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BACKGROUND: Recent studies indicate that the bioavailability of anthocyanins is extremely low. One of the possible reasons could be their binding to proteins. Therefore, the binding affinity of cyanidin-3-glucoside (Cy3glc) to HSA and alpha-amylase was investigated by the quenching of protein tryptophan fluorescence. From data obtained, the binding constants and the free Gibbs energy were calculated. The changes in conformation of the proteins tested were studied with circular dichroism and the influence of binding on alpha-amylase activity determined. RESULTS: Cy3glc quenched the tryptophan fluorescence and upon ligand binding a change in protein structure was observed related to the corresponding decrease in the et-amylase activity. The association constants of 25 to 77 x 10(3) L mol(-1) were calculated for different proteins, indicating weak interactions of non-covalent nature. Competitive binding with HSA in the presence of 8-anilino-1-naphthalene sulfonic acid suggest involvement of hydrophobic interactions, in the case of HSA the possible site being subdomain IIA. CONCLUSION: The strongest affinity of Cy3glc for HSA being at pH 7 underlines its potential in transport and distribution of the phenolic compounds in organisms. An influence on salivary amylase activity is possible when drinking berry juices with high anthocyanins content.
Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting- incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics.
Isoflavone intake is associated with various properties beneficial to human health which are related to their antioxidant activity, for example, to their ability to increase LDL oxidation resistance. However, the distribution of isoflavones among plasma lipoproteins has not yet been elucidated in vivo. Therefore, the objective of the present study was to investigate the association between daidzein (DAI) and lipoproteins in human plasma upon administration of the aglycone and glucoside form. Five men aged 22-30 years participated in a randomised, double-blind study in cross-over design. After ingestion of DAI and daidzein-7-O-beta-D-glucoside (DG) (1 mg DAI aglycone equivalents/kg body weight) blood samples were drawn before isoflavone administration as well as 1, 2, 3, 4.5, 6, 8, 10, 12, 24 and 48 h post-dose. Concentrations of DAI in the different lipoprotein fractions (chylomicrons, VLDL, LDL, HDL) and in the non-lipoprotein fraction were analysed using isotope dilution capillary GUMS. The lipoprotein fraction profiles were similar for all subjects and resembled those obtained for plasma in our previously published study. The lipoprotein distribution based on the area under the concentration-time profiles from 0 h to infinity in the different fractions were irrespective of the administered form: non-lipoprotein fraction (53%) > LDL (20%) > HDL (14%) > VLDL (9-5%) > chylomicrons (2-5%). Of DAI present in plasma, 47% was associated to lipoproteins. Concentrations in the different lipoprotein fractions as well as in the non-lipoprotein fraction were always higher after the ingestion of DG than of DAI. Taken together, these results demonstrate an association between isoflavones and plasma lipoproteins in vivo.
Vitamin A (VA) deficiency in very low birth weight (VLBW) infants is associated with an increased risk for disorders related to kidney and lung maturation and function. VA losses through increased urinary retinol (ROH) excretion might contribute to this deficiency risk. The mechanism accounting for ROH loss in the urine has not yet been clarified. The aim of this study was to assess the excretion of ROH, retinol-binding protein 4 (RBP4) and transthyretin (TTR) in urine from VLBW infants in comparison with that in term infants in relation to kidney function. Urine specimens were collected from 15 VLBW infants (birth weight < 1,500 g) as well as from 20 term infants during the first 2 days after birth. ROH in urine was detectable in 14 of the 15 VLBW infants at a median concentration of 234 nmol/g creatinine. In the group of term infants, 17 of the 20 excreted ROH, but at an approximately five-times lower concentration (P<0.001). Excretion of RBP4 and TTR was also much higher in VLBW infants (both P<0.001). The urinary ROH excretion in VLBW infants may be related to the impaired tubular handling of its carrier proteins RBP4 and TTR. Thus, ROH excretion might contribute to an increased risk of VA deficiency, especially in VLBW infants.
The health promoting effects of a grapevine-shoot extract named Vineatrol (R) 30, which contains resveratrol (Resv) as well as considerable amounts of Resv oligomers, have recently been investigated. In the present study, we analyzed the free radical scavenging capacity, the ability to inhibit lipid peroxidation, and the capacity to enhance the human glutathione peroxidase 1 (GPx) and the human superoxide dismutase 1 (SOD) gene promoter activities of Vineatrol (R) 30. Vineatrol (R) 30 was able to scavenge the 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation and led to concentration-dependent inhibition of lipid peroxidation, Vineatrol (R) 30 not being superior to Resv alone in both cases. Vineatrol (R) 30 also enhanced the gene promoter activities of human GPx and SOD expressed in V79 cells, whereas this effect could not be demonstrated for Resv. In summary, the results presented in this study show that the Vineatrol (R) 30 grapevine-shoot extract is a free radical scavenger and potent antioxidant at non- eytotoxic concentrations.
This paper provides an overview of analytical techniques used to determine isoflavones (IFs) in foods and biological fluids with main emphasis on sample preparation methods. Factors influencing the content of IFs in food including processing and natural variability are summarized and an insight into IF databases is given. Comparisons of dietary intake of IFs in Asian and Western populations, in special subgroups like vegetarians, vegans, and infants are made and our knowledge on their absorption, distribution, metabolism, and excretion by the human body is presented. The influences of the gut microflora, age, gender, background diet, food matrix, and the chemical nature of the IFs on the metabolism of IFs are described. Potential mechanisms by which IFs may exert their actions are reviewed, and genetic polymorphism as determinants of biological response to soy IFs is discussed. The effects of IFs on a range of health outcomes including atherosclerosis, breast, intestinal, and prostate cancers, menopausal symptoms, bone health, and cognition are reviewed on the basis of the available in vitro, in vivo animal and human data.
The influence of a commercial production process for dehydrated potato flakes on the content of free phenolic compounds, total phenolics, and glycoalkaloids in potatoes during the subsequent processing steps was determined. Processing byproducts, such as potato peel (steam peeling), mashed potato residues, and side streams (blanching and cooking waters), have also been investigated. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify caffeic acid, gallic acid, ferulic acid, p-coumaric acid, p-hydoxybenzoic acid, protocatechuic acid, vanillic acid, catechin, and three isomers of caffeoylquinic acid: chlorogenic, neochlorogenic and cryptochlorogenic acid. Determination of the glycoalkaloids a-solanine and alpha-chaconine was performed by using a high- performance thin-layer chromatography (HPTLC) method. The deliverables reveal that processing potatoes to potato flakes remarkably diminishes the content of the analyzed compounds, mainly due to peeling and leaching. The influence of thermal exposure is less significant. About 43% of the initial phenolic acids and 10% of the glycoalkaloids remain after processing. The results of the total phenolic content assay by Folin-Ciocalteu reagent are proportional to the content of phenolic compounds determined by HPLC. Steam peeling has a higher influence on glycoalkaloid losses compared to that on phenolics. The highest amounts of phenolic compounds and glycoalkaloids were found in peeling byproduct. During processing, the amount of chlorogenic acid decreased, whereas the concentration of neochlorogenic acid increased due to isomerization. The impact of the results on potato processing technology is discussed.
