Biomedical subjects

W Voelter

Publications and source records attributed to W Voelter.

At least 19 recordsLinked to original sources

PubMed2006

Synthesis of a new series of heterocyclic scaffolds for medicinal purposes.

A new series of substituted 8-fluro-4H-pyrimido[2,1-b] [1,3]benzothiazole-4-ones () substituted 7-methyl-4H-isoxazolo[2,3-a]pyrimidin-4-ones, and substituted 2-methyl-5,6,7,8-tetrahydro-9H-isoxazolo[2,3-a]pyridopyrimidin-9-ones, compounds I-VII, have been prepared via condensation of beta-keto esters with 2-aminopyridine derivatives, in the presence of polyphosphoric acid. The same technique has also been used to prepare diazepine compounds, VIII-X, by condensation of a gamma-keto ester with 2-aminopyridine derivatives. Details of synthetic procedures are shown. The new compounds have been characterized by elemental analysis, GC-MS, FT-IR and NMR spectrometry. Antibacterial, antifungal and anticancer (cytotoxic) activities, for three of these compounds, have been investigated and are presented.

Anti-Bacterial Agents↗

PubMed2004

Demonstration of antibodies to the chitin-binding mistletoe lectin (cbML) in tumor patients before and during therapy with an aqueous mistletoe extract.

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.

Adjuvants, Immunologic↗

PubMed2004

Carbohydrate moieties of molluscan Rapana venosa hemocyanin.

Using Zn2+ ions as new method, several FUs have been isolated from molluscan Hc Rapana venosa without formation of non-functional proteolytic side products. N-terminal sequences of these fragments in comparison with FUS from other gastropodan Hcs show a very high degree of structural identity. Four Fus, purified from enzyme-treated structural subunits RvH1 and RvH2 (RvH1-a, RvH1-f, RvH2-a and RvH2-e) show identical N-terminal sequences compared to fragments isolated after treatment with Zn2+ ions. However, in some cases trypsin cleaves RvH chains at different positions if compared to the Zn2+ treatment. To analyze the oligosaccharide composition of two FUS from the first structural subunit of Rapana Hc, RvH1-a and RvH1-f, several techniques were applied: capillary electrophoresis, MALDI-MS, ESI-MS in combination with glycosidase digestions. On basis of these results and the determined amino acid sequence two N-linkage sites were identified in the FU RvH1-a, but only one in the FU RvH1-f.

Amino Acid Sequence↗

PubMed2004

Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103 improves ram spermatozoa functions in vitro.

In this study we determined the effect of reactive oxygen species (ROS) generation during incubation in media at 39 degrees C on ram spermatozoa and the protection by exogenously added antioxidant enzyme, superoxide dismutase (SOD). A novel Cu/Zn-SOD, isolated from the fungal strain Humicola lutea 103 (HLSOD), was used. Our results point out that the levels of both, superoxide anion radicals (*O2-) and H2O2, increase approximately 8-10- and 2-3-fold, respectively, during incubation of spermatozoa. Enhanced ROS generation coincided with reduction of motility, independently of the type of diluted medium. Addition of HLSOD (30, 60 and 120 U ml(-1) sperm) improved sperm functions, maintaining almost initial percentages of motile spermatozoa and increasing the values of mean cytochemical coefficient. At the same time, a significant diminution of *O2- and H2O2 content in the presence of antioxidant enzyme was established. The results suggest that HLSOD is an effective *O2- scavenger in semen that leads to protection of sperm functions.

Animals↗

PubMed2002

Crystallisation under microgravity of mistletoe lectin I from Viscum album with adenine monophosphate and the crystal structure at 1.9 A resolution.

The crystal structure of the ribosome-inactivating protein (RIP) mistletoe lectin I (ML-I) from Viscum album in complex with adenine has been refined to 1.9 A resolution. High quality crystals of the ML-I complex were obtained by the method of vapour diffusion using the high density protein crystal growth system (HDPCG) on the international space station, mission ISS 6A. Hexagonal crystals were grown during three months under microgravity conditions. Diffraction data to 1.9A were collected applying synchrotron radiation and cryo- techniques. The structure was refined subsequently to analyse the structure of ML-I and particularly the active site conformation, complexed by adenine that mimics the RNA substrate binding.

Adenosine Monophosphate↗

PubMed2002

Rapana thomasiana hemocyanin (RtH): dissociation and reassociation behavior of two isoforms, RtH1 and RtH2.

