Structure and Characterization of Eriphia verrucosa Hemocyanin.

Arthropod hemocyanins (Hcs) are a family of large extracellular oxygen-transporting proteins with high molecular mass and hexameric or multi-hexameric molecular assembly. This study reports for the first time the isolation and characterization of the structure of an arthropod hemocyanin from crab Eriphia verrucosa (EvH) living in the Black Sea. Its oligomeric quaternary structure is based on different arrangements of a basic 6 × 75 kDa hexameric unit, and four of them (EvH1, EvH2, EvH3, and EvH4) were identified using ion-exchange chromatography. Subunit 3 (EvH3) shows high similarity scores (75.0, 87.5, 91.7, and 75.0 %, respectively) by comparison of the N-terminal sequence of subunit 1 from Cancer pagurus of the North Sea (Cp1), subunits 3 and 6 of Cancer magister (Cm3 and Cm6), and subunit 2 of Carcinus aestuarii (CaSS2), respectively. Moreover, a partial cDNA sequence (1309 bp) of E. verrucosa hemocyanin encoding a protein of 435 amino acids was isolated. The deduced amino acid sequence shows a high degree of similarity with subunits 3, 4, 5, and 6 of C. magister (81-84 %). Most of the hemocyanins are glycosylated, and three putative O-linkage sites were identified in the partial amino acid sequence of EvH at positions 444-446, 478-480, and 547-549, respectively. The higher stability of native Hc in comparison to its subunit EvH4 as determined by circular dichroism (CD) could be explained with the formation of a stabilizing quaternary structure.

In vitro hypoxic cytotoxicity and hypoxic radiosensitization. Efficacy of the novel 2-nitroimidazole N,N,N-tris[2-(2-nitro-1H-imidazol-1-yl)ethyl]amine.

BACKGROUND AND PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: Our in vitro findings suggest that PRC might be a promising new 2-nitroimidazole for improving radiation therapy of hypoxic tumors in vivo.

6-Nitrobenzimidazole derivatives: potential phosphodiesterase inhibitors: synthesis and structure-activity relationship.

6-Nitrobenzimidazole derivatives (1-30) synthesized and their phosphodiesterase inhibitory activities determined. Out of thirty tested compounds, ten showed a varying degrees of phosphodiesterase inhibition with IC(50) values between 1.5±0.043 and 294.0±16.7 µM. Compounds 30 (IC(50)=1.5±0.043 µM), 1 (IC(50)=2.4±0.049 µM), 11 (IC(50)=5.7±0.113 µM), 13 (IC(50)=6.4±0.148 µM), 14 (IC(50)=10.5±0.51 µM), 9 (IC(50)=11.49±0.08 µM), 3 (IC(50)=63.1±1.48 µM), 10 (IC(50)=120.0±4.47 µM), and 6 (IC(50)=153.2±5.6 µM) showed excellent phosphodiesterase inhibitory activity, much superior to the standard EDTA (IC(50)=274±0.007 µM), and thus are potential molecules for the development of a new class of phosphodiesterase inhibitors. A structure-activity relationship is evaluated. All compounds are characterized by spectroscopic parameters.

The mistletoe lectin I--phloretamide structure reveals a new function of plant lectins.

The X-ray structure at 2.7A resolution of the complex between the European mistletoe lectin I (Viscum album, ML-I) and the plant growth hormone, 3-(p-hydroxyphenyl)-propionic acid amide (phloretamide, PA) from xylem sap has revealed the binding of PA at the so far undescribed hydrophobic cavity located between the two subunits of this ribosome-inhibiting protein. No such cavity is observed in related lectins. The binding of PA is achieved through interactions with the non-conserved residues Val228A, Leu230A, Arg388B, and the C-terminal Pro510B. It is conceivable that binding of PA to ML-I is part of a defence mechanism of the parasite against the host, whereby the parasite prevents the growth hormone of the host from interfering with its own regulatory system. The specific binding of PA to ML-I indicates that heterodimeric RIPs are multifunctional proteins whose functions in the cell have not yet been fully recognized and analyzed.

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.

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.

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.

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.

Combined protective effect of a fungal Cu/Zn-containing superoxide dismutase and rimantadine hydrochloride in experimental murine influenza a virus infection.

The combined protective effect of a novel naturally glycosylated Cu/Zn-containing superoxide dismutase, produced by the fungus Humicula lutea (HL-SOD) strain 103, and the selective anti-influenza drug rimantadine hydrochloride (Rim) was evaluated in experimental virus infection in mice, induced with influenza virus A/Aichi/2/68 (H3N2). A combined application of HL-SOD and Rim in doses, which by themselves did not protect significantly mice against the infection, resulted in a synergistically increased protection, determined on the basis of protective indices. Lung virus titers, lung weights and consolidation and mortality rates were all decreased significantly, while survival times were prolonged.

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.

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.

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.

Viviparus ater hemocyanin: investigation of the dioxygen-binding site and stability of the oxy- and apo-forms.

The active site of Viviparus ater (mollusc) hemocyanin was investigated using the fact that the binding of dioxygen to the binuclear copper-containing sites of hemocyanins is connected with the appearance of specific dichroic bands which are very sensitive to changes in the structrure and polarity of the environment. Oxy-Viviparus ater hemocyanin exhibits near UV and visible circular dichroism spectra different from those of other molluscan and arthropodan hemocyanins. These differences are due probably to variations in the geometry or charge distribution in the dioxygen binding sites of the compared proteins. The thermostability of Viviparus ater hemocyanin and the significance of the copper-dioxygen system for the stability were also investigated. "Melting" temperatures, Tm, of 77 degrees C for the oxy-hemocyanin and 57 degrees C for the apo-protein were calculated from the denaturation curves which demonstrates the considerable role of the binuclear active site for the thermostability. Viviparus ater hemocyanin is more thermostable than other hemocyanins for which data are published.

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.

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.

[Protein analysis of enzyme tablets]. / Proteinanalytische Untersuchungen an Enzymtabletten.

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.

Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo.

Angiogenesis is an ordered process requiring the inter-play of numerous cellular and humoral factors. Studies over the past 20 years have identified several growth factors, cytokines, and enzymes that promote blood vessel formation. Most have revealed how individual factors promote an angiogenic phenotype in endothelial cells in vitro or contribute to blood vessel formation in vivo. However, the fundamental question that remains unanswered is how the cellular microenvironment contributes to angiogenesis. Fibrocytes are a recently characterized mesenchymal cell type isolated from peripheral blood that rapidly enter subcutaneously implanted wound chambers and sites of tissue injury. Here we describe the induction of an angiogenic phenotype in microvascular endothelial cells in vitro and promotion of angiogenesis in vivo by cultured fibrocytes. Fibrocytes constitutively secrete extracellular matrix-degrading enzymes, primarily matrix metalloproteinase 9, which promotes endothelial cell invasion. In addition, fibrocytes secrete several proangiogenic factors including VEGF, bFGF, IL-8, PDGF, and hematopoietic growth factors that promote endothelial cell migration, proliferation, and/or tube formation. By contrast, they do not produce representative antiangiogenic factors. Finally, both autologous fibrocytes and fibrocyte-conditioned media were found to induce blood vessel formation in vivo using the Matrigel angiogenesis model.

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.

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.

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.