beta-Carotene functions independently of vitamin A in the reproductive performance of dairy cows. The concentrations of beta-carotene in plasma decrease during the dry period, and reach a nadir in about the first week postpartum. This coincides with a negative energy balance, which affects the onset of the first ovulation in early postpartum cows. Thus, we hypothesised that plasma beta-carotene concentrations during the peripartum period may affect ovulation in the first follicular wave postpartum in dairy cows. The aim of the present study was to investigate changes in the profiles of plasma beta-carotene concentrations during the peripartum period in ovulatory and anovulatory cows during the first follicular wave postpartum. We used 22 multiparous Holstein cows, which were fed a total mixed ration consisting of grass, corn silage and concentrate, and collected blood samples for beta-carotene and progesterone analysis from week 3 prepartum to week 3 postpartum when the period of day 0-6 after parturition was regarded as the parturient week (week 0). The first ovulation was confirmed using the profile of plasma progesterone concentrations and colour Doppler ultrasound. Thirteen cows ovulated during the first postpartum follicular wave. Parity, the dry-off period, calving interval, mastitis episodes, and actual 305 days' milk yield during the previous lactation, and milk composition in the last month during the previous lactation in this study did not differ between ovulatory and anovulatory cows. Differences in the plasma beta-carotene profile were observed between ovulatory and anovulatory cows. Plasma P-carotene concentrations at week 3 prepartum were greater in ovulatory cows (2.97 +/- 0.24mg/L) than in anovulatory cows (1.53 +/- 0.14mg/L; P < 0.001), after that its concentrations in ovulatory cows decreased and reached the lowest level at week I postpartum, although its concentrations in anovulatory cows remained unchanged. No differences in plasma P-carotene concentrations between the two groups were observed postpartum. The present study indicates for the first time that the lower P-carotene concentrations in plasma during the prepartum period is associated with anovulation during the first follicular wave postpartum.
Effects of isoflavones on estrogen sensitive tissues are discussed controversially This study was designed to investigate tissue specific effects of an isoflavone exposure through different periods of life in female Wistar rats and to compare the effects of genistein (GEN) to those of mixed dietary isoflavones, GEN and daidzein (DAI). One group received an isoflavone-free diet (IDD). another was fed an isoflavone-rich diet (IRD) and the third group an IDD supplemented with GEN (GEN(d)) prior to mating. throughout pregnancy and up to weaning The offspring were kept on the respective diets during growth. puberty and adulthood The weight of the uterus, the height of the uterine and vaginal epithelium, the bone mineral density of the tibia, and the expression of the estrogen sensitive gene CaBP9K in the liver were determined. At d21, the uterine weight, the uterine epithelium and the expression of CaBP9K in the liver were significantly stimulated in GEN(d) animals compared to IDD and IRD Interestingly, bone mineral density was increased in GEN(d) and in IRD animals Around puberty (d50) neither uterine wet weights nor trabecular bone density differed significantly among the isoflavone groups and the IDD control. At d80 nosignificant differences in uterine weight were observed among IDD. GEN(d) and IRD animals. However, bone mineral density was increased in GEN(d) and IRD animals In summary, Our results demonstrate that lifelong dietary exposure to isoflavones can affect estrogen sensitive tissues, apparently in a tissue selective manner With respect to health risk and benefit our data indicate that an increased bone mineral density can be achieved by lifelong exposure to an IRD. which, in contrast to GEN supplementation, does not seem to stimulate the proliferation of the uterine epithelium
Aggravation by prostaglandin e-2 of interleukin-6-dependent insulin resistance in hepatocytes
(2009)
Hepatic insulin resistance is a major contributor to fasting hyperglycemia in patients with metabolic syndrome and type 2 diabetes. Circumstantial evidence suggests that cyclooxygenase products in addition to cytokines might contribute to insulin resistance. However, direct evidence for a role of prostaglandins in the development of hepatic insulin resistance is lacking. Therefore, the impact of prostaglandin E-2 (PGE(2)) alone and in combination with interleukin-6 (IL-6) on insulin signaling was studied in primary hepatocyte cultures. Rat hepatocytes were incubated with IL-6 and/or PGE(2) and subsequently with insulin. Glycogen synthesis was monitored by radiochemical analysis; the activation state of proteins of the insulin receptor signal chain was analyzed by western blot with phosphospecific antibodies. In hepatocytes, insulin-stimulated glycogen synthesis and insulin-dependent phosphorylation of Akt-kinase were attenuated synergistically by prior incubation with IL-6 and/or PGE(2) while insulin receptor autophosphorylation was barely affected. IL-6 but not PGE(2) induced suppressors of cytokine signaling (SOCS3). PGE(2) but not IL-6 activated extracellular signal-regulated kinase 1/2 (ERK1/2) persistently. Inhibition of ERK1/2 activation by PD98059 abolished the PGE(2)-dependent but not the IL-6-dependent attenuation of insulin signaling. In HepG2 cells expressing a recombinant EP3-receptor, PGE(2) pre-incubation activated ERK1/2, caused a serine phosphorylation of insulin receptor substrate 1 (IRS1), and reduced the insulin-dependent Akt-phosphorylation. Conclusion: PGE(2) might contribute to hepatic insulin resistance via an EP3-receptor-dependent ERK1/2 activation resulting in a serine phosphorylation of insulin receptor substrate, thereby preventing an insulin-dependent activation of Akt and glycogen synthesis. Since different molecular mechanisms appear to be employed, PGE(2) may synergize with IL-6, which interrupted the insulin receptor signal chain, principally by an induction of SOCS, namely SOCS3.
Background Canine primary immune-mediated hemolytic anemia (IMHA) is associated with a high-mortality rate. C- reactive protein (CRP) is the most important acute-phase protein in dogs and may have value as a marker of prognosis or response to treatment in IMHA. Objective The objectives of this study were to evaluate serum CRP concentration in dogs with primary IMHA at presentation and during treatment, to assess potential differences based on survival time, and to compare CRP with other laboratory parameters of inflammation and prognosis. Methods Inclusion criteria for primary IMHA were anemia (PCV < 0.30 L/L), a positive Coombs' test or persistent autoagglutination of erythrocytes, and the exclusion of underlying diseases by other diagnostic tests. Dogs were divided into 2 groups based on survival: dogs that were still alive 14 days after start of treatment (group 1) and dogs that died or were euthanized before day 14 (group 2). Serum CRP concentration, a CBC, and a biochemistry profile were performed on days 0, 3, 8, and 14. Serum CRP also was determined in 25 clinically healthy dogs. Results CRP concentration in the 25 clinically healthy dogs ranged from 0- 8.9 mu g/mL (median 2.2 mu g/mL). Thirty dogs were diagnosed with primary IMHA, 24 in group 1 and 6 in group 2. On day 0, CRP concentration in dogs in both groups (median 224 mu g/mL) was increased above the reference interval. In group 1 dogs, median CRP concentration was 242 mu g/mL on day 0, 69 mu g/mL on day 3, 35 mu g/mL on day 8, and 2 mu g/mL on day 14. In group 2 dogs, median CRP concentration was 194 mu g/mL on day 0, 119 mu g/mL on day 3, and 41 mu g/mL on day 8; only 1 dog in group 2 survived to day 8. There was a significant correlation between CRP and total WBC concentrations on days 0 and 3 (r=-.598, P=.003). Conclusions Serum CRP concentration was markedly increased in dogs with primary IMHA. CRP concentration did not differ based on patient survival, but might be a marker for long-term monitoring of these patients.