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin isoforms, termed RtH1 and RtH2. The two subunit types, purified by ion exchange chromatography, were used for macromolecular reassociation studies. In vitro reassociation was achieved with Tris-saline stabilizing buffer at pH 7.4, containing 100mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation was monitored, and the different oligomeric forms of RtH1 and RtH2 were studied by transmission electron microscopy, using samples negatively stained with 1% (w/v) uranyl acetate or 5% (w/v) ammonium molybdate containing 1% (w/v) trehalose at pH 7.0. The two subunits reassociate to produce characteristic didecamers, oligomeric and polymeric forms depending on the dissociated material and the reassociation conditions (i.e. divalent ion concentration, duration). In contrast to the didecamers of the freshly isolated RtH preparations, RtH1 and RtH2 show after 2 weeks' reassociation a clear tendency to generate multidecameric structures. The behavior of the native RtH1 and RtH2 during reassociation in the presence of 100mM calcium and magnesium chloride corresponds to the reported common oligomerization characteristics of KLH1/HtH1 and KLH2/HtH2, respectively. It is important to note that during the reassociation of the RtH isoforms: (I) no smaller diameter tubular polymers (ca. 25-27nm) were formed from the subunits as well as from the decamers; (II) multidecamers with one or more 'nucleating' didecamers were detected in addition to the multidecamers, composed of didecamers with associated decamers at one or both ends.

Amino Acid Sequence↗

PubMed2001

Peptide HER2(776-788) represents a naturally processed broad MHC class II-restricted T cell epitope.

HER2/neu-derived peptides inducing MHC class II-restricted CD4+ T helper lymphocyte (Th) responses, although critical for tumour rejection, are not thoroughly characterized. Here, we report the generation and characterization of CD4+ T cell clones specifically recognizing a HER-2/neu-derived peptide (776-788) [designated HER2(776-788)]. Such clones yielded specific proliferative and cytokine [gamma-interferon(IFN)-gamma] responses when challenged with autologous dendritic cells (DCs) loaded with HER2(776-788). By performing blocking studies with monoclonal antibodies (MAbs) and by using DCs from allogeneic donors sharing certain HLA-DR alleles, we found that HER2(776-788) is a promiscuous peptide presented, at least, by DRB5*0101, DRB1*0701 and DRB1*0405 alleles. One TCRV beta 6.7+ clone recognized the HLA-DRB5*0101+ FM3 melanoma cell line transfected with a full length HER-2/neu cDNA. Moreover, this clone recognized the HER-2/neu+ SKBR3 breast cancer cell line induced to express HLA-DR, thus demonstrating that HER2(776-788) represents a naturally processed and presented epitope. Our data demonstrate that helper peptide HER2(776-788) represents a promiscuous epitope binding to at least three HLA-DR alleles, thus offering a broad population coverage. The use of antigenic peptides presented by major histocompatibility complex (MHC) class II in addition to those presented by class I may improve the therapeutic efficacy of active immunization.

Antigen Presentation↗

PubMed2001

Preliminary X-ray diffraction studies of the external functional unit RtH2-e from the Rapana thomasiana.

The 'external' oxygenated functional unit RtH2-e of the Rapana hemocyanin subunit RHSS2 was isolated and crystallized. X-ray intensity data to 3.3 A resolution have been collected at 100 K and the structure has been solved using the molecular-replacement method. The space group is assigned to be the tetragonal P4(3)2(1)2, with unit-cell parameters a = b = 105.5, c = 375.0 A.

Animals↗

PubMed2001

Primary structure, isoforms, and molecular modeling of a chitin-binding mistletoe lectin.

From mistletoe Viscum album L. extracts three chitin-binding lectin isoforms, cbML1, cbML2, and cbML3, were isolated and their primary structure determined. All three cbML isoforms are composed of two protein chains of 48 or 49 amino acid residues, linked by an intermolecular disulfide bond. The sequence of each single cbML chain is characterized by a relatively high number of cysteine and glycine residues, 9 and 6, respectively, and contains four intramolecular disulfide bridges. On the basis of the combined interpretation of sequencing and MALDI MS data, the following results for the three cbML isoforms were obtained: the first one consists of two identical truncated polypeptide chains (1--48), the second is a heterodimer, containing one truncated (1--48) and one full-length chain (1--49), and the third is composed of two full length chains (1--49). The cbML sequence shows 55% identity to hevein, a single-chain chitin-binding protein of 43 amino acids, one of the most predominant proteins in natural rubber latex. On the basis of the NMR data on hevein from Hevea brasiliensis the three-dimensional structure of cbML3 was modelled. The 26 sequence changes between cbML3 and hevein were accommodated with only little perturbation in the main chain folding. A comparison of the primary structures of cbML3 and hevein is shown and the effects of the sequence changes are discussed. Differences have been identified in the loop region of the molecule and the potential interface region of cbML3 supporting the dimer formation. The high-affinity chitin-binding site seems to be highly conserved.

