. The structures of the proteins as determined by circular dichroism indicate changes in the tertiary structure with the secondary structure remaining intact
Megalin-mediated reuptake of retinol in the kidneys of mice is essential for vitamin A homeostasis
(2005)
The reuptake of retinol (ROH) and retinol-binding protein (RBP) in the kidneys is mediated by the endocytic receptor megalin, suggesting an important role for this receptor in vitamin A (VA) metabolism. We examined the extent to which megalin deficiency may affect urinary ROH excretion, levels of ROH and RBP in plasma, as well as storage of VA in liver and kidney. For this purpose, mice with a kidney-specific megalin gene defect (megalin(lox/lox):; apoE(Cre)) and control mice (megalin(lox/lox)) were fed either a basal diet containing 4500 retinol equivalents (RE)/kg diet or a diet without VA during experimental periods of 42 and 84 d. Urinary ROH excretion was observed only in megalin(lox/lox); apoE(Cre) mice (P < 0.0001, 2-way ANOVA) and not in the controls. Plasma ROH and RBP differed only by diet (P < 0.05), but not genotype (P = 0.615). A major effect of megalin deficiency, however, was evident in retinyl ester levels in the liver (P < 0.05), which were similar to 37% lower than those in megalin(lox/lox) controls (P < 0.05, Student's t test) during the 84-d period of dietary VA deprivation. Kidney levels of VA were not affected by the receptor gene defect. The findings demonstrate that urinary ROH excretion caused by megalin deficiency requires accelerated mobilization of hepatic VA stores to maintain normal plasma ROH levels, which suggests that megalin plays an essential role in systemic VA homeostasis
The term proteinuria is taken to mean abnormally high protein excretion in the urine. Proteinuria is the consequence of glomerular filtration of plasma proteins, their subsequent reabsorption by the proximal tubular cells and secretion of protein by the tubular cells and distal urinary tract. In physiological conditions, the structural integry of the glomerular filtration barrier prevents the abnormal passage of albumin (molecular mass 66 kDa) and high-molecular- weight proteins (> 66 kDa),whereas the passage of low-molecular-weight proteins (< 66 kDa) is almost completely unrestricted. Proteins that arrive the tubular lumen are reabsorbed by endocytosis after binding to the megalin-cubilin complex. An increased load of proteins in the tubular lumen leads to the saturation of the reabsorptive mechanism and higher urinary protein excretion. Proteinuria can originate from prerenal, renal and postrenal causes. Elevated tubular protein concentrations have been recognized to be toxic to tubular cells and associated with the progression of chronic renal disease. Therefore, the quantitative and qualitative evaluation of proteinuria is important for the diagnosis of renal disease
Studies in vitro show important interactions among vitamin A, lactoferrin, and insulin-like growth factor (IGF) binding proteins (IGFBP) and, thus, the IGF system. As a consequence, mammary gland epithelial cell proliferation and apoptosis during the bovine dry period and potential milk yield may be affected. We have studied effects of feeding vitamin A (550,000 IU/d) that exceed daily requirements about 8-fold for up to 2 mo to dairy cows during the dry period on concentrations of retinol and its metabolites in plasma and milk, milk lactoferrin, plasma and milk IGF-I and IGFBP- 3, lactoferrin and IGF-I mRNA levels in mammary gland tissue, mammary gland apoptosis, and 100-d milk yield in the ensuing lactation. In the group supplemented with vitamin A, the peripartal decrease of plasma retinol was delayed and attenuated, and colostral retinol plus retinylester concentration was enhanced, but colostral beta-carotene concentration decreased. The retinoic acid isomer 9,13-dicis retinoic acid that coeluted with 13-cis retinoic acid, was the predominant circulating retinoic acid and was higher in GrA than the control group. Plasma IGFBP-3 concentrations were positively correlated with plasma retinol concentrations (r = 0.51), but there were no group differences. Numbers of apoptotic epithelial cells in mammary epithelium were higher at drying off and parturition than in the middle of the dry period, coinciding with high concentrations of IGF-I and lactoferrin in mammary secretions. At parturition, numbers of apoptotic cells in mammary gland biopsies in cows supplemented with vitamin A were higher than in control cows. In conclusion, supplementation of dairy cows during the dry period with high amounts of vitamin A did not significantly modify concentrations of lactoferrin, IGFBP-3, and IGF-I in plasma and in mammary secretions, but slightly decreased energy-corrected 100-d milk yield and milk fat yield, possibly because of enhanced apoptic rates of mammary cells
During processing and storage, phenolic compounds (PCs) may react with food protein bound amino acids (AAs). Such reactions have been reported to change physicochemical and to decrease in vitro digestion properties of proteins. A rat growth and nitrogen (N) balance study was conducted to prove whether derivatization with chlorogenic acid (CA) affects the nutritional quality of beta-lactoglobulin (beta-LG). Test diets (10% protein level) contained nonderivatized beta-LG (LG, treated under omission of CA), low derivatization level beta-LG (LGL), high derivatization level beta-LG (LGH), or casein supplemented with L-methionine (0.3% of diet; C+met) as an internal standard. An additional group received untreated beta-LG supplemented with pure CA (1.03% of diet; LG+CA). The AA composition of test proteins, plasma AAs, and liver glutathione (GSH) concentrations were determined. Protein digestibility-corrected amino acid score (PDCAAS) was calculated using human or rat AA requirement patterns and rat fecal digestibility values. N excretion was significantly higher in feces and lower in urine of rats fed with LGH as compared to LG and LGL. Consequently, true N digestibility (TND) was significantly lower with LGH as compared to LG and LGL. The lower content of methionine, cysteine, lysine, and tryptophan in LGH corresponded to a reduced TND. Net protein utilization (NPU) was not different between treated beta-LG fed diet groups but was lower than in LG+CA and C+met fed groups. Only at a relatively high level of derivatization with CA, the otherwise good nutritional quality of beta-LG is affected so that TND is reduced, while NPU still remains unaffected. Derivatization of beta-LG with CA does not seem to lead to an additional deficiency in a specific indispensable AA in growing rats fed with 10% protein
The human FP-R (F2alpha prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu(132) in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132-->Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.