Background There is a high mortality rate in patients with systemic inflammatory response syndrome (SIRS) or sepsis. Therefore, an early diagnosis and prognostic assessment is important for optimal therapeutic intervention. The objective of the study was to evaluate if baseline values and changes in serum C-reactive protein (CRP) might predict survival in dogs with SIRS and sepsis. Design Prospective study; July 2004 to July 2005. Setting Small Animal Clinic, Berlin, Clinic of Small Animal Medicine, Munich. Animals Sixty-one dogs. Measurements and Main Results For the CRP analysis blood was drawn on day 0, 1, and 2; CRP was measured using a commercial ELISA test kit. Thirteen dogs suffered from nonseptic SIRS and 48 dogs from sepsis. The 14-day survival rate was 61% (69% nonseptic SIRS, 58% sepsis). Serum CRP was higher in sick dogs compared with controls (P < 0.001). Over the 3-day period surviving dogs (n=31) displayed a significantly greater decrease in CRP than nonsurvivors (n=10) (P=0.001). No correlation was found between the initial CRP concentrations and the survival rate. The changes in CRP corresponded to the survival rate (P=0.01). Conclusion There was no significant relationship between the survival rate in dogs with nonseptic SIRS or sepsis and the initial serum CRP concentrations. There was a correlation between decreasing CRP concentrations and recovery from disease. However, the changes in CRP concentrations over a 3-day period correctly predicted survival in 94% of dogs and death in 30% of the dogs (false positive rate 22%).
Retinol-binding protein 4 (RBP4) is an adipokine bound in plasma to transthyretin (TTR), which prevents its glomerular filtration and subsequent catabolism in the kidney. Alterations of this interaction have been Suggested to be implicated in the elevation of RBP4 that are thought to contribute to the development Of insulin resistance associated with obesity and type 2 diabetes mellitus (T2DM). However, the factors linking RBP4 to TTR in humans are not clear. Therefore, this Study evaluated parameters influencing the RBP4-TTR interaction and their relation to obesity and T2DM. The RBP4 and TTR levels were quantified in plasma of 16 lean controls, 28 overweight controls, and 14 overweight T2DM patients by enzyme-linked immunosorbent assay. Transthyretin isoforms involved in RBP4 binding were determined by linear matrix-assisted laser desorption/ionization-time of flight-mass spectrometry after RBP4 coimmunoprecipitation. Holo-RBP4 (retinol-bound) and apo-RBP4 (retinol-free) were assessed by immunoblotting using nondenaturating polyacrylamide gel electrophoresis. Plasma levels of both RBP4 and TTR did not differ among the groups of lean controls, overweight controls, and overweight T2DM subjects. Using RBP4 immunoprecipitation, 4 mass signals were observed for TTR representing native, S-cysteinylated, S-cysteinglycinylated, and S-glutathionylated TTR. No differences in peak intensity of TTR isoforms were observed among the groups. Moreover, no differences in the ratio of holo- and apo-RBP4 were evident. The results suggest that circulating RBP4 and TTR were not affected by human obesity or T2DM, which might be attributed to the absence of alterations of TTR isoforms and the ratio of holo- and apo-RBP4 that might modify the TTR-RBP4 interaction.
Background: Retinol-binding protein 4 (RBP4) levels are elevated in the serum of patients with kidney dysfunction. We recently showed that RBP4 isoforms including apo-RBP4 (RBP4 not bound to retinol) and RBP4 truncated at the C-terminus (RBP4-L, RBP4-LL) are increased in the serum of patients with kidney diseases but not in serum of patients with various liver diseases. The aim of this study was to investigate the effect of renal replacement therapy on RBP4 isoforms. Methods: We investigated serum levels of RBP4, apo-RBP4, holo-RBP4, RBP4-L, RBP4-LL, retinol and transthyretin (TTR) in 18 hemodialysis (HD) patients, 30 patients after renal transplantation (RTx) and in 35 healthy controls. RBP4 and TTR levels were measured by enzyme-linked immunosorbent assay, apo- and holo-RBP4 by native electrophoresis, retinol by high performance liquid chromatography and RBP4-L and RBP4-LL were analyzed by mass spectrometry. Results: HD and RTx patients had elevated RBP4, apo-RBP4 and RBP4-LL levels compared to controls. RTx patients had elevated amounts of RBP4-L compared to controls and elevated RBP4 and apo-RBP4 levels compared to HD patients. Conclusion: The results demonstrate a strong correlation between kidney function and RBP4 isoforms and provide data for investigating the relation of RBP4 and insulin resistance in these patients.
To offer the best choice of healthy and acceptable food to the consumer a coordination of plant breeding, food processing and nutrition science is required. Here the nutritional aspects of the high oleic/low linolenic (HOLLi) varieties of rapeseed with a low alpha-linolenic acid content of about 3% are reviewed. The content of alpha-linolenic acid amounting to around 9% is the hallmark of the positive nutritional value of the original (erucic acid free) 00 varieties of rapeseed oil ("canola" quality in North America). n-3 fatty acids are endowed with the property to protect the cardiovascular system from chronic disease and the consumption of food containing n-3 fatty acids is explicitly recommended by national and international nutritional and medical authorities. Although the use of HOLLi with a low n-3 fatty acid content can be unavoidable for specific purposes, because of technological and health considerations the continuous future consumption of the original rapeseed oil with around 9% of alpha-linolenic acid by the consumer should have high priority from the standpoint of public health. To pursue this aim confusion of the consumer must be avoided by creating a new name and a new brand for HOLLi varieties.