Amino Acid Sequence↗

PubMed2001

Human HDAC7 histone deacetylase activity is associated with HDAC3 in vivo.

Histone deacetylases (HDACs) are part of transcriptional corepressor complexes and play key roles in regulating chromatin structure. Three different classes of human HDACs have been defined based on their homology to HDACs found in Saccharomyces cerevisiae: RPD3 (class I), HDA1 (class II), and SIR2 (class III). Here we describe the identification and functional characterization of HDAC7, a new member of the human class II HDAC family. Although HDAC7 is localized mostly to the cell nucleus, it is also found in the cytoplasm, suggesting nucleocytoplasmic shuttling. The HDAC activity of HDAC7 maps to a carboxyl-terminal domain and is dependent on the interaction with the class I HDAC, HDAC3, in the cell nucleus. Cytoplasmic HDAC7 that is not bound to HDAC3 is enzymatically inactive. We provide evidence that the transcriptional corepressors SMRT and N-CoR could serve as critical mediators of HDAC7 activity by binding class II HDACs and HDAC3 by two distinct repressor domains. Different class II HDACs reside in the cell nucleus in stable and autonomous complexes with enzymatic activity, but the enzymatic activities associated with HDAC7 and HDAC4 rely on shared cofactors, including HDAC3 and SMRT/N-CoR.

Amino Acid Sequence↗

PubMed2001

Purification, characterization, and primary structure of four depressant insect-selective neurotoxin analogs from scorpion (Buthus sindicus) venom.

Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the scorpion Buthus sindicus were purified to homogeneity in a single step using reverse-phase HPLC. The molecular masses of the purified toxins were 6820.9, 6892.4, 6714.7, and 6657.1 Da, respectively, as determined by mass spectrometry. These long-chain neurotoxins were potent against insects with half lethal dose values of 67, 81, 103, and 78 ng/100 mg larva and 138, 160, 163, and 142 ng/100 mg cockroach, respectively, but were not lethal to mice even at the highest applied dose of 10 microg/20 g mouse. When injected into blowfly larvae (Sarcophaga falculata), Bs-dprIT1 to 4 induced classical manifestations of depressant toxins, i.e., a slow depressant flaccid paralysis. The primary structures of Bs-dprIT 1 to 4 revealed high sequence homology (60-75%) with other depressant insect toxins isolated from scorpion venoms. Despite the high sequence conservation, Bs-dprIT1 to 4 showed some remarkable features such as (i) the presence of methionine (Met(6) in Bs-dprIT1 and Met(24) in Bs-dprIT2 to 4) and histidine (His(53) and His(57) in Bs-dprIT1) residues, i.e., amino acid residues that are uncommon to this type of toxin; (ii) the substitution of two highly conserved tryptophan residues (Trp43 --> Ala and Trp53 --> His) in the sequence of Bs-dprIT1; and (iii) the occurrence of more positively charged amino acid residues at the C-terminal end than in other depressant insect toxins. Multiple sequence alignment, sequence analysis, sequence-based structure prediction, and 3D homology modeling studies revealed a protein fold and secondary structural elements similar to those of other scorpion toxins affecting sodium channel activation. The electrostatic potential calculated on the surface of the predicted 3D model of Bs-dprIT1 revealed a significant positive patch in the region of the toxin that is supposed to bind to the sodium channel.

Amino Acid Sequence↗

PubMed2001

Carbohydrate composition of Carcinus aestuarii hemocyanin.

