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A simple method was devised to fabricate and anchor organosiloxane coatings along low-surface-energy plastics without resorting to chemical pre-treatment of the polymer surface. Polypropylene tubes were purposely distended by exposure to toluene-based solutions. An established solvent of many organic polymers and compounds, toluene not only perfused into the plastic via gradual intercalation of accessible polymer chains, but it also facilitated the entry of less compatible reagents as co-perfusates. The reagents, typically aminoalkyl si lanes, were activated either concomitant to perfitsion of polypro-pylene or during a subsequent hydrolysis step. Freed silanol groups, interacting along and beneath the Surface of the plastic, reacted to form a network held together by siloxane bonds. The type of network formed and presented herein was interpreted as an amorphous aminopropylsiloxane-silanol hydrate film, with overall thickness partially submerged and caught in the matrix of the plastic. This concept to intertwine a growing organosiloxane network and a compliant matrix precluded the conventional methods normally used to improve bonding on hydrophobic plastics. A comparison of ninhydrin color yields suggested that films were more effectively and conveniently synthesized when aminopropyltri-methoxysilane was applied in water-saturated toluene. It follows that by integrating low-temperature hydrothermal processing into this strategy, inorganic-organic hybrids or inorganic oxide films may be formed oil pre-molded plastics and feature traits such as covalent-like fastness and gas impermeability.

A water-soluble extract of dry-cured ham was fractionated by gel filtration chromatography. Collected fractions (below 1200 Da) were sensory-analyzed and subjected to amino acid analysis with and without previous acid hydrolysis, in order to determine the amino acid and peptide distribution. Those fractions with the highest peptide amount were further separated by reverse-phase and cation-exchange high-performance liquid chromatography in order to isolate the peptides and sequence them. The results demonstrated the presence of small peptides, mainly dipeptides. The combination of these peptides and free amino acids may contribute to the characteristic taste in the savory fractions of dry-cured ham.

Zahlreiche Patienten mit Tumorerkrankungen nutzen neben schulmedizinischen auch alternative Heilmethoden. Die Misteltherapie ist in der Alternativmedizin eine der am haufigsten verwendeten Therapieformen und wird von Schulmedizinern heftig diskutiert. Zwar haben Mistelextrakte nachweislich eine immunmodulatorische und zytotoxische Wirkung, die bisher publizierten Studienergebnisse rechtfertigen jedoch nur bedingt ihren Einsatz im Sinne einer primaren oder sekundaren Chemopravention.

Tumor hypoxia is a major problem in radiation therapy of solid tumors because of the radiosensitizing effect of oxygen. Nitroimidazole-containing compounds are oxygen mimetics accumulating in hypoxic tumor areas. However, the broad use of 2-nitroimidazoles as a hypoxic radiosensitizer is limited by their partially low efficacy and/or high neurotoxicity.Here, we characterized the in vitro hypoxic cytotoxicity and hypoxic radiosensitizing efficacy of N,N,N-tris [2-(2-nitro-1H-imidazol-1-yl)ethyl]amine (PRC) in a hypoxia-sensitive lymphoma and a hypoxia-resistant glioblastoma cell line by colony formation assay and flow cytometry.PRC exerted high hypoxic cytotoxic and radiosensitizing action on both cell lines at almost absent toxicity under normoxic conditions. In particular, under hypoxia, but not normoxia, PRC targeted the mitochondria resulting in oxidative stress, G(2)/M cell cycle arrest, and triggering of the intrinsic apoptosis pathway.Our in vitro findings suggest that PRC might be a promising new 2-nitroimidazole for improving radiation therapy of hypoxic tumors in vivo.

β-Thymosins, a group of highly homologous peptides consisting of about 40 amino acid residues, were found to be distributed from mammals up to echinoderms. Althogh they have first been isolated from mammalian thymus tissue preparations, their occurrance is not organ-specific and they are present even in different types of cells. For thymosinβ 4 several biological activities have been reported, stating that this peptide acts as a thymus peptide hormone and is also involved in the neuroendocrine and immune system. However, it was recently demonstrated that thymosinβ 4 has actin-sequestering properties and therefore might play an important role in the regulation of the microfilament system. This fact gives a new outlook on the real biological function ofβ-thymosins.