The aim of the study was to investigate the influence of oral rehydration solutions (ORS) on milk clotting, abomasal pH, electrolyte concentrations, and osmolality, as well as on the acid-base status in blood of suckling calves, as treatment with ORS is the most common therapy of diarrhea in calves to correct dehydration and metabolic acidosis. Oral rehydration solutions are suspected to inhibit abomasal clotting of milk; however, it is recommended to continue feeding cow's milk or milk replacer (MR) to diarrheic calves to prevent body weight losses. Three calves with abomasal cannulas were fed MR, MR-ORS mixtures, or water-ORS mixtures, respectively. Samples of abomasal fluid were taken before and after feeding at various time points, and pH, electrolyte concentrations, and osmolality were measured. The interference of ORS with milk clotting was examined in vivo and in vitro. To evaluate the effects of ORS on systemic acid-base status, the Stewart variables strong ion difference ([SID]), acid total ([A(tot)]), and partial pressure of CO2 (pCO(2)) were quantified in venous blood samples drawn before and after feeding. Calves reached higher abomasal pH values when fed with MR-ORS mixtures than when fed MR. Preprandial pH values were re-established after 4 to 6 h. Oral rehydration solutions prepared in water increased the abomasal fluid pH only for 1 to 2 h. Oral rehydration solutions with high [SID3] ([Na+] + [K+] - [Cl-]) values produced significantly higher abomasal pH values and area under the curve data of the pH time course. Caseinomacropeptide, an indicator of successful enzymatic milk clotting, could be identified in every sample of abomasal fluid after feeding MR-ORS mixtures. The MR-ORS mixtures with [SID3] values >= 92 mmol/L increased serum [SID3] but did not change venous blood pH. Oral rehydration solutions do not interfere with milk clotting in the abomasum and can, therefore, be administered with milk. In this study, MR-ORS mixtures with high [SID3] values caused an increase of serum [SID3] in healthy suckling calves and may be an effective treatment for metabolic acidosis in calves suffering from diarrhea.
Mechanismen zur Detoxifizierung von Benzo[a]pyren-Metaboliten in der gastrointestinalen Barriere
(2009)
Chronic kidney disease and type 2 diabetes mellitus as factors influencing retinol-binding protein 4
(2009)
Die Bedeutung Eisen-Schwefel-Cluster-assoziierter Mechanismen für Mutagenese und Kanzerogenese
(2009)
Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.
Heterocyclic aromatic amines (HCAs) are formed in meat cooked at high temperatures for a long time or over an open flame. In this context 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant HCA in cooked meat, has been suggested to be involved in colon and prostate carcinogenesis. In the latter case it has been reported that: (1) roughly 50% of Fischer F344 male rats treated with PhIP develop carcinomas in the ventral prostate lobe at 1 year of age; (2) inflammation precedes prostatic intraepithelial neoplasia in PhIP-fed rats; (3) inflammation specifically occurs in the ventral prostate lobe of PhIP-fed rats. To test whether PhIP by itself leads to inflammation in the colon and whether a human-relevant concentration of PhIP is able to induce preneoplastic lesions in the colon, male F344 rats were fed 0.1 or 100 ppm PhIP for up to 10 months and thereafter the colon tissue was analyzed histochemically. In none of the experimental groups signs of acute or chronic colonic inflammation were observed. 0.1 ppm PhIP leads to the development of hyperplastic and dysplastic lesions in the colon of single animals, but the incidence of these lesions does not reach a statistical significance. In contrast, in rats fed 100 ppm PhIP for 10 months hyperplastic and dysplastic colonic lesions were induced in a statistically significant number of animals. It is concluded that: (1) the induction of preneoplastic lesions in rat colon by PhIP is not preceded or accompanied by an inflammatory process; (2) a human-relevant concentration of PhIP alone is not sufficient to initiate colon carcinogenesis in rats.
Eine besondere Rolle im Fremdstoffmetabolismus hat die SULT1A1 beim Menschen aufgrund der hohen Expression und breiten Gewebeverteilung. Während die humane SULT1A1 in sehr vielen Geweben exprimiert wird, wurde die murine SULT1A1 vor allem in der Leber, Lunge und Colon gefunden. Neben der Gewebeverteilung spielt auch der Polymorphismus im humanen SULT1A1-Gen eine bedeutende Rolle. Der häufigste Polymorphismus in diesem Gen führt zu einer Aminosäuresubstitution von Arginin zu Histidin an Position 213. Die Genvariante mit Histidin (auch als SULT1A1*2 bezeichnet) codiert für ein Protein mit einer geringen Enzymaktivität und einer reduzierten Enzymmenge in Thrombocyten. Über den Einfluss dieser allelischen Varianten in anderen Geweben ist bislang wenig bekannt. In vorausgegangenen epidemiologischen Studien wurden mögliche Korrelationen zwischen den Genvarianten und der Krebsentstehung in verschiedenen Geweben untersucht. Diese Daten liefern jedoch widersprüchliche Ergebnisse zum Krebsrisiko. Aufgrund der strittigen epidemiologischen Daten sollten Tiermodelle generiert werden, um die häufigsten SULT1A1-Allele hinsichtlich der Empfindlichkeit gegenüber Nahrungs- und Umweltkanzerogenen zu untersuchen. Zur Erzeugung transgener (tg) Mauslinien wurde mittels Mikroinjektion der codierenden Genbereich und große flankierende Humansequenzen stromaufwärts und stromabwärts in das Mausgenom integriert. Es wurden mehrere Mauslinien hergestellt. Zwei davon, die Mauslinie 31 mit dem SULT1A1*1-Allel und die Mauslinie 28 mit dem SULT1A1*2-Allel, wurden eingehend analysiert. In beiden Linien wurde eine identische Kopienzahl des Transgens ermittelt. Proteinbiochemische Charakterisierungen zeigten eine weitgehend dem Menschen entsprechende Gewebeverteilung und zelluläre und subzelluläre Lokalisation der humanen SULT1A1 in der Linie (Li) 28. In Li 31 wurden Unterschiede zu Li 28 sowohl in der Gewebeverteilung als auch in der zellulären Lokalisation des exprimierten humanen Proteins ermittelt. Dabei war die Expression auf Proteinebene in der SULT1A1*2-tg Linie generell stärker als in der SULT1A1*1-Linie. Dieses Ergebnis war überraschend, denn in humanen Thrombocyten führt das SULT1A1*1-Allel zu einem höheren Gehalt an SULT1A1-Protein als das SULT1A1*2-Allel. Zur Analyse der unterschiedlichen Proteinexpressionen in den tg Mauslinien wurde die cDNA und der 5´-flankierende Bereich des SULT1A1-Gens sequenziert. In beiden tg Linien entsprach die Sequenz der cDNA der Referenzsequenz aus der Gendatenbank (Pubmed). In der 5´-flankierenden Region wurden bekannte Polymorphismen analysiert und unterschiedliche Haplotypen in den tg Linien an den Positionen -624 und -396 ermittelt. Dabei wurde in der Li 31 der Haplotyp detektiert, der in der Literatur mit einer höheren SULT1A1-Enzymaktivität beschrieben wird. Der mögliche Zusammenhang zwischen Transkriptionsrate und Proteinexpression wurde in RNA-Expressionsanalysen im codierenden und 5´-nicht codierenden Bereich (mit den alternativen Exons 1B und 1A) untersucht. Im codierenden Bereich und im Exon 1B konnte in den untersuchten Organen eine höhere RNA-Expression in der Li 28 im Vergleich zur Li 31 ermittelt werden. Außer in der Lunge wurde für Exon 1B eine identische RNA-Expression detektiert. RNA, die Exon 1A enthielt, wurde in allen untersuchten Organen der Li 28, aber nur in der Lunge bei der Li 31 gefunden. In beiden tg Linien konnten mit den Exon 1A-Primern jedoch auch größere PCR-Produkte ermittelt werden. Dieser Unterschied im Exon 1A und mögliche Spleißvarianten könnten damit für die unterschiedliche Proteinexpression des humanen SULT1A1-Proteins in den beiden tg Mauslinien sein. Die in dieser Arbeit generierten und charakterisierten tg Mausmodelle wurden in einer toxikologischen Studie eingesetzt. Es wurde das heterozyklische aromatische Amin 2-Amino-1-methyl-6-phenylimidazo-[4,5-b]pyridin (PhIP) verwendet. PhIP wird beim Erhitzen und Braten von Fleisch und Fisch gebildet und könnte mit der erhöhten Krebsentstehung im Colon in der westlichen Welt im Zusammenhang stehen. Mittels 32P-Postlabelling sollte der Einfluss der zusätzlichen Expression der humanen SULT-Proteine auf die PhIP-DNA-Adduktbildung analysiert werden. Dabei wurden mehr DNA-Addukte in den tg Tieren als in den Wildtyp-Mäusen ermittelt. Die Konzentration der gebildeten DNA-Addukte korrelierte mit der Expressionsstärke des humanen SULT1A1-Proteins in den tg Mäusen. An den in dieser Arbeit generierten tg Mauslinien mit den häufigsten allelischen Varianten des SULT1A1-Gens konnten Unterschiede auf RNA- und Protein-Ebene ermittelt werden. Zudem konnte gezeigt werden, dass die Expression der humanen SULT1A1 eine Auswirkung sowohl auf die Stärke als auch das Zielgewebe der DNA-Adduktbildung in vivo hat.
