A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples.

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-L-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.

Breakdown products of neoglucobrassicin inhibit activation of Nrf2 target genes mediated by myrosinase-derived glucoraphanin hydrolysis products.

Glucosinolates (GLSs) present in Brassica vegetables serve as precursors for biologically active metabolites, which are released by myrosinase and induce phase 2 enzymes via the activation of Nrf2. Thus, GLSs are generally considered beneficial. The pattern of GLSs in plants is various, and contents of individual GLSs change with growth phase and culture conditions. Whereas some GLSs, for example, glucoraphanin (GRA), the precursor of sulforaphane (SFN), are intensively studied, functions of others such as the indole GLS neoglucobrassicin (nGBS) are rather unknown as are functions of combinations thereof. We therefore investigated myrosinase-treated GRA, nGBS and synthetic SFN for their ability to induce NAD(P)H:quinone oxidoreductase 1 (NQO1) as typical phase 2 enzyme, and glutathione peroxidase 2 (GPx2) as novel Nrf2 target in HepG2 cells. Breakdown products of nGBS potently inhibit both GRA-mediated stimulation of NQO1 enzyme and Gpx2 promoter activity. Inhibition of promoter activity depends on the presence of an intact xenobiotic responsive element (XRE) and is also observed with benzo[a]pyrene, a typical ligand of the aryl hydrocarbon receptor (AhR), suggesting that suppressive effects of nGBS are mediated via AhR/XRE pathway. Thus, the AhR/XRE pathway can negatively interfere with the Nrf2/ARE pathway which has consequences for dietary recommendations and, therefore, needs further investigation.

Pyridone dipeptide backbone scan to elucidate structural properties of a flexible peptide segment.

Whereas the C-terminal fragment of neuropeptide Y (NPY) has been structurally well-defined both in solution and as membrane-bound, detailed structural information regarding the proline-rich N-terminus is still missing. The systematic variation of each position by a conformationally constrained pyridone dipeptide building block within the amino terminal segment of NPY leads to a systematic receptor subtype selectivity of the neuropeptide. Thereby, the systematic dipeptide scan proved superior to the traditional L-Ala scan because it showed how to modify the N-terminus in order to obtain increasingly more Y1 or Y5 receptor selective ligands. NMR and CD spectroscopic analyses were used to characterize the stepwise rigidification of the N-terminus of NPY when up to three dipeptide building blocks were incorporated by solid-phase peptide synthesis. The pyridone dipeptide increases the hydrophobicity of the amino terminus of NPY, and this allows the tuning of the membrane affinity of NPY. The amphiphilic C-terminal helix of 3-fold-substituted NPY thus becomes visible by selective line broadening in the (1)H NMR. Accordingly, we could structurally characterize protein segments that are too flexible for other methods.

Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex.

For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days.

Characteristic single glucosinolates from Moringa oleifera: Induction of detoxifying enzymes and lack of genotoxic activity in various model systems.

Leaves of Moringa oleifera are used by tribes as biological cancer medicine. Scientific investigations with M. oleifera conducted so far have almost exclusively used total plant extracts. Studies on the activity of single compounds are missing. Therefore, the biological effects of the two main aromatic multi-glycosylated glucosinolates of M. oleifera were investigated in the present study. The cytotoxic effects of M. oleifera glucosinolates were identified for HepG2 cells (NRU assay), for V79-MZ cells (HPRT assay, SCE assay), and for two Salmonella typhimurium strains (Ames test). Genotoxic effects of these glucosinolates were not observed (Ames test, HPRT assay, and SCE assay). Reporter gene assays revealed a significant increase in the ARE-dependent promoter activity of NQO1 and GPx2 indicating an activation of the Nrf2 pathway by M. oleifera glucosinolates. Since both enzymes can also be induced via activation of the AhR, plasmids containing promoters of both enzymes mutated in the respective binding sites (pGL3enh-hNQO1-ARE, pGL3enh-hNQO1-XRE, pGL3bas-hGPX2-mutARE, pGL3bas-hGPX2-mutXRE) were transfected. Analyses revealed that the majority of the stimulating effects was mediated by the ARE motif, whereas the XRE motif played only a minor role. The stimulating effects of M. oleifera glucosinolates could be demonstrated both at the transcriptional (reporter gene assay, real time-PCR) and translational levels (enzyme activity) making them interesting compounds for further investigation.

Glucosinolates from pak choi and broccoli induce enzymes and inhibit inflammation and colon cancer differently.

High consumption of Brassica vegetables is considered to prevent especially colon carcinogenesis. The content and pattern of glucosinolates (GSLs) can highly vary among different Brassica vegetables and may, thus, affect the outcome of Brassica intervention studies. Therefore, we aimed to feed mice with diets containing plant materials of the Brassica vegetables broccoli and pak choi. Further enrichment of the diets by adding GSL extracts allowed us to analyze the impact of different amounts (GSL-poor versus GSL-rich) and different patterns (broccoli versus pak choi) of GSLs on inflammation and tumor development in a model of inflammation-triggered colon carcinogenesis (AOM/DSS model). Serum albumin adducts were analyzed to confirm the up-take and bioactivation of GSLs after feeding the Brassica diets for four weeks. In agreement with their high glucoraphanin content, broccoli diets induced the formation of sulforaphane-lysine adducts. Levels of 1-methoxyindolyl-3-methyl-histidine adducts derived from neoglucobrassicin were the highest in the GSL-rich pak choi group. In the colon, the GSL-rich broccoli and the GSL-rich pak choi diet up-regulated the expression of different sets of typical Nrf2 target genes like Nqo1, Gstm1, Srxn1, and GPx2. GSL-rich pak choi induced the AhR target gene Cyp1a1 but did not affect Ugt1a1 expression. Both colitis and tumor number were drastically reduced after feeding the GSL-rich pak choi diet while the other three diets had no effect. GSLs can act anti-inflammatory and anti-carcinogenic but both effects depend on the specific amount and pattern of GSLs within a vegetable. Thus, a high Brassica consumption cannot be generally considered to be cancer-preventive.

alpha-Tocopherol enhances degranulation in RBL-2H3 mast cells.