Using hydrophilicity, surface probability, backbone flexibility and secondary structure parameters, antigenic sites of R-thymosins (TR), ubiquitin and gp41 envelope glycoprotein fragments of HIV- 1 are determined. Corresponding antigenic sites of TR,, TR,, ubiquitin and gp41 (583-623) are produced either by tryptic cleavage from the natural peptides or by solid phase peptide synthesis (FmocBu' strategy, Ecosyn P batch synthesizer, EppendorfBiotronik, Maintal, Germany). The predicted antigenic fragments allow the generation of specific antibodies for the determination of body fluid or tissue levels and immunostaining experiments, or, in the case of gp41-HIV fragments, the antigens can be used directly for the diagnosis of AIDS patients.

Radioimmunoassays specific for the N and C termini of human prothymosin alpha and the N terminus of human parathymosin alpha were employed for the measurement of the levels of alpha-thymosins in human thymus, spleen, and liver during normal growth and intestine and breast in malignant growth. A differential expression of the two alpha-thymosins was observed in thymus (prothymosin alpha-rich) and liver (parathymosin alpha-rich). A decline in the levels of both alpha-thymosins was found with age, with prothymosin alpha in thymus showing the sharpest change (15- to 30-fold). The levels of both alpha-thymosins were higher in malignant tissues as compared with healthy ones. In breast cancer, in particular, the mean increase for prothymosin alpha and parathymosin alpha was 17.9- and 11.5-fold, respectively. The major crossreactive material was characterized in all cases as intact prothymosin alpha and parathymosin alpha. These results suggest an in vivo relationship of the expression of alpha-thymosins with the human tissue cell proliferation activity.

Anhydro sugars have proved to be versatile chiral synthons for the synthesis of chiral heterocycles including pyradazines, oxazines, triazines, chiral morpholines, tetrahydroquinoxalines, benzodioxins, oxazolidinones, episulfides, thiazolines, and cyclic trithiocarbonates. Selected examples are described in this chapter.

Mistletoe extracts exert immunomodulatory properties in vivo and in vitro, and these effects have been related mainly to mistletoe lectin 1 (ML-1). Recently, a new chitin-binding mistletoe lectin (cbML) has been isolated and structurally characterized in these extracts. Aim of the present study was, therefore, to evaluate whether this cbML also affects immunocompetent cells and can for instance activate B-cells to produce anti-cbML-specific antibodies. Sera from patients with different tumors who were treated with the mistletoe extract ABNOBAviscum Mali (AM) 4 for at least 18 weeks were analysed before therapy and after 3, 6, 9, 12, 18, and 24 weeks. Sera were tested by ELISA against ML-1, -3, and cbML, isolated from a single mistletoe plant collected from an apple tree (Malus domestica). Eight of the 26 patients (31%) had IgG anti-cbML antibodies already before therapy, while only four had anti-ML-1 and -3 antibodies. Of the 18 anti-cbML negative patients before therapy 54% developed these antibodies during therapy, and there was a significant increase in anti-cbML antibody titers. In contrast, anti-ML-1 or -3-antibodies developed in almost 100% of the 25 patients being negative before therapy. These data indicate that cbML can induce immunological responses in patients treated with mistletoe extracts, although it seems to have lower antigenicity. Interestingly, anti-cbML antibodies can be observed in a low incidence also in individuals, not having yet received mistletoe therapy.

Aerial parts of Delphinium fissum subsp. anatolicum yielded two new hetisan-type alkaloids, fissumine and delfissinol, in addition to two known C 20 , and one C 19 , diterpene alkaloids, hetisine, hetisinone and lycoctonine, respectively.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.

Enzyme tablets with butyrylcholine esterase (CHE) and peroxidase (POD) partly lose enzymatic activity during compaction at a pressure of 495 MPa. Compared to solutions of the original enzyme, no changes of ultraviolet absorbance and fluorescence intensity in the tablet solutions were found. Only small changes were observed in the far ultraviolet circular dichroism spectra. Neither missing nor additional bands were detected with polyacrylamide gel electrophoresis. Heated (150 degrees C) solid starting material with CHE and POD showed still part of its original enzymatic activity. The ultraviolet absorbance increased with continued heating until precipitation occurred. The circular dichroism spectra are changed clearly.

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.