Der ubiquitär exprimierte, multifunktionale Glucosetransporter GLUT8 gehört zur Klasse III der Familie der passiven Glucosetransporter, die aus insgesamt 14 Proteinen besteht. Die fünf Mitglieder der Klasse IIII unterscheiden sich strukturell leicht von den Mitgliedern der Klasse I und II (Joost und Thorens, 2001). GLUT8 besitzt ein N-terminales Dileucin-Motiv, das Teil eines [DE]XXXL[LI] Motivs ist, welches für die Sortierung des Transporters in späte Endosomen und Lysosomen verantwortlich ist (Augustin et al., 2005). Da bis heute kein Signal identifiziert wurde, das eine Translokation des Transporters zur Plasmamembran auslöst, wird eine intrazelluläre Funktion von GLUT8 vermutet (Widmer et al., 2005). Im Rahmen der vorliegenden Arbeit wurde die intrazelluläre Funktion des Transporters in der Regulation der Glucosehomöostase des Körpers durch Analyse einer Slc2a8-knockout-Maus untersucht. Die homozygote Deletion des Transporters erbrachte lebensfähige Nachkommen, die sich augenscheinlich nicht von ihren Wildtyp-Geschwistern unterschieden. Allerdings wurde bei Verpaarungen heterozygoter Mäuse eine verminderte Anzahl an Slc2a8-/--Nachkommen beobachtet, die signifikant von der erwarteten Mendel’schen Verteilung abwich. Da Slc2a8 die höchste mRNA-Expression in den Testes aufwies und die Überprüfung der Fertilität mittels verschiedener homozygoter Verpaarungen eine Störung der weiblichen Fortpflanzungsfähigkeit ausschloss, wurden die Spermatozoen der Slc2a8-/--Mäuse eingehender untersucht. Als Ursache für die verringerte Anzahl von Slc2a8-/--Geburten wurde eine verminderte Prozentzahl motiler Slc2a8-/--Spermien ermittelt, die durch eine unzureichende mitochondriale Kondensation in den Spermien bedingt war. Diese Veränderung war mit einem reduzierten mitochondrialen Membranpotential assoziiert, was eine verminderte ATP-Produktion nach sich zog. Somit scheint GLUT8 in den Spermien an einem intrazellulären Transportprozess beteiligt zu sein, der einen Einfluss auf die oxidative Phosphorylierung der Mitochondrien ausübt. Im Gehirn wurde Slc2a8 besonders stark im Hippocampus exprimiert, der in der Regulation von körperlicher Aktivität, Explorationsverhalten, Erinnerungs- und Lernprozessen sowie Angst- und Stressreaktionen eine Rolle spielt. Außerdem wurde GLUT8 im Hypothalamus nachgewiesen, der unter anderem an der Regulation der Nahrungsaufnahme beteiligt ist. Die Slc2a8-/--Mäuse zeigten im Vergleich zu ihren Slc2a8+/+-Geschwistern eine signifikant gesteigerte körperliche Aktivität, die zusammen mit der von Membrez et al. (2006) publizierten erhöhten Zellproliferation im Hippocampus auf eine Nährstoffunterversorgung dieses Areals hindeutet. Die Nahrungsaufnahme war in Abwesenheit von GLUT8 nicht verändert, was zusammen mit dem nur geringfügig niedrigeren Körpergewicht der Slc2a8-/--Mäuse eine Funktion von GLUT8 im Glucose-sensing der Glucose-sensitiven Neurone des Gehirns ausschließt. Das leicht reduzierte Körpergewicht der Slc2a8-/--Mäuse ließ sich keinem bestimmten Organ- oder Gewebetyp zuordnen, sondern schien durch eine marginale Gewichtsreduktion aller untersuchten Gewebe bedingt zu sein. Zusammen mit den erniedrigten Blutglucosespiegeln und der anscheinend gesteigerten Lebenserwartung zeigten die Slc2a8-/--Mäuse Symptome einer leichten Nährstoffunterversorgung. GLUT8 scheint daher am Transport von Zuckerderivaten, die während des lysosomalen/endosomalen Abbaus von Glykoproteinen anfallen, beteiligt zu sein. Die so wiederaufbereiteten Zucker dienen dem Körper offenbar als zusätzliche Energiequelle.