1 Two isoforms of the rat prostaglandin E-2 receptor, rEP3 alpha-R and rEP3 beta-R, differ only in their C- terminal domain. To analyze the function of the rEP3-R C-terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C-terminal domain of the rEP3 alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3 alpha-ST341-349A-R) were stably expressed in HEK293 cells. 2 All rEP3-R receptors showed a similar ligand- binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation. 3 Repeated exposure of cells expressing the rEP3 alpha-R isoform to PGE(2) reduced the agonist induced inhibition of forskolin-stimulated cAMP-formation by 50% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi- response as well as plasma membrane localization of the rEP3 beta-R and the rEP3 alpha-ST341-349A-R were not affected by prior agonist-stimulation. 4 Agonist-stimulation of HEK293-rEP3 alpha-R cells induced a time- and dose-dependent phosphorylation of the receptor most likely by G protein-coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist-stimulation the rEP3 beta-R was not phosphorylated and the rEP3 alpha-ST341-349A-R was phosphorylated only weakly. 5 These results led to the hypothesis that agonist-induced desensitization of the rEP3 alpha-R isoform is mediated most likely by a GRK-dependent phosphorylation of Ser/Thr residues 341 - 349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization
An imbalance between formation and detoxification of oxygen radicals leads to oxidant stress that may increase in more intense oxidative metabolism caused by a high intake of metabolizable energy to provide metabolic intermediates for the milk synthesis and secretion. This hypothesis was tested using dairy cows and the concentration of hydroperoxides in lipids (LHP) extracted from circulative lipoprotein particles of low and very low density (LDL and VLDL/chylomicrons) as oxidant stress indicator. The particles were prepared by ultracentrifugation of serum obtained by coccygeal bleeding (13 cows, 1. parity, n=8 and 2. parity, n=5, lactation stage, 53 +/- 1.4 days post partum) and purified by precipitation. Concentrations of LHP-LDL/mg Lipoprotein correlated significantly with daily milk yield (r = 0.73, P = 0.004) or daily milk energy output (r = 0.77, P = 0.003) in contrast to LHP of VLDL/chylomicron particles. Thus, some evidence was obtained for an almost linear, positive relationship between milk productivity and oxidant stress occurring in LDL
OBJECTIVE - We studied the association of digestible carbohydrates, fiber intake, glycemic index, and glycemic load with insulin sensitivity (S-I), fasting insulin, acute insulin response (AIR), disposition index, BMI, and waist circumference. RESEARCH DESIGN AND METHODS - Data on 979 adults with normal (67%) and impaired (33%) glucose tolerance from the Insulin Resistance Atherosclerosis Study (1992-1994) were analyzed. Usual dietary intake was assessed via a 114- item interviewer-administered food frequency questionnaire from which nutrient intakes were estimated. Published glycemic index values were assigned to food items and average dietary glycemic index and glycemic load calculated per subject. S-I and AIR were determined by frequently sampled intravenous glucose tolerance test. Disposition index was calculated by multiplying S-I with AIR. Multiple linear regression modeling was employed. RESULTS - No association was observed between glycemic index and S-I fasting insulin, AIR, disposition index, BMI, or waist circumference after adjustment for demographic characteristics or family history of diabetes, energy expenditure, and smoking. Associations observed for digestible carbohydrates and glycemic load, respectively, with S-I insulin secretion, and adiposity (adjusted for demographics and main confounders) were entirely explained by energy intake. In contrast, fiber was associated positively with S-I and disposition index and inversely with fasting insulin, BMI, and waist circumference but not with AIR. CONCLUSION - Carbohydrates as reflected in glycemic index and glycemic load may not be related to measures of insulin sensitivity, insulin secretion, and adiposity. Fiber intake may not only have beneficial effects. on insulin sensitivity and adiposity, but also on pancreatic functionality
Vitamin E : elucidation of the mechanism of side chain degradation and gene regulatory functions
(2005)