A thermostable D-xylose-glucose isomerase was isolated from the thermophilic strain Streptomyces thermovulgaris 127, var. 7-86, as a result of mutagenic treatment by gamma-irradiation of the parent strain, by precipitation and sequential chromatographies on DEAE-Sephadex A50, TSK-gel, FPLC-Mono Q/HR, and Superose 12 columns. The N-terminal amino acid sequence and amino acid analysis shows 73-92% homology with xylose-glucose isomerases from other sources. The native molecular mass, determined by gel filtration on a Superose 12 column, is 180 kDa, and 44.6 and 45 kDa were calculated, based on amino acid analysis and 10% SDS-PAGE, respectively. Both, the activity and stability of the enzyme were investigated toward pH, temperature, and denaturation with guanidine hydrochloride. The enzyme activity showed a clear pH optimum between pH 7.2 and 9.0 with D-glucose and 7.4 and 8.3 with D-xylose as substrates, respectively. The enzyme is active up to 60-85 degrees C at pH 7.0, using D-glucose, and up to 50-60 degrees C at pH 7.6, using D-xylose as substrates. The activation energy (Ea = 46 kJ x mol(-1)) and the critical temperature (Tc = 60 degrees C) were determined by fluorescence spectroscopy. Tc is in close coincidence with the melting temperature of denaturation (Tm = 59 degrees C), determined by circular dichroism (CD) spectroscopy. The free energy of stabilization in water after denaturation with Gdn.HCl was calculated to be 12 k x mol(-1). The specific activity (km values) for D-xylose-glucose isomerase at 70 degrees C toward different substrates, D-xylose, D-glucose, and D-ribose, were determined to be 4.4, 55.5, and 13.3 mM, respectively.

It was shown that agents inducing phagocytosis (zymosan, lectins) cause changes in the number of receptors responsible for fast neutrophil reaction (chemotaxis or respiratory burst) or inhibit the binding of the agonist to its receptor. Among lectins are ribosome-inactivating proteins of type II ricin and agglutinin ricin, which penetrate the cell by binding to mannose and galactose receptors. It was shown that ribosome-inactivating proteins of type II can exhibit the properties of the antagonist of the receptor N-formylmethionylleucylphenylalanine. Ricin is more effective in modulating the respiratory burst induced by the chemotactic peptide than agglutinin ricin. The modulating effect of ribosome-inactivating proteins of type II on neutrophils is likely to be mediated by their interaction with galactose rather than mannose receptors. Presumably, the affinity of ribosome-inactivating proteins to galactose receptors increases with increasing amount of saccharides bound to the protein molecule. The modulating effect of ribosome-inactivating proteins of type II on the respiratory burst of neutrophils induced the chemotactic peptide is due to the structural peculiarities of these proteins.

In the course of the study of the primary structures and molecular mechanisms of action of immunologically active compounds of the nervous system we have isolated from the soluble fraction of total bovine brain two heat-stable proteins. The purification procedure was mainly based on DEAE-Servacel ion-exchange chromatography and reversed-phase HPLC. The proteins were identified by the N-terminal Edman microsequence analysis and database searching as macrophage migration inhibitory factor (MIF). The N-terminal sequences for MIF1 and MIF2 were found to be identical. According to mass spectral analysis, the molecular masses for MIF1 and MIF2 were determined respectively as 12,369.21 and 12,299.7 Da. In addition, we have also isolated a third peptide having the same N-terminal sequence and Mr 9,496.2 that seems to be a proteolytic fragment of MIF. Using p-hydroxyphenylpyruvate as a substrate, we have not revealed tautomerase activity of either MIF1 or MIF2. As both the immunologic and enzymatic activities were reported to be expressed by the oligomeric structure of MIF, we suggest that the present study may give additional information on MIF in terms of structural properties of this protein. A comparatively simple purification procedure is presented that may be widely used for simultaneous isolation in one run of MIF isoforms.

The ability of viscum at different concentrations to modulate the respiratory burst in neutrophils, induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine was studied. This does not exclude the possibility that viscum can interact with the receptor of this peptide. The analysis of the primary structure of viscum revealed elements structurally analogous to the chemotactic peptide. It is assumed that viscum can exhibit the properties an antagonist of the receptor of N-formyl-methionyl-leucyl-phenylalanine, and the mechanism of action of viscum depends on its concentration.