Vitamin E wird immer noch als das wichtigste lipophile Antioxidanz in biologischen Membranen betrachtet. In den letzten Jahren hat sich jedoch der Schwerpunkt der Vitamin E-Forschung hin zu den nicht-antioxidativen Funktionen verlagert. Besonderes Interesse gilt dabei dem α-Tocopherol, der häufigsten Vitamin E-Form im Gewebe von Säugetieren, und seiner Rolle bei der Regulation der Genexpression. Das Ziel dieser Dissertation war die Untersuchung der genregulatorischen Funktionen von α-Tocoperol und die Identifizierung α-Tocopherol-sensitiver Gene in vivo. Zu diesem Zweck wurden Mäuse mit verschiedenen Mengen α-Tocopherol gefüttert. Die Analyse der hepatischen Genexpression mit Hilfe von DNA-Microarrays identifizierte 387 α-Tocopherol-sensitive Gene. Funktionelle Clusteranalysen der differentiell exprimierten Gene zeigten einen Einfluss von α-Tocooherol auf zelluläre Transportprozesse. Besonders solche Gene, die an vesikulären Transportvorgängen beteiligt sind, wurden größtenteils durch α-Tocopherol hochreguliert. Für Syntaxin 1C, Vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and Syntaxin binding protein 1 konnte eine erhöhte Expression mittels real time PCR bestätigt werden. Ein funktioneller Einfluss von α-Tocopherol auf vesikuläre Transportprozesse konnte mit Hilfe des in vitro β-Hexosaminidase Assays in der sekretorischen Mastzelllinie RBL-2H3 gezeigt werden. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer konzentrationsabhängigen Erhöhung der PMA/Ionomycin-stimulierten Sekretion der β-Hexosaminidase. Eine erhöhte Expression ausgewählter Gene, die an der Degranulation beteiligt sind, konnte nicht beobachtet werden. Damit schien ein direkter genregulatorischer Effekt von α-Tocopherol eher unwahrscheinlich. Da eine erhöhte Sekretion auch mit β-Tocopherol aber nicht mit Trolox, einem hydrophilen Vitamin E-Analogon, gefunden wurde, wurde vermutet, dass α-Tocopherol die Degranulation möglicherweise durch seine membranständige Lokalisation beeinflussen könnte. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer veränderten Verteilung des Gangliosids GM1, einem Lipid raft Marker. Es wird angenommen, dass diese Membranmikrodomänen als Plattformen für Signaltransduktionsvorgänge fungieren. Ein möglicher Einfluss von Vitamin E auf die Rekrutierung/Translokation von Signalproteinen in Membranmikrodomänen könnte die beobachteten Effekte erklären. Eine Rolle von α-Tocopherol im vesikulären Transport könnte nicht nur seine eigene Absorption und seinen Transport beeinflussen, sondern auch eine Erklärung für die bei schwerer Vitamin E-Defizienz auftretenden neuronalen Dysfunktionen bieten. Im zweiten Teil der Arbeit wurde die α-Tocopheroltransferprotein (Ttpa) Knockout-Maus als genetisches Modell für Vitamin E-Defizienz verwendet, um den Effekt von Ttpa auf die Genexpression und die Gewebeverteilung von α-Tocopherol zu analysieren. Ttpa ist ein cytosolisches Protein, das für die selektive Retention von α-Tocopherol in der Leber verantwortlich ist. Die Ttpa-Defizienz resultierte in sehr geringen α-Tocopherol-Konzentrationen im Plasma und den extrahepatischen Geweben. Die Analyse der α-Tocopherol-Gehalte im Gehirn wies auf eine Rolle von Ttpa bei der α-Tocopherol-Aufnahme ins Gehirn hin.
Untersuchung des Recyclings Kaede-fusionierter Corticotropin-Releasing-Factor Rezeptoren Typ 1
(2009)
Aktivierte G-Protein-gekoppelte Rezeptoren (GPCR) werden schnell desensitisiert, internalisiert und anschließend entweder lysosomal degradiert oder zur Plasmamembran (PM) recycelt. Zur Resensitisierung der Zellen tragen neben recycelten auch neusynthetisierte Rezeptoren bei. Die Überlagerung beider Prozesse erschwert die Untersuchung des Rezeptorrecyclings. In dieser Arbeit sollte mit Hilfe des photokonvertierbaren Fluoreszenzproteins Kaede eine Technik entwickelt werden, mit der es möglich ist Recycling- von Neusyntheseprozessen zu trennen und das Recycling von GPCR mikroskopisch in Echtzeit zu beobachten. Als Modellproteine wurden der Vasopressin-1a-Rezeptor V1aR (recycelnder Rezeptor), der Vasopressin-2-Rezeptor V2R (degradierter Rezeptor) und der Corticotropin-Releasing Factor-Rezeptor Typ 1 (CRF1R) verwendet, wobei bei Letzterem untersucht werden sollte, ob er nach Stimulation zur PM zurücktransportiert wird. Da Kaede als fluoreszierendes Protein mit den GPCR fusioniert wird, wurde zunächst überprüft, ob es die Eigenschaften der Rezeptoren verändert und generell für Transportstudien geeignet ist. Eventuell könnte die bereits publizierte Tetramerisierung von Kaede seine Anwendung verhindern oder erschweren. Mittels Fluoreszenz-Korrelationsspektroskopie konnte gezeigt werden, dass Kaede nicht tetramerisiert, wenn es an ein Membranprotein fusioniert ist. Außerdem konnte in in vitro- und Zellkulturexperimenten belegt werden, dass die native und die photokonvertierte Form von Kaede gleichermaßen stabil sind. Darüber hinaus zeigten Kaede-fusionierte GPCR sowohl in Kolokalisationsstudien als auch in Agonistbindungs- und Rezeptoraktivierungsexperimenten die gleichen Eigenschaften wie CFP- bzw. die unfusionierte Rezeptoren. Lediglich die Expression der Kaede-fusionierten Rezeptoren war geringer. Parallel wurde anhand der bereits publizierten Kaede-Struktur versucht, die Tetramerisierung des Proteins durch den Austausch interagierender Aminosäuren zu unterbinden. Die eingeführten Mutationen bewirkten aber eine Fehlfaltung des Proteins und damit den Verlust der Fluoreszenz. Da zuvor gezeigt werden konnte, dass Kaede-fusionierte Membranproteine nicht tetramerisieren und nicht die Eigenschaften der fusionierten Proteine verändern, war monomerisiertes Kaede zur Untersuchung des Rezeptorrecyclings nicht notwendig. Im zweiten Teil der Arbeit wurde mit Hilfe von Kaede-Fusionsproteinen und mikroskopischer Testsysteme das noch unbekannte Recyclingverhalten des CRF1R untersucht. Hierfür wurden die Kaede-fusionierten Rezeptoren in eukaryotischen Zellen exprimiert und mit Agonisten internalisiert. Die internalisierten Rezeptoren wurden in Endosomen selektiv mit UV-Strahlung photokonvertiert. Anschließend wurde der Transport der photokonvertierten Form verfolgt. Sowohl beim CRF1R als auch beim V1aR wurden Signale in der PM detektiert, beim V2R hingegen nicht. Dies zeigt, dass es sich beim CRF1R um einen recycelnden Rezeptor handelt. Die als Kontrolle eingesetzten Rezeptoren verhielten sich in diesem Experiment wie erwartet: Der V1aR wurde zur PM zurücktransportiert, der V2R nicht. Diese Ergebnisse konnten mit Hilfe biochemischer und durchflusscytometrischer Experimente bestätigt werden. Die Internalisierung des CRF1R verläuft Clathrin-vermittelt in Anwesenheit von β-Arrestin. Je nach Stabilität der β Arrestin-Interaktion unterscheidet man zwei Klassen von Rezeptoren: Klasse A-Rezeptoren interagieren transient mit β Arrestin und können recyceln. Im Gegensatz dazu gehen Klasse B-Rezeptoren eine stabile Interaktion mit β Arrestin ein und werden nach Internalisierung degradiert. In mikroskopischen Untersuchungen konnte für die aktivierten CRF1R und V1aR eine Rekrutierung von β Arrestin zur PM und eine transiente Interaktion mit β Arrestin gezeigt werden (Klasse A-Rezeptoren). Für den V2R wurde dagegen eine stabile Interaktion mit β Arrestin beobachtet (Klasse B-Rezeptor). Diese Daten stützen die Ergebnisse des Kaede-basierten Recyclingversuchs und zeigen, dass der CRF1R ein recycelnder Rezeptor ist. Ferner wurde untersucht, ob der CRF1R zu den schnell oder langsam recycelnden Rezeptoren zählt. Schnell recycelnde Rezeptoren werden direkt aus frühen Endosomen, langsam recycelnde hingegen über das Trans-Golgi-Netzwerk (TGN) bzw. über Recycling-Endosomen zur PM transportiert. Als Marker für das TGN oder die Recycling-Endosomen wurde Rab11 verwendet. In Kolokalisationsstudien konnte gezeigt werden, dass der CRF1R den langsam recycelnden Rezeptoren zugeordnet werden kann. Zusammenfassend konnte in dieser Arbeit belegt werden, dass Kaede als Fusionspartner für Membranproteine genutzt werden kann um deren Transport in Echtzeit zu studieren. Damit wurde erstmals eine mikroskopische Methode etabliert, die es erlaubt recycelnde von neusynthetisierten Rezeptoren zu unterscheiden. Mit Hilfe dieser Methode war es möglich zu zeigen, dass der CRF1R ein recycelnder Rezeptor ist.