Based on the observation that 3 months alpha-tocopherol supplementation caused an up-regulation of the mRNA of vesicular transport proteins in livers of mice, the functional relevance was investigated in RBL-2H3 cells, a model for mast cell degranulation. In total, 24 h incubation with 100 muM alpha-tocopherol enhanced the basal and phorbol-12-myristyl-13-acetate/ionomycin-stimulated release of beta-hexosaminidase and cathepsin D as measured by enzymatic analysis as well as Western blotting and immunocytochemistry, respectively. beta-Tocopherol exerted the same effect, whereas alpha-tocopheryl phosphate and trolox were inactive, indicating that both the side chain and the 6-OH group at the chroman ring are essential for activation of degranulation. alpha-Tocopherol did not induce mRNA expression of soluble NSF-attachment protein receptor (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins, such as N-ethylmaleimide sensitive fusion protein, complexin-2, SNAP23 or syntaxin-3, in the RBL-2H3 cell model. In view of the well known alpha-tocopherol-mediated activation of protein phosphatases, which regulate soluble NSF-attachment protein receptor activities by dephosphorylation, underlying mechanisms are discussed in terms of preventing oxidative inactivation of protein phosphatases and so far unknown functions in certain membrane domains.

Towards understanding the free and receptor bound conformation of neuropeptide Y by fluorescence resonance energy transfer studies.

Despite a considerable sequence identity of the three mammalian hormones of the neuropeptide Y family, namely neuropeptide Y, peptide YY and pancreatic polypeptide, their structure in solution is described to be different. A so-called pancreatic polypeptide-fold has been identified for pancreatic polypeptide, whereas the structure of the N-terminal segment of neuropeptide Y is unknown. This element is important for the binding of neuropeptide Y to two of its relevant receptors, Y(1) and Y(5), but not to the Y(2) receptor subtype. In this study now, three doubly fluorescent-labeled analogs of neuropeptide Y have been synthesized that still bind to the Y(5) receptor with high affinity to investigate the conformation in solution and, for the first time, to probe the conformational changes upon binding of the ligand to its receptor in cell membrane preparations. The results obtained from the fluorescence resonance energy transfer investigations clearly show considerable differences in transfer efficiency that depend both on the solvent as well as on the peptide concentration. However, the studies do not support a pancreatic polypeptide-like folding of neuropeptide Y in the presence of membranes that express the human Y(5) receptor subtype.

Stabilization of the compactification volume by quantum corrections.

We discuss prospects for stabilizing the volume modulus of N = 1 supersymmetric type IIB orientifold compactifications using only perturbative corrections to the Kähler potential. Concretely, we consider the known string loop corrections and tree-level alpha' corrections. They break the no-scale structure of the potential, which otherwise prohibits stabilizing the volume modulus. We argue that when combined, these corrections provide enough flexibility to stabilize the volume of the internal space without nonperturbative effects, although we are not able to present a completely explicit example within the limited set of currently available models. Furthermore, a certain amount of fine-tuning is needed to obtain a minimum at large volume.

Induction of cellular immune responses against carcinoembryonic antigen in patients with metastatic tumors after vaccination with altered peptide ligand-loaded dendritic cells.

PURPOSE: Dendritic cells (DCs) are characterized by their extraordinary capacity to induce T-cell responses, providing the opportunity of DC-based cancer vaccination protocols. In the present study, we conducted a phase I/II clinical trial to determine the capability of DCs differentiated from immunomagnetically isolated CD14+ monocytes and pulsed with a carcinoembryonic antigen-derived altered peptide (CEAalt) to induce specific CD8+ T cells in cancer patients. EXPERIMENTAL DESIGN: Nine patients with CEA-positive colorectal cancer (n=7) or lung cancer (n=2) were enrolled in this study. Autologous CD14+ monocytes were isolated by large-scale immunomagnetic separation and differentiated to mature DCs in sufficient numbers and at high purity. After incubation with the CEAalt peptide and keyhole limpet hemocyanin, DCs were administered to patients intravenously at dose levels of 1 x 10(7) and 5 x 10(7) cells. Patients received four immunizations every second week. RESULTS: ELISPOT analysis revealed a vaccine-induced increase in the number of CEAalt peptide-specific Interferon (IFN)-gamma producing CD8+ T cells in five of nine patients and of CD8+ T lymphocytes recognizing the native CEA peptide in three of nine patients. In addition, CD8+ T lymphocytes derived from one patient exhibiting an immunological response after vaccination efficiently lysed peptide-loaded T2 cells and tumor cells. Immunization was well tolerated by all patients without severe signs of toxicity. CONCLUSION: Vaccination with CEAalt-pulsed DCs derived from immunomagnetically isolated CD14+ monocytes efficiently expand peptide-specific CD8+ T lymphocytes in vivo and may be a promising alternative for cancer immunotherapy.