For more than 80 years vitamin E has been in the focus of scientific research. Most of the progress concerning non-antioxidant functions, nevertheless, has only arisen from publications during the last decade. Most recently, the metabolic pathway of vitamin E has been almost completely elucidated. Vitamin E is metabolized by truncation of its side chain. The initial step of an omega-hydroxylation is carried out by cytochromes P450 (CYPs). This was evidenced by the inhibition of the metabolism of alpha-tocopherol by ketoconozole, an inhibitor of CYP3A expression, whereas rifampicin, an inducer of CYP3A expression increased the metabolism of alpha-tocopherol. Although the degradation pathway is identical for all tocopherols and tocotrienols, there is a marked difference in the amount of the release of metabolites from the individual vitamin E forms in cell culture as well as in experimental animals and in humans. Recent findings not only proposed an CYP3A4-mediated degradation of vitamin E but also suggested an induction of the metabolizing enzymes by vitamin E itself. In order to investigate how vitamin E is able to influence the expression of metabolizing enzymes like CYP3A4, a pregnane X receptor (PXR)-based reporter gene assay was chosen. PXR is a nuclear receptor which regulates the transcription of genes, e.g., CYP3A4, by binding to specific DNA response elements. And indeed, as shown here, vitamin E is able to influence the expression of CYP3A via PXR in an in vitro reporter gene assay. Tocotrienols showed the highest activity followed by delta- and alpha-tocopherol. An up-regulation of Cyp3a11 mRNA, the murine homolog of the human CYP3A4, could also be confirmed in an animal experiment. The PXR-mediated change in gene expression displayed the first evidence of a direct transcriptional activity of vitamin E. PXR regulates the expression of genes involved in xenobiotic detoxification, including oxidation, conjugation, and transport. CYP3A, e.g., is involved in the oxidative metabolism of numerous currently used drugs. This opens a discussion of possible side effects of vitamin E, but the extent to which supranutritional doses of vitamin E modulate these pathways in humans has yet to be determined. Additionally, as there is arising evidence that vitamin E's essentiality is more likely to be based on gene regulation than on antioxidant functions, it appeared necessary to further investigate the ability of vitamin E to influence gene expression. Mice were divided in three groups with diets (i) deficient in alpha-tocopherol, (ii) adequate in alpha-tocopherol supply and (iii) with a supranutritional dosage of alpha-tocopherol. After three months, half of each group was supplemented via a gastric tube with a supranutritional dosage of gamma-tocotrienol per day for 7 days. Livers were analyzed for vitamin E content and liver RNA was prepared for hybridization using cDNA array and oligonucleotide array technology. A significant change in gene expression was observed by alpha-tocopherol but not by gamma-tocotrienol and only using the oligonucleotide array but not using the cDNA array. The latter effect is most probably due to the limited number of genes represented on a cDNA array, the lacking gamma-tocotrienol effect is obviously caused by a rapid degradation, which might prevent bioefficacy of gamma-tocotrienol. Alpha-tocopherol changed the expression of various genes. The most striking observation was an up-regulation of genes, which code for proteins involved in synaptic transmitter release and calcium signal transduction. Synapsin, synaptotagmin, synaptophysin, synaptobrevin, RAB3A, complexin 1, Snap25, ionotropic glutamate receptors (alpha 2 and zeta 1) were shown to be up-regulated in the supranutritional group compared to the deficient group. The up-regulation of synaptic genes shown in this work are not only supported by the strong concentration of genes which all are involved in the process of vesicular transport of neurotransmitters, but were also confirmed by a recent publication. However, a confirmation by real time PCR in neuronal tissue like brain is now required to explain the effect of vitamin E on neurological functionality. The change in expression of genes coding for synaptic proteins by vitamin E is of principal interest thus far, since the only human disease directly originating from an inadequate vitamin E status is ataxia with isolated vitamin E deficiency. Therefore, with the results of this work, an explanation for the observed neurological symptoms associated with vitamin E deficiency can be presented for the first time.
The structure of interaction products resulting from the reaction of unmodified glucose with benzyl isothiocyanate is reported. Prior to their identification, the main products of this reaction were isolated using solid- phase extraction (SPE) as well as preparative HPLC. They were then identified by NMR and MS as 3-benzyl-4-hydroxy-5-(D- arabino-1,2,3,4-tetrahydroxybutyl)- 1,3-oxazolidine-2-thione, 3-benzyl-4-hydroxy-4-hydroxymethyl-5-(D-erythro-1,2,3- trihydroxypropyl)- 1,3-oxazolidine-2-thione, N-benzyl-(D-gluco-4,5-dihydroxy-6-hydroxymethyl-tetrahydropyrano)[2,3-b] oxazolidine-2-thione and 3-benzyl-4-(N-benzyl amino)-5-(D-arabino-1,2,3,4-tetrahydroxybutyl)-1,3-thiazolidine-2-thione . The identity of the last compound was secured by X-ray crystal structure data. (C) 2004 Elsevier Ltd. All rights reserved