Dietary antioxidants are believed to play an important role in the prevention and treatment of a variety of diseases associated with oxidative stress. Although there is a wide range of dietary antioxidants, the bulk of the research to date has been focused on the nutrient antioxidants vitamin C, E, and carotenoids. Certain relatively uncommon antioxidants such as lipoic acid (LA), and phenolic compounds such as (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), have not been extensively investigated although they may exert greater antioxidant potency than that of carotenoids and vitamins. Extracts from selected plants and plant byproducts may represent rich sources for one or more of such antioxidants and therefore exhibit higher effects than a single antioxidant due to the synergistic effects produced between such antioxidants. However, in the last decade a number of epidemiological, animal and in vitro studies have suggested a protective and therapeutic potency of these antioxidants in a broad range of diseases such as cancer, diabetes, atherosclerosis, cataract and acute and chronic neurological disorders. Inflammation, the response of the host toward any infection or injury, plays a central role in the development of many chronic diseases. Several evidences demonstrated the rise of different types of cancer from sites of inflammation. This suggests that active oxygen species and some cytokines generated in the inflamed tissues can cause injury to DNA and ultimately lead to carcinogenesis. Diethylnitrosamine (DEN) is one of the most important environmental carcinogens, present in a variety of foods, alcoholic beverages, tobacco smoke and it can be synthesized endogenously. In addition to the liver it can induce carcinogenesis in other organs like kidney, trachea, lung, esophagus, fore stomach, and nasal cavity. Several epidemiological and laboratory studies indicate that nitroso compounds including DEN may induce hyperplasia and chronic inflammation which is closely associated with the development of hepatocellular carcinoma. Despite increasing evidence on the potential of antioxidants in modulating the etiology of chronic diseases, little is known about their role in inflammation and acute phase response (APR). Therefore the aim of the present work was to study the protective effect of water and solvent extracts of eight plant and plant byproducts including green tea, artichoke, spinach, broccoli, onion and eggplant, orange and potato peels as well as eight antioxidants agents including EC, EGC, ECG, EGCG, ascorbic acid (AA), acetylcysteine (NAC), α-LA, and alpha-tocopherol (α-TOC) toward acute inflammation induced by interleukin-6 (IL-6) and hepatotoxicity induced by DEN in vitro. The negative acute phase proteins (APP), transthyretin (TTR) and retinol-binding protein (RBP) were used as inflammatory biomarkers analyzed by ELISA, whereas neutral red assay was used for evaluating the cytotoxicity. All experiments were performed in vitro using human hepatocarcinoma cell line (HepG2). Additionally the antioxidant activity was measured by TEAC and FRAP assays, phenolic content was measured by Folin–Ciocalteu and characterized by HPLC. Moreover, the microheterogeneity of TTR was detected using immunoprecipitation assay combined with SELDI-TOF MS. Results of present study showed that HepG2 cells provide a simple, sensitive in vitro system for studying the regulation of the negative APP, TTR and RBP under free and inflammatory condition. IL-6, a potent proinflammatory cytokine, in a concentration of 25 ng/ml was able to reduce TTR and RBP secretion by approximately 50-60% after 24h of incubation. With exception of broccoli and water extract of onion which showed pro-inflammatory effects in this study, all other plant extracts, at specific concentrations, were able to elevate TTR secretion in normal condition and even under treatment of IL-6 where the effect was quite lower. Green tea followed by artichoke and potato peel exhibited the highest elevation in TTR concentration which reached 1.1 and 2.5 folds of control in presence and absences of IL-6 respectively. In general Plant extracts were ordered according their anti-inflammatory potency as following: in water extracts; green tea > artichoke > potato peel > orange peel > spinach > eggplant peel, where in solvent extracts; green tea > artichoke > potato peel > spinach > eggplant peel > onion > orange peel. The antiinflammatory effect of water extracts of green tea, artichoke and orange peel were significantly higher than their corresponding solvent extracts whereas water extracts of eggplant-, potato peels and spinach showed lower effect than their solvent extracts. On the other hand α-LA followed by EGCG and ECG exhibited the highest elevation in TTR concentration compared to other antioxidants. The relation between the anti-inflammatory potential and antioxidants activity and phenolic content for the investigated substances was generally weak. This may suggest the involvement of other mechanisms than antioxidants properties for the observed effect. TTR secreted by HepG2 cells has a molecular structure quite similar to the purified standard and serum TTR in which all the three main variants are contained including native, S-cystinylated and Sglutathionylated TTR. Interestingly, a variant with molecular mass of 13453.8 + 8.3 Da has been detected only in TTR secreted by HepG2. Among all investigated antioxidants and plant extracts, six substances were able to elevate the native preferable TTR variant. The potency of these substances can be ordered as following α-LA > NAC > onion > AA > EGCG > green tea. A weak correlation between elevation on TTR and shifting to the native form was observed. Similar weak correlation has also been observed between antioxidants activity and elevation in native TTR. Although DEN was able to induce cell death in a concentration dependent manner, it requires considerably higher concentrations for its effects especially after 24h. This may be attributed to a lack in cytochrome P450 enzymes produced by HepG2. At selected concentrations some antioxidants and plant extracts significantly attenuate DEN cytotoxicity as following: spinach > α-LA > artichoke > orange peel > eggplant peel > α-TOC > onion > AA. Contrary all other substances especially green tea, broccoli, potato peel, and ECG stimulate DEN toxicity. In conclusion, this study demonstrated that selected antioxidants and plant extracts may attenuate the inflammatory process, not only by their antioxidants potency but also by other mechanisms which remain unclear. They may also play a vital role on stabilizing the tetramic structure of TTR and thereby prevent amyloidosis diseases. Lipoic acid represents in this study unique function against inflammation and hepatotoxicity. Despite the protective effect demonstrated by investigated substances, attention should also be given to the pro-oxidant and potential cytotoxic effects produced at higher concentrations.