The hemocyanin of the crab Carcinus aestuarii contains a carbohydrate moiety that represents 1.6% of protein mass. This carbohydrate content is higher than that exhibited by other arthropod hemocyanins so far investigated. By combination of FPLC ion exchange chromatography and reverse-phase HPLC, the native oligomeric protein can be resolved into three major and one minor electrophoretically pure fractions that are found to be homogeneous by N-terminal sequencing and correspond to the subunit polypeptide chains. Sugar analysis on the different subunits reveals that the subunit referred to as Ca2 is glycosylated, with a carbohydrate content of 6.3%. By Ca2 trypsin digestion, separation of glycopeptides, and amino acid sequencing, three consensus sequences for O-glycosylation and one for N-glycosylation were found. MALDI-MS was applied for the determination of the molecular masses of the various glycopeptides and peptides after removal of carbohydrates by neuraminidase and alpha-N-acetylgalactosaminidase.

Animals↗

PubMed2001

Isolation and spectroscopic characterization of the structural subunits of keyhole limpet hemocyanin.

Keyhole limpet hemocyanin is a respiratory glycoprotein of high molecular weight from the gastropod mollusc Megathura crenulata. Two subunits, KLH1 and KLH2, were isolated using ion exchange chromatography and their physical properties are compared with the parent molecule. The various proteins are characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The conformational stability of the native aggregate and its isolated structural subunits are also studied by circular dichroism and fluorescence spectroscopy as a function of temperature, as well as in the presence of guanidinium hydrochloride and urea. The associated subunits in the hemocyanin aggregates increase considerably the melting temperature to 67 degrees C and the free energy of stabilization in water, DeltaG(H(2)O)(D), towards guanidinium hydrochloride is higher for the decamer as compared to the isolated subunits; this difference can be accounted for by the stabilizing effects of intra-subunit interactions exerted within the oligomer. The copper-dioxygen complex at the active site additionally stabilizes the molecule, and removing of the copper ions increases the tryptophan emission and the quantum yield of the fluorescence.

Animals↗

PubMed2001

Differences in the specificities of the highly alkalophilic proteinases Savinase and Esperase imposed by changes in the rigidity and geometry of the substrate binding sites.

Savinase and Esperase are closely related highly alkalophilic proteinases produced by Bacillus lentus. They are suitable couple for investigating the structural basis of proteinase specificity due to the identity of the catalytic and the differences in the substrate binding sites. Two of the substitutions in these sites are very important: T129P and G131P. The two prolines provide an extra rigidity of the Savinase-binding site. The substitutions S166N and Q191T in the S1 recognition loop change the binding geometry of the substrate P1 residue. The geometry of S1 in Esperase is more favorable for binding and catalysis in comparison to that in Savinase. Differences in P3 specificity are probably created by the substitution V104L, which influences the conformation of S3. Leu in position 104 is more favorable for the binding of Phe to S4 than Val. The lower affinity and catalytic efficiency as well as more narrow proteolytic specificity of Savinase in comparison to those of Esperase are explained with the extra rigidity and unfavorable changes in geometry of the substrate binding site of the first enzyme.

Alkalies↗

PubMed2001

The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold.

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain-like fold in solution, with two perpendicular, antiparallel, three- and five-stranded beta-sheets. In contrast to known structures with the SH3 domain fold, MIA is a single-domain protein, and contains an additional antiparallel beta-sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain-like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin alpha4beta1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.

Amino Acid Sequence↗

PubMed2001

A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies.

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.

Amino Acid Sequence↗

PubMed2001

Chalcone derivatives antagonize interactions between the human oncoprotein MDM2 and p53.

The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivation domain. The p53 gene is inactivated in many human tumors either by mutations or by binding to oncogenic proteins. In some tumors, such as soft tissue sarcomas, overexpression of MDM2 inactivates an otherwise intact p53, disabling the genome integrity checkpoint and allowing cell cycle progression of defective cells. Disruption of the MDM2/p53 interaction leads to increased p53 levels and restored p53 transcriptional activity, indicating restoration of the genome integrity check and therapeutic potential for MDM2/p53 binding antagonists. Here, we show by multidimensional NMR spectroscopy that chalcones (1,3-diphenyl-2-propen-1-ones) are MDM2 inhibitors that bind to a subsite of the p53 binding cleft of human MDM2. Biochemical experiments showed that these compounds can disrupt the MDM2/p53 protein complex, releasing p53 from both the p53/MDM2 and DNA-bound p53/MDM2 complexes. These results thus offer a starting basis for structure-based drug design of cancer therapeutics.

Antineoplastic Agents↗

PubMed2001

Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.

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.

Alkalies↗