Die funktionelle Bedeutung des Coxsackie- und Adenovirus Rezeptors (CAR) im kolorektalen Karzinom
(2009)
Der Coxsackie- und Adenovirus Rezeptor (CAR) ist als Bestandteil von Tight Junctions (TJ) an interzellulären Adhäsionsprozessen beteiligt und scheint eine wichtige Rolle in der Karzinogenese zu spielen. Diese ist jedoch insbesondere bei Entstehung von Darmkrebs weitgehend unklar. Ziel der Arbeit war es daher, die funktionelle Bedeutung, mögliche Interaktionspartner sowie die Expressionsregulation von CAR im kolorektalen Karzinom zu analysieren. In den Zelllinien CaCo2, Colo205, DLD1, HCT116, HT29, SW480 und T84 konnte die Expression von CAR (mRNA und Protein) nachgewiesen werden. Nach stabiler CAR-Überexpression durch Transfektion von CARcDNA in DLD1, HCT116 und SW480 wurde das Zellwachstum gehemmt und eine Abnahme von Migration und Invasion induziert. Eine stabile CAR-Inhibition nach Transfektion von CARsiRNA führte in diesen Zelllinien zum Anstieg der Proliferation sowie zu verstärkter Migrations- und Invasionsaktivität, die in DLD1 mit morphologischen Änderungen einhergingen. Eine Genexpressionsanalyse der Zelllinie DLD1 mit CAR-Inhibition identifizierte α-Catenin als das am stärksten regulierte Gen. Obwohl keine direkte Interaktion beider Proteine detektiert werden konnte, führte eine stabile Re-Expression von α-Catenin in DLD1 mit stabiler CAR-Inhibition zu einer deutlichen Reduktion von Proliferation, Migration und Invasion sowie zu einem Rückgang der zellmorphologischen Änderungen. Um den Einfluss von Differenzierung auf die Regulation der CAR-Expression zu untersuchen, erfolgte eine Behandlung aller Zelllinien mit Natriumbutyrat. Dies führte in fünf der sieben Zelllinien zu einer Aktivierung des CAR-Promotors sowie zu einer gesteigerten Expression und Immunoreaktivität von CAR an der Zelloberfläche. Die Zelllinie CaCo2 zeigte nach spontaner Differenzierung durch 21-tägiges Wachstum post Konfluenz ebenfalls eine verstärkte CAR-mRNA-Expression sowie eine erhöhte CAR-Präsenz an der Zelloberfläche. Die gewonnenen Daten konnten die funktionelle Bedeutung von CAR für die Kolonkarzinogenese sowie den Einfluss von α-Catenin auf diese Funktion deutlich machen. Es wurde gezeigt, dass die Expressionsregulation sowie die subzelluläre Verteilung von CAR durch den zellulären Differenzierungsstatus beeinflusst werden kann.
The fat-soluble vitamin A, which is chemically referred to retinol (ROH), is known to be essential for the process of vision, the immune system but also for cell differentiation and proliferation. Recently, ROH itself has been reported to be involved in adipogenesis and a ROH transport protein, the retinol-binding protein 4 (RBP4), in insulin resistance and type 2 diabetes. However, there is still considerable scientific debate about this relation. With the increasing amount of studies investigating the relation of ROH in obesity and type 2 diabetes, basic research is an essential prerequisite for interpreting these results. This thesis enhances the knowledge on this relation by reviewing ROH metabolism on extra- and intracellular level. Aim 1: In the blood stream ROH is transported in a complex with RBP4 and a second protein, transthyretin (TTR), to the target cells. The levels of RBP4 and TTR are influenced by several factors but mainly by liver and kidney function. The reason for that is that liver and the kidneys are the sites of RBP4 synthesis and catabolism, respectively. Interestingly, obesity and type 2 diabetes involve disorders of the liver and the kidneys. Therefore the aim was to investigate factors that influence RBP4 and TTR levels in relation to obesity and type 2 diabetes (Part 1). Aim 2: Once arrived in the target cell ROH is bound to cellular retinol-binding protein type I (CRBP-I) and metabolised: ROH can either be stored as retinylesters or it can be oxidised to retinoic acid (RA). By acting as a transcription factor in the nucleus RA may influence processes such as adipogenesis. Therefore vitamin A has been postulated to be involved in obesity and type 2 diabetes. CRBP-I is known to mediate the storage of ROH in the liver, but the extra-hepatic metabolism and the functions of CRBP-I are not well known. This has been investigated in Part 2 of this work. Material & Methods: RBP4 and TTR levels were investigated by ELISA in serum samples of human subjects with overweight, type 2 diabetes, kidney or liver dysfunction. Molecular alterations of the RBP4 and TTR protein structure were analysed by MALDI-TOF mass spectrometry. The functions of intracellular CRBP-I were investigated in CRBP-I knock-out mice in liver and extra-hepatic tissues by measuring ROH levels as well as the levels of its storage form, the retinylesters, using reverse phase HPLC. The postprandial uptake of ROH into tissues was analysed using labelled ROH. The mRNA levels of enzymes that metabolize ROH were examined by real-time polymerase chain reaction (RCR). Results: The previous published results showing increased RBP4 levels in type 2 diabetic patients could not be confirmed in this work. However, it could be shown that during kidney dysfunction RBP4 levels are increased and that RBP4 and TTR levels are decreased during liver dysfunction. The important new finding of this work is that increased RBP4 levels in type 2 diabetic mice were increased when kidney function was decreased. Thus an increase in RBP4 levels in type 2 diabetes may be the effect of a reduced kidney function which is common in type 2 diabetes. Interestingly, during severe kidney dysfunction the molecular structure of RBP4 and TTR was altered in a specific manner which was not the case during liver diseases and type 2 diabetes. This underlines the important function of the kidneys in RBP4 metabolism. CRBP-I has been confirmed to be responsible for the ROH storage in the liver since CRBP-I knock-out mice had decreased ROH and retinylesters (the storage form of ROH) levels in the liver. Interestingly, in the adipose tissue (the second largest ROH storage tissue in the body) ROH and retinylesters levels were higher in the CRBP-I knock-out compared to the wild-type mice. It could be shown in this work that a different ROH binding protein, cellular retinol-binding protein type III, is upregulated in CRBP-I knock-out mice. Moreover enzymes were identified which mediate very efficiently ROH esterification in the adipose tissue of the knock-out mice. In the pancreas there was a higher postprandial ROH uptake in the CRBP-I knock-out compard to wild-type mice. Even under a vitamin A deficient diet the knock-out animals had ROH and retinylesters levels which were comparable to wild-type animals. These results underline the important role of ROH for insulin secretion in the pancreas. Summing up, there is evidence that RBP4 levels are more determined by kidney function than by type 2 diabetes and that specific molecular modifications occur during kidney dysfunction. The results in adipose tissue and pancreas of CRBP-I knock-out mice support the hypothesis that ROH plays an important role in glucose and lipid